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MCB 12OL MOLECULAR BIOLOGY amp BIOCHEMISTRY LABORATORY MANUAL WINTER 2014 2 523 rquot 39I l 2 lquot L S l nm l ill JrIn FE 39 p Phycocyanobilin Department of Molecular amp Cellular Biology College of Biological Sciences University of California at Davis Copyright UC Regents Davis campus 200514 All Rights Reserved May not be redistributed without prior written consent Of course instructor TABLE of CONTENTS Page Teaching Labs Safety Rules and Guidelines MCB 120L l40L 160L Experiment 0 Pipetting Spectrophotometry pH meter Single and multichannel pipettesspectrophotometers39 doublebeam microplate reader and nanodrop pH meter operation 0 Experiment 1 Biological Buffers Properties and preparations Experiment 2 Protein Assays Properties and applications of the Bradford and A280 protein assays 21 Experiment 3 Enzyme Kinetics Alkaline Phosphatase reaction rates as a function of pH enzyme concentration and substrate concentration and the effects of inhibitors specific activity of Lactate Dehydrogense 3 Experiment 4 Isolation amp Ampli cation of a Protein Coding Gene by the Polymerase Chain Reaction Amplification of a GAF 4 Domain Restriction Enzyme amp A garose Gel Analysis Primer design for directional cloning amp creating an Intein tsion protein Experiment 5 Expression amp Puri cation of a Recombinant Protein Nostoc GAF4 puri cation via Intein affinity column 51 Experiment 6 Electrophoresis of Proteins by SDSPAGE Analysis of the purification of the GAF4 Domain from Exp 5 61 Experiment 7 Expression amp Westem blot Analysis of a Recombinant Protein Investigate the expression of the Synechocystis Biliverdin Reductase Bvrd Primer design for directional cloning amp creating a streptag epitope fusion protein 71 Experiment 8 Introduction to Internet Proteomics Tools 8 Appendix Study Problems designed to help understand concepts Online in the class Smartsite Teaching Labs Safety Rules and Guidelines MCB 120L 140L 160L General Rules These are UC Safety regulations that must be followed and students will be ask to leave the lab as necessary if these rules are not met 1 No food or drinks allowed in lab 2 Dress Code a Closed toe shoes and closed heel shoes tops of the feet must be covered b Long pants past the knee when seated c Long hair should be tied back especially when working with flames 3 No pipetting by mouth use mechanical pipetting devices 4 Do not throw glass waste into the regular trash cans Any and all broken glass must be discarded in the quotglass waste boxes 5 Know the location of fire extinguishers in the lab To use a fire extinguisher pull out the metal pin near the handles on top point the nozzle at the flame and squeeze the handles 6 Know the location of emergency showers and eye wash stations in the lab 7 Emergency evacuation procedures eg if the fire alarm goes off turn off all flames turn off equipment evacuate to the paved area outside the building on the north side of SLB by Biobrew DO NOT LEAVE THE AREA until the instructor checks the class list to ensure all students made it out of the building 8 Any student TA or instructor may access the Materials Safety Data Sheet MSDS for any chemical at i39 iquotcp fec1emxvatlth 2eiquot39lti1e239 xIs 3c quots1ei cEssEboai39cl General Guidelines Wearing Gloves and Safety Glasses The wearing of gloves and safety glasses are not mandatory however wearing them is encouraged if you wish to do so There will be procedures requiring gloves and or safety glasses and the Instructor will inform and instruct the class when and why certain procedures require them Lab coats are not required 1 The Instructors and Staff involved in the MCB teaching labs whenever possible have eliminated or greatly reduced the use of hazardous and carcinogenic chemicals Also the protocols have been tailored to eliminate or greatly reduce hazards from spilling splashing breaking etc mostly by using small volumes and directing the use of pipets for acquiring solutions instead of pouring from large bottles 2 Use of Safety Glasses These are onesizefitsall so secure them as best you can They are impact resistant and block UV light You may wish to clean the glasses before use if so then please use the INTRODUCTION TO THE LABORATORY 1 Reproducible pipetting PipetmanTM single sample pipettes Finnpipette multicharmel pipettes 2 Spectrophometry Theory and Operation Schimadzu Spectrophotometer Photometric and Spectrum functions Microplate Reader Basic Operations Nanodrop Spectrophotometer Basic Operations 3 Calibrating the pH meter Day 1 Each student is to complete all of the exercises individually A REPRODUCIBLE PIPETTING GRAVIMETRIC ANALYSIS 1 Pipetman Micropipets Speci cations Accuracy Accuracy means the closeness with which the dispensed volume approximates the volume set on the pipette Accuracy is speci ed as mean error the maximum amount by which the mean value of a large number of replicate measurements of the same volume will deviate from the set volume Precision Precision means the scatter of individual measurements around the mean of a large number of replicate measurements of the same volume how close the values are to one another Speci cations for precision are generally tighter than for accuracy In most experiments where sample measurements are compared to standards the precision speci cation will detennine the accuracy of results as long as both samples and standards are measured with the same Pipetmarz PIPETMANTM SPECIFICATIONS Volume Increment Accuracy Precision Model pl pl mean error repeatability Relative Absolute ul Relative Absolute ul 2 5 01 2 004 P20 10 002 1 01 05 005 20 1 02 03 006 50 1 05 04 02 P200 100 02 08 08 025 025 200 08 16 015 03 100 3 3 06 06 P1000 500 2 08 4 02 1 1000 08 8 013 1 3 2 Operation Practice pipetting with a Pipetman 1 1 Adjust the P1000 pipetting device to 09 ml 900141 Always dial down to the desired volume 2 Attach a disposable tip to the end of the pipette shaft and press on rmly to ensure an airtight seal 3 Depress the plunger to the FIRST POSITIVE STOP and hold in place 4 Holding the pipette vertically immerse the end of the disposable tip into a beaker of water With the tip immersed in water gently release the plunger and allow the pipette tip to ll with liquid Never let it snap up by just letting go of the plungerpushbutton Wait a few seconds to make sure that the full volume of liquid is drawn up into the pipette Note be sure air bubbles are not drawn up into the pipette 5 To dispense the sample place the tip against the wall of a 15 ml plastic microcentri lge tube in a tube rack then depress the plunger slowly to the FIRST POSITIVE STOP and continue depressing the plunger through to the SECOND FULL STOP position to fully eject the liquid in the pipette tip Note placing the pipette tip in contact with the plastic surface of the microcentri lge tube is critical to reproducible pipetting The contact tends to leave about the same amount of liquid on the pipette tip at each stage so that the volume transferred is reproducible 6 With the plunger still fully depressed withdraw the pipette from the microcentri ige tube 7 Allow the plunger to slowly return to the TOP POSITION prior to pipetting another sample or discarding the used pipette tip 3 Gravimetric calibration Each of the six Pipetrnanm in the drawers will be checked for accuracy and precision one student checks a P 1000 a P200 and a P20 while another student sharing the bench checks the other three Below is a Table for recording the calibration of the pipettes 1 Turnon the balance and wait for it to read zero if it does not automatically read zero after a few moments then press tare to zero the balance 2 Place a small weighing boat on the analytical balance and use the tare feature to zero the balance 3 Using the P l 000 Pipetman measure 1000 ml of dH2O into the weighing boat Record the weight of the water One ml of water should weigh 1000 g Then check the other two pipettes at their maximum settings 4 The precision limits listed in the Speci cations table above are ideal ie brand new and carefully used these Pipetman pipettors are heavily used and often by novices Therefore if any of the pipettes does not dispense a volume within i 10 of the volume being measured eg set at 500 141 should dispense volume within d 50 ul bring the pipette to the instructor the instructor may examine it and then ask that the calibration be repeated If the problem persists then bring it back to the instructor who will send it for repair If at any time the pipette volumes are not accurate then check the pipette again if an error in accuracy is validated bring the pipette to the instructor for repair CALIBRATION of PIPETMANTM PIPETTES Date P1000 P200 P20 Expected Actual mg Expected Actual mg Expected Actual mg mg ul mg H1 mg M1 1000 200 20 500 100 10 If needed to check the accuracy of a pipet later in the course 1000 200 20 500 100 10 1000 200 20 500 100 10 B REPRODUCIBLE PIPETTING SPECTROPHOTOMETRIC ANALYSIS 1 Theory Relationship between Absorbance and Concentration The absorbance A of a lightabsorbing solution also called optical density OD is de ned as log ItIo 1 where I0 is the intensity of the incident monochromatic light and It is the residual intensity after passing through an absorbing solution ie the transmitted light A solution containing a light absorbing substance may be so concentrated as to not permit any light to pass through or extremely little amount of light to pass such conditions are not useful for spectrophotometric measurements Ideally a solution of a light absorbing substance will be dilute enough where absorbance depends on the concentration of tl1e absorbing material c and the length of the optical light path through the solution 1 according to the equation AoLlc 2 Aalc 3 where on and 3 are proportionality constants of a particular lightabsorbing species being measured these equations are known as the BeerLambert Law or Beer s Law When c is de ned for example in units of mgml then on is used and is called an absorption coefficient having units of mgml 1cmquot1 When c is de ned in units of molesliter ie M then the symbol a is used having units of M 1cm 1 or mMquot1cmquot39 or ulV1391cm39l and is called the molar absorption coefficient or molar extinction coefficient The molar absorption coef cient 8 is adopted as the standard unit for comparison whereas on is used for a substance whose molecular weight is indeterminate such as a solution of RNA or DNA The absorption coef cients vary with temperature pH ionic strength and type of solvent along with the wavelength of the incident light which should always be indicated The light path I for a given instrtunent and cuvette are generally known Thus the concentration of a solution of an absorbing substance can be calculated from a single absorbance measurement and knowledge of the an absorption coef cient value This is apparent from the simple rearrangement of equations 2 or 3 to yield cAat or cAel 4 Equations 2 or 3 are more convenient to use in analytical work than is equation 1 It states that the absorbance is directly ie linearly proportional to the concentration of the absorbing compound Beer s Law does not hold for concentrated solutions as absorbance approaches 20 where 99 of the incident light is absorbed with very little transmitted light that is dif cult to measure accurately Because of this the concentration range that is linearly related to absorbance must be determined experimentally for each substance Also note that any wavelength where light is absorbed is potentially available for determining the concentration of a substance It is preferable to use the point of maximum absorbance for greatest accuracy The use of any wavelength generally depends on two factors the absorbance value at a given concentration and whether or not other substances in the solution are absorbing light at that wavelength High quality doublebeam spectrophotometers such as the Shimadzu instrument used in this laboratory and most conventional spectrophotometers in research labs employ cuvettes with a 1cm light path for measuring the absorbance of solutions Reliable readings can be made up to an absorbance of 20 but it is 04 generally advisable not to exceed absorbance values of 15 Two other spectrophotometers also are used in this laboratory a Microplate Reader and the Nanodrop Spectrophotometer each are discussed below 2 Operation of the Shimadzu UVVIS Spectrophotometer 1 Reproducible pipetting using spectrophotometric measurements 0 Place ve l5 ml plastic microcentri ige tubes in a rack and pipet 980 pl of dH2O into each tube 0 Introduce 20 ul of 01 bromphenol blue dye solution into each tube using a P20 Eject the dye beneath the water surface Mix the contents of each tube thoroughly 0 Shimadzu spectrophotometer select the Photometric Mode and set the wavelength at 591 nm Obtain g plastic cuvettes 15 ml volume 0 Place one cuvette of water in the reference back compartment This cuvette will remain as the reference cuvette 0 Place a cuvette of water in the front compartment this cuvette is the sample cuvette Then select the autozero function The autozero function electronically sets the transmitted light signals from the reference and sample cuvettes to the same value The spectrophotometer will now only read the transmitted light of the sample introduced in the sample cuvette I Transfer the samples one at a time to the sample cuvette and measure the absorbance of each solution at 591 nm in the spectrophotometer It is good practice to return the contents of the cuvette to its microcentrifuge tube for later measurements if a mistake was discovered Be sure to rinse wash the sample cuvette between each reading often one good shake is sufficient to remove excess solution remaining in the cuvette before adding the next sample to be measured Enter the values in the Table 01 below for Trial 1 Calculate the mean m standard deviation sd and percentage error sdm x 100 for Trial 1 The A591 values should agree within a standard deviation of 1 0025 or lower the percentage error should be 5 or lower if not then repeat this exercise ie Trial 2 Table 01 Testing the reproducibility of pipetting Test Tube A591 A591 rial 1 rial 2 if m n tandard Deviation sd e Error Standard Deviation for measurements of less than 20 samples is de ned as 1 2 1 N1 1 where x are the individual absorbance measurements m is the mean absorbance value and N is the total number of samples measured For calculators with programmable statistic functions choose the n1 key for the standard deviation A calculator or MS Excel can be used 2 Veri cation that 591 nm was a good wavelength to measure absorbance of bromphenol blue 0 Follow the directions for performing Overlay Spectra on page 0 13 for bromphenol blue 0 Prepare one milliliter of a 120 dilution of bromphenol blue Transfer the diluted sample to a cuvette run the spectrum 0 Use the cursor arrows on the spectrophotometer to identify and record the following values 9 max Abs at l max Abs at 595 nm 0 Prepare one milliliter of 110 dilution of bromphenol blue 0 Transfer the diluted sample to a cuvette run the spectrum Use the cursor arrows on the spectrophotometer to identify and record the following values 7Lmax Abs at 7L max Abs at 595 nm 0 Last Print the spectrum by choosing the Print function there should be two spectra on the same spectral graph Tips For Good Pipetting 1 Avoid multiple pipettings of a single substance into a tube whenever possible Each pipetting introduces an error If 40 ul of a substance is needed do not perform two 20 ul pipettings with the P20 instead perform a single 40 ul pipetting from the P200 2 Always pick up the tubes and watch to see that the sample is being properly delivered into the tube 3 Always keep the pipette in the vertical position Tilting the pipette even slightly allows liquid to ow into the pipette which will ruin the pipette and contaminate the next sample Tips For Good Spectrophotometry 1 Always auto zero the spectrophotometer with two cuvettes containing water in the front and back cuvette compartments The water used in the cuvettes should be from the same beaker of water as the water used in the experimental procedure This is especially important when reading in the UV range 2 The back cuvette should contain water In the event of having reagents with color the absorbance of the reagent alone should be read just as all the other samples in the assay are read known as the reagent blank The reagent blank does not contain the experimental variable to be measured that interacts with the reagent such as protein in a protein assay the reagent blank is the control sample for the contribution to the absorbance due to the reagent itself After measuring the reagent blanks and the experimental samples corrected absorbance values are obtained by subtracting the average reagent blank value from each of the sample tubes in the assay Abs comted Abs experimental Abs ban 06 Name Turn in this page with writeup attached at the end of the lab period for Day 1 DATA ANALYSIS AND POINTS FOR DISCUSSION 1 Report the best results from Table 01 in the table below Test Tube Best result trial A591 1 2 3 4 5 can m Standard Deviation Sd Percentage Error 2 Attach the printed overlay spectra of the bromphenol blue Solution i Was 591 nm the best wavelength choice to measure dye absorbance in the exercise ii What other wavelengths could be used iii IS 595 nm a good wavelength for measuring bromphenol blue Why or why not iv Do the absorbance values recorded at 591 nm and or 595 nm follow Beer s Law Why or why not Day 2 Each student is to complete all of the exercises individually C REPRODUCIBLE MULTICHANNEL PIPETTING AND MICROPLATE SPECTROPHOTOMETRIC ANALYSIS 1 Multichannel Pipettes Thermo Electron Corp FINN PIPETTE Speci cations There are two multichannel pipettes used in this course which are set out in the lab when needed One has a pipetting volume range of 5 pl to 50 pl and the other has a pipetting volume range 30 pl to 300 pl MULTICHANNEL PIPETTE SPECIFICATIONS Range Increment Volume Accuracy Precision model sd pl 550 pl 0 pl 50 pl 030 pl l06 015 5 pl 015 pl 30 0125 30300 pl 1 pl 300 pl I18 pl l06 06 30 pl 045 pl ll5 0108 sd Standard Deviation 2 Operation Important For the 550 pl pipette MI pYI l 0 F I I OI H H p 0 zH I the upper yellow knob dials the ones and tens place from 5 to 50 pl the lower knob dials the tenths place between 00 to 09 pl For the 30300 pl pipette IF A E P9E E p D 9 1 quot 1 the upper orange knob dials the tens and hundreds place from 30 to 300 pl the lower knob dials the ones place between 00 to 90 pl Upper Orange Knob for the 30300 pl pipette Rr II falt1 39 quot 3 i ll quot 3939 5 7 Ia I 39iquotI 39 E I quot J L 3939 quotquot quot J 39 quot e 1 pa gt3 n gg P0 Me Ev ig 1 p r 0N 3939s 1 a 1 p p lt2 3 15 I 3 3 ili 3 ans 2 i39E FE 3Jig iii 5 Upper Yellow Knob for the 550 pl pipette Lower Knob Each multichannel pipette holds eight tips 1 Obtain a plastic trough dispense several milliliters of dH20 into the trough 2 Practice simultaneously setting the eight tips on the pipettes from the box of yellow tips place rmly on the eight tips remove gently from box Do not pound the pipette onto the tips 3 Set the pipettes to any midrange volume and draw up water from the trough Visually inspect the tips and ask whether or not the volumes in each of the eight tips are equivalent if not practice setting the tips on 09 6 Using the 30300 pl multichannel pipette transfer 125 pl of the solution from Column 1 to Column 2 Rows A D mix well with repeated pipette action careful not to introduce bubbles best accomplished by drawing up the solution 39om the bottom of the well and dispensing the solution near the top of the well Repeat the 125 pl transfer and mixing om Column 2 to Colunm 3 and then from Column 3 to Colunm 4 for Rows A D After mixing Column 4 remove the last 125 pl and discard 7 Using the 550 pl multichannel pipette transfer 25 pl of the solution from Column 7 to Colunm 8 Rows A D mix well with repeated pipette action care il not to introduce bubbles best accomplished by drawing up the solution om the bottom of the well and dispensing the solution near the top of the well Repeat the 25 pl transfer and mixing from Column 8 to Colunm 9 and then from Column 9 to Column 10 for Rows A D After mixing Column 10 remove the last 25 pl and discard 8 In colunms 5 and 11 transfer 125 pl dH2O to each well using the 30300 pl multichannel pipette 9 Each well has an equal volume of 125 pl thus equal path lengths for the transmitted light to pass from the top of the solution through the bottom of the plate 10 Take the microplate to the microplate reader place the microplate in the holder on instrument 11 At the computer controlling the microplate reader select the program BRADFORDSEE from the Sessions tab found on the upper right corner or if the Bradford program is already open go to 12 The BRADFORDSEE program measures at 595 nm close to the 591 absorption maxima for bromophenol blue determined above from the spectnun 12 Click on the START button found along the top tool bar If the START button is not visible then click on the Procedure tab found along the right edge of the program screen The plate will be shaken the absorbances will be read and the results will be printed all automatically If the program is already open it might ask if the previous results should be saved select CONTINUE 13 Take away the microplate and the printout of the results 14 Wash the microplates they are reused a shake the contents of the assay out of the wells into a sink b rinse with warm tap water and vigorously shake the water out of the wells c rinse lightly with 50 ethanol a couple of times and vigorously shake the ethanol out of the wells d rinse with warm tap water a couple of times and vigorously shake the water out of the wells e repeat c amp d until no blue is visible LASTLY rinse with dH2O D Nanodrop Spectrophotometer Basic Operation 1 Nanodrop Spectrophotometer The Nanodrop spectrophotometer measures UV and visible absorbances directly in a 2 pl sample volume The sample is loaded onto a ber optic cable and the absorbance is measured across 1 mm gap distance ie the path length is 1 mm for this instrument This spectrophotometer is fast and obviously saves sample volumes The other major advantage of using this spectrophotometer is that solutions resulting in unacceptable absorbances greater than 20 in a conventional 1 cm path length cuvette might result in acceptable absorbances with a the 1 mm path length without having to dilute the sample apply algebra to the Beer s Law equation Abs sol for 1 cm will result in Abs10 ecl for 1 mm 010 2 Procedure 1 The user is set to Default in the Nanodrop startup menu and UVVis is selected The instructor or TA will initialize the instrument Initialization is necessary only at the beginning of the lab period 2 In the upper left hand corner of the screen note the display UVVis Module Before measuring a sample scru the pedestals of the Nanodrop briskly with a Kimwipe Please treat the Nanodrop gently DO NOT lift the arm by the exible ber optic cable DO NOT bang the arm down onto the bottom pedestal Lower the arm gently 3 Measure the absorbance of a diluted bromphenol blue sample prepare a sample by pipetting 980 pl of dH2O into a 15 ml microfuge tube using the P1000 and introduce 20 ul of 01 bromphenol blue dye solution into the tube using an appropriately adjusted P20 Mix well a Load 2 ul of dH2O onto the bottom pedestal Lower the ber optic cable arm gently b Find the button labeled Blank on the UVVis screen and click it The machine will create a meniscus between the two arms and display a blank screen Find the box marked 9 1 on the right hand side of the screen and enter 91 into it Find the box marked L 2 and enter 280 into it c Wipe the water off the pedestals with a Kimwipe Load 2 pl of the diluted bromphenol blue sample onto the lower pedestal Click the button labeled Measure When the machine displays the spectrum record Absorbance at 591 mn measured at a 1 mm path length Also on the spectrum is a red line which is the absorbance measured at a 01 mm path length d After measuring the sample clean the pedestals with H20 and a Kimwipe e Select Print Screen to obtain a printout of the spectrum results E CALIBRATING THE PH METER 1 Using the pH Meter The electrode of the pH meter is extremely sensitive and fragile It should be handled with care to avoid mechanical damage and should not be allowed to become dry The electrode should be rinsed only by directing a stream of distilled water from a wash bottle onto the sides of the electrode The electrode should not be wiped dry with a tissue Instead the last drop of liquid on the tip of the electrode can be removed by touching it with a Kimwipe After use the electrodes should be left in a slightly acidic aqueous solution such as 001 N HCl or saturated KCl 2 Procedure for Calibrating the pH Meter 1 The pH meter is programmed to receive pH standard buffer solutions in a certain order Turn power ON Press the 1 key this enters the calibration mode of the pH meter Choose quotB1quot by pressing either the T or 1 keys and then press quotReadquot Bl should read 700 400 1001 and 168 These are the pH buffer standards that the machine expects 2 Fill 3 small plastic cups nearly to the top one each with the standard pH buffers 400 700 and 1001 The plug is part of the KCl bridge contact between the plug and test solution is required to complete the electrical circuit 011 3 Take the pH electrode out of the KCl storing solution rinse it with water and place the electrode in the pH 700 buffer standard Press quotCalquot calibrate The decimal in the pH reading will ash as the instrument measures this pH standard When it stops ashing take the pH electrode out of the solution rinse with water and place the electrode in the pH 400 standard Press quotCalquot and wait for the reading to stop ashing The electrode is now calibrated between pH 4 to 7 4 Press quotReadquot The pH meter is now reading the actual pH record the pH in the Table below Press pHmV This merely switches between reading pH and mV Record the mV in the Table below 5 Rinse the electrode with water and then place it in the pH 7 standard Press Read and record the pH and mV in the Table below 6 To calibrate between pH 7 to 10 press Cal When it stops ashing take the pH electrode out of the solution rinse with water and place the electrode in the pH 1000 standard Press quotCalquot and wait for the reading to stop ashing The electrode is now calibrated between pH 7 to 10 Table 02 pH and Voltjge First reading Second reading Average reading pH mV pH mV pH mV pH 4 Standard pH 7 Standard I I I pH 10 Standard 7 Press Read and record the pH and mV in the Table 02 8 Rinse the electrode with water and then place it in the pH 7 standard Press Read and record the pH and mV in the Table below 9 Repeat the entire calibration and reading a second time record data in Table 02 below 4 Calibration of pH Meter for Experiments Calibrate the pH meter every day that you use it Calibrate the pH meter using the standard buffers Standard buffers at pH 7 and pH 10 are used if the solution to be measured is expected to be pH 2 7 and standard buffers at pH 7 and pH 4 are used if the solution to be measured is expected to be pH 5 7 This pH meter automatically measures the temperature of the solution However it is not necessary to check the voltage readings every time you calibrate the pH meter Name the lab period for Day 2 DATA ANALYSIS AND POINTS FOR DISCUSSION 1 Microplate reading of the bromphenol dilution series i Attach the microplate printout 012 Turn in this page with writeup attached at the end of ii Calculate the dilution Series for each of the bromophenol blue dilutions created in the microplate For example the 150 dilution in Column 1 was diluted 12 into Colunm 2 when 125 pl of the dye was transferred from Column 1 to 125 pl H20 in Column 2 for a 1 100 dilution 150 x 12 and the 150 dilution in Column 7 was diluted 16 in Colunm 8 when 25 ul of the dye was transferred from Column 7 to 125 pl H20 in Column 8 for a 1300 dilution 150 x 16 Record the nal dilutions for each Column in Table 03 below iii Calculate the average corrected absorbance for each dilution series record in Table 03 below Plot the Corrected Average Absorbance versus the Dilution use MS Excel or graph paper iv Do the average corrected absorbances follow Beer s Law Table 0 3 Bromphenol Blue Dilutions Microplate Protocol 1 2 3 4 5 6 7 10 11 12 nal dilutions 150 1100 H20 U50 1300 H20 average abs ave corrected abs 00 00 2 Nanodrop result Compare the absorbance values at 591 nm for the diluted bromphenol blue dye obtained om the Nanodrop spectrophotometer and the Shirnadzu Spectrophotometer Brie y explain the differences between these values 3 Prepare a plot i From the data in Table 02 plot Voltage yaxis dependent variable units mV versus pH xaxis independent variable units pH ii Is there a linear relationship between pH and voltage Table 02 pH and Voltage Average reading pH mV pH 4 Standard pH 7 Standard pH 10 Standard 0 13 DIRECTIONS FOR PERFORMING OVERLAY SPECTRA USING THE SHIMADZU UV160 With the overlay option in the Spectrum Mode of this spectrophotometer two or more spectra are plotted on the same output Spectra are read from longer to shorter wavelengths since shorter wavelengths with higher energy can be more destructive to the absorbing substance than the longer wavelengths a Choose quotSpectrumquot mode from the main menu b Set wavelength range from high to low wavelength as directed bromphenol blue 700 nm to 400 nm use plastic cuvettes pnitrophenol 550 nm to 260 nm use quartz cuvettes BSA lysozyme amp gelatin 350 nm to 260 nm use quartz cuvettes Bradford reagent 1 BSA 800 nm to 400 nm use plastic cuvettes NADH NAD 400 nm to 220 mn use quartz cuvettes c Set absorbance scale 000 to 200 d Choose 6 Display mode from menu Push 6 until Overlay function is displayed Leave other parameters at default settings e Perform a baseline correction essentially autozero across the entire spectrum of wavelengths Place the cuvettes with water in the spectrophotometer then on the softkey pad press the F1 key f Leave the reference cuvette with water in the reference chamber Place the first sample in the sample cuvette chamber and press quotstartquot This produces the rst spectrum g Use the cursors arrows to manually locate the wavelength of peak absorbance and any other desired wavelengths for absorbance readings The wavelength and its associated absorbance are displayed at the upper right corner of the screen Write these data in the lab notebook Once the next step b is performed data from the current spectrum cannot be reacquired 11 Place the second sample in the sample cuvette chamber and press quotstartquot which removes the cursor arrow function Press start a second time which produces the second overlay spectrum Once the scan is nished go to g above to manually nd the peak wavelength and its absorbance Repeat start sequence for a third sample if required i Once all of the samples for the overlay spectra have been collected on screen and the desired absorbances obtained press quotprintquot on the keypad to print a hard copy of the overlay spectra j If the peaks are too low lt 10 then rerun the spectra with the scale from 000 to 100 k Label each spectrum with the name of the compound or with the treatment of the compound 11 Experiment 1 BIOLOGICAL BUFFERS Objectives 1 Prepare buffers by three methods measure their pHs 2 Examine buffer capacity 3 Determine the pH of buffers using a pH indicator dye Introduction A large fraction of the constituents of cells are weak acids and some are weak bases for example proteins and amino acids nucleic acids and nucleotides fatty acids and most metabolic intermediates Since the acquisition of a proton can cause an unchargedbase to take on a positive charge ie NH3 H gt NH4 or can neutralize a negative charge ie RCOO H ltgt RCOOH the ionic forms of the many molecules that exist in a cell are very much dependent on the intracellular pH For experiments in vitro the pH must be set and maintained at a value that will assure appropriate levels of the quotbiologically activequot ionic forms of the molecules being examined The maintenance of pH is accomplished by the introduction of a buffer into the biochemical solution Buffers resist changes in pH by xing the ratios of protonated and unprotonated forms of all ionizable groups within the solution The relation between pH and the ratios of the protonated and unprotonated forms of weak acids and weak bases is described by the HendersonHasselbalch equation The HendersonHasselbalch equation is revisited here because it is not only experimentally important in the design of buffers but also is central to the understanding of many laboratory procedures for example separating and identifying molecules determining pKa values moderating chemical reactivities etc The dissociation of a Bronsted protonic general acid HA is represented by the chemical equation HA2 lt H AZquot 1 where z and zI are the net charges on the conjugate acid HAZ and A24 its conjugate base Examples of Bronsted acids are Bronsted Acid 2 value NH4 1 CH3COOH 0 1I3NCH2COO39 0 P044 2 Note especially in the case of zwitterionic glycine that z is the algebraic sum of the charges on that species The z superscripts have been omitted from equations 2 to 5 for clarity The Law of Mass Action establishes a quantitative relationship between the chemical activities of an acid and its dissociation products Ka 213 a 113 A 1 2 3HA HA 39YHA 12 where Ka is a constant at constant temperature and pressure and a is the activity of the species Activity is a measure of the reactivity of a species and can be equated to the concentration multiplied by the activity coef cient 7 of the species Under ideal conditions of dilute solutions the activity coef cient is 10 thus the activity is equated to the concentration of a species Substituting in concentrations of the conjugate base A and conjugate acid HA and rearranging the equation results in the following expression K 0 39 a J 3 HA where K3 is the apparent equilibrium constant and K3 is a function of all ionizable species present in the buffer solution ionic strength interactions between species and possibly by temperature The pKa values for common biological buffers is given at the end of this section see Table 11 The most useful relationship that applies to buffer behavior is derived from equation 3 by taking the logarithms of both sides A logKa39loga log 4 HA Since by de nition pH log aH and pKa log Ka39 5 6 equation 4 can be rearranged to give the familiar HendersonHasselbalch equation A pH pK3910g 7 FM Since K3 is approximately constant it follows from equation 7 that the pH depends primarily on the A HA ratio and very little on the total buffer concentration buffer total A HA Furthermore equation 7 indicates that a particular buffer mctions within a pH range near its pKa a simple mathematical exercise illustrates this if the pH of a solution is 2 pH units or more below the pKa the concentration of the conjugate base A approaches 0 and the conjugate acid HA approaches 100 Conversely a pH that is 2 pH units above or more above the pKa shows the concentrations of the conjugate base and conjugate acid approaches 100 and 0 respectively In such situations where the buffer has been fully titrated by acid or base to either 100 of its conjugate base or conjugate acid the buffering capacity of the buffer has been exceeded ie the buffer is no longer functioning as a buffer Choice of a Buffer and Buffer Capacity First a buffer must not participate in not inhibit nor stimulate the biological process being examined For many buffers available commercially this is not a major problem A common buffer for tissue homogenization is a phosphate buffer but this is an inappropriate buffer for many other biochemical purposes since phosphate is a substrate or product in many cellular events A buffer is next chosen for the pH range it will maintain and is selected by its pKa Looking again at the HendersonHasselbalch equation 7 the pH pKa when HA A which means the buffer has an equal capacity to resist pH changes in both the acid and base directions Thus the pKa of the buffer is matched as closely as possible to the desired pH needed by the experiment The concentration of a buffer depends on its use A buffer is said to have suf cient bu er capacity when its concentration is suf cient to resist changes in pH with additions of acid or base Buffer capacity is dependent on both i the total buffer concentration and ii the ratio of its conjugate acid and conjugate 13 base If the pH is above the pKa of the buffer then there is less buffer capacity in the base direction ie ability to absorb addition of base and greater buffer capacity in the acid direction ie ability to absorb addition of acid conversely if the pH is below the pKa of the buffer then there is less buffer capacity in the acid direction and greater buffer capacity in the base direction When at the same pH values a buffer at a lower concentration has less buffer capacity in both the acid and base directions than for the same buffer at a higher concentration Hence for practical considerations the concentration of the conjugate base form of the buffer A approximates the buffer capacity in the acid direction ie the amount of A available to titrate additional H and the concentration of the conjugate acid form of the buffer HA approximates the buffer capacity in the base direction ie the amount of HA available to titrate additional OHquot So what concentration of buffer should be used When a cell homogenate is prepared the entire contents of the cell is released and a relatively higher concentration of a buffer is required typically between 02 M and 05 M whereas a puri ed protein is stored in a much lower buffer concentration typically between 2 mM and 20 mM A formal treatment of what is called the practical buffer capacity is found on pages 46 53 in Segel s Biochemical Calculations The de nition of practical buffer capacity is the amount of acid or base required to lower or raise the pH by 1 pH unit On page 50 of Segel s Biochemical Calculations the following formulas for the practical buffer capacity are found 0 BCa buffer capacity in the acid direction 9HA A l0HA A BCb buffer capacity in the base direction 9HA A 10A HA Buffer preparation Buffers are prepared in one of three ways 1 Titration of a weak acid with a strong base until the appropriate pH is reached Starting with the buffer that is 100 in the conjugate acid HA HA NaOH gt HA A 2 Titration of a weak base with a strong acid until the appropriate pH is reached Starting with the buffer that is 100 in the conjugate base form A A HCl gt HA A 3 Mix a conjugate weak acid with its conjugate weak base until the appropriate pH is reached The weak acid and weak base forms in equal concentrations A HA are mixed to achieve the proper A HA ratio that reaches the desired pH Each of these methods will be performed and are outlined in the experiment section below No matter which method is used once a buffer is prepared the nal and actual pH must be recorded pH and Ionizable species Spectrophotometric T itrations With some lightabsorbing compounds ionization of the functional group alters the spectral properties of the compound The pKa of such compounds can be easily determined using spectrophotometry instead of using a pH meter Also since many such compounds are dyes a change in pH is observable by a change in color These are referred to as pH indicator dyes and were used extensively in solution or in pH paper before the advent of reliable pH meters One such indicator dye is phenol red that is used extensively in mammalian cell culture media Phenol red has a pKa of 800 and an acidic solution is easily seen as yellow in color while a basic pH is red or bright pink If a slow growing culture of mammalian cells having phenol red present in the media becomes infected by rapidly growing bacteria the metabolic waste exuded by the bacteria will tum the solution acidic thus yellow The visual inspection of 14 mammalian cell cultures is an important routine in industrial and university laboratories Thus understanding the effect of a solution pH on a dye is an important lesson for life science research Ho Px A 0H d I SO Figure 11 Structure of phenol red In this experiment the pKa of an indicator dye pnitrophenol a weak acid will be determined In visible light between 400 nm to 700 nm the acid form of pnitrophenol is colorless and the anionic form pnitrophenolate ion is yellow When a known concentration of the pnitrophenol is placed in buffer solutions at different pH values the relative concentrations of the protonated and unprotonated anionic forms can be calculated from the absorbance measured at an appropriate wavelength The absorption maximum of pnitrophenolate is 400 nm OZN OH 4 OZN M M X O H PquotitF0Ph9 0l pnitrophenolate colorless A gt 400 nm yellow Amax at 400 nm Figure 12 Dissociation of pnitrophenol pKa 715 The concentration of the anionic form can be calculated from an absorbance near the absorption maxima of 400 nm If the total amount of the dye is known the amount remaining in the protonated form at a given pH can be determined by subtracting the amount calculated to be in the anionic form from the total Idyeltotal A HA Given the pH the concentration of HA pnitrophenol and the concentration of A pnitrophenolate it is possible to calculate the pKa using the HendersonHasselbalch equation 7 above since the pKa is the only unknown quantity in the following equation References 1 Freifelder D 1982 Physical Biochemistry 2nd ed Chapter 4 p 118127 W H Freeman and Co San Francisco 2 Cooper T 1977 The Tools of Biochemistry Chapter 1 John Wiley amp Sons New York 3 Segel I H 1976 Biochemical Calculations 2nd ed Chapter 1 John Wiley amp Sons New York 4 Good N E Winget G D Winte W Connolly T N Izawa S and Singh R M 1966 Biochemistry 5 467 MES ACES TES tricine bicine 5 Bates R G and Hetzer H B 1961 J Phys Chem 65 667 Tris 6 Lewis J C 1966 Anal Biochem 14 495 BIS Tris 7 httpenwikipediaorgwikiPhenolred 39 15 Experimental A PREPARATION or THREE BUFFERS 1 Preparation of a Tris buffer 01 M TrisHCI pH 90 A solution of Tris buffer is typically prepared from crystaline Tris in its base form The required concentration of Tris buffer is prepared by weighing out the proper mass of Tris or by diluting a stock of concentrated Trisbase solution to the needed concentration The Tris is then titrated to the required pH HI CCH2OH3NH lt gt CCH20H3NH3 Tris pKa 321 Trisquot 1 Calculate the theoretical volumes of 10 M Tris base 10 N HCl or 02 N NaOH and water needed to prepare 50 ml of a 01 M Tris buffer pH 90 10 M Tris base H20 10 N HCl or 02N NaOH 2 In an 80 ml beaker or 100 ml or 150 ml beaker add the calculated volume of 10 M Tris base and then add dH2O to approximately 40 ml NOT 50 ml so there is volmne remaining to titrate the buffer with HCl or NaOH 3 Using the pH meter slowly add 10 N HCl or 02 N NaOH to the Tris solution using a glass pipette or P1000 Pipetman until the pH is 90 measure and record the amount added Use a magnetic stir bar for continuous mixing Note avoid breaking the electrode with the moving magnetic stir bar 4 In the space below record the amount of 10 N HCl or 02 N NaOH actually required to adjust the buffer pH to 90 Does the measured amount agree with the calculated amount Measured amount added 5 Adjust the volume to 50 ml with water in a graduated cylinder and recheck the pH 11 Preparation of an Acetate buffer 01 M acetate pH 57 An acetate buffer is prepared from crystalline sodium or potassium acetate and brought to the appropriate pH by titration or is prepared from acetic acid and brought to the appropriate pH by titration CH3COOH lt gt CH3COO H acetic acid pKa 476 acetate 1 Calculate the theoretical volumes of 20 M acetic acid 10 N HCl or 02 N NaOH and water needed to prepare 50 ml of a 01 M acetate buffer pH 57 20 M acetic acid H20 10 N HCl or 02 N NaOH 2 In an 80 ml beaker or 100 ml or 150 ml beaker add the calculated volume of acetic acid and then add dH2O to approximately 10 ml NOT 50 ml so there is volume remaining to titrate the buffer with HCl or NaOH 1 7 B BUFFER CAPACITY Effect of dilution on buffer pH and buffer capacity 1 Beginning with the 010 M Tris buffer prepared in Part A prepare the set of decreasing concentrations of Tris buffers outlined in Table B1 Prepare 10 ml of each dilution 2 Measure and record the pH of the undiluted buffer and the various dilutions in Table B1 TABLE B1 Test Tube Buffer nal Tris M Measured pH 1 010 M Tris undiluted 010 M 2 010 M Tris diluted 12 3 010 M Tris diluted If 10 3 Recalibrate the pH meter 4 7 Pipet 4 ml of each of the three buffers prepared in Table B1 into clean test tubes add 1 ml 005 N HCl cover with Para lm invert to mix and record the pH in Table B2 TABLE B2 nal if Buffer 39I3g1N Measured pH 4 4 ml of 010 M Tris undiluted 1 ml 5 4 ml of 010 M Tris diluted U2 1 ml 6 4 ml of0l0 M Tris diluted U10 1 ml 4 Recalibrate the pH meter 7 10 Pipet 4 ml of each of the three buffers prepared in Table B1 into clean test tubes add 1 ml 005 N NaOH cover with Para lm invert to mix and record the pH in Table B3 TABLE B3 nal 318 Buffer p 0 l Measured pH 7 4 ml of 010 M Tris undiluted 1 ml 8 4 ml of 010 M Tris diluted 12 1 ml 9 4 ml of 010 M Tris diluted 110 1 ml 18 C SPECTROPHOTOMETRIC DETERMINATION OF BUFFER pH USING THE pH INDICATOR DYE PNITROPHENOL The pH of the phosphate buffer prepared in Part A will be determined using the pH indicator dye pnitrophenol pKa 715 in a microplate method The limitations of using a spectrophotometric analysis and the application of the Henderson Hasselbalch equation will be examined by attempting to detennine the pH of the acetate and Tris buffers also prepared in Part A Overlay Spectra of the pnitrophenolate and the pnitrophenol form of the pNitrophenol dye 1 Prepare two 1 10 dilutions of pnitrophenol one in HC1 and the other in NaOH as follows a mix 100 pl of 05mM pnitrophenol 900 pl 01 N HC1 b mix 100 pl of O5mM pnitrophenol 900 pl 01 M NaOH 2 Run a spectrum of each 1 1 O diluted pnitrophenol solution with quartz cuvettes using the Spectral Overlay Method Directions for the Overlay Method is on page 013 There should be two spectral curves on the same graph Make sure each curve is labeled 3 Follow the directions for the Overlay Method and record the wavelength and Abs for each solution into Table C1 TABLE C1 Abs max 1 nm Abs at it max Abs at it 405nm pnitrophenolate basic solution pnitrophenol acidic solution the absorbance maximum is a wavelength at maximum absorbance Determination of the pH of the three bujfers prepared in Part A using the pH indicator dye pnitrophenol Preparation of the pnitrophenol dilution series refer to Table C2 for the microplate template 1 Obtain 1 ml 05mM pnitrophenol 2 Pour approximately 5 ml dH2O into a trough Using the multichannel pipette transfer 125 pl of the dH2O to Row A Columns 2 5 and 8 11 3 Pipette 250 pl 05 mM pnitrophenol into Row A Column 1 and Row A Column 7 4 Transfer 125 pl of pnitrophenol from Row A Colunm 1 to Row A Colurrm 2 and mix well repeat the 125 pl transfer successively along Row A through to Column 5 mixing well after each transfer 5 Repeat step 4 above for Row A Columns 7 11 19 Table C2 Microplate template for the determination of buffer pH using pnitrophenol The dilutions indicated in Rows C D E and F are the nal dilutions of the 05 mM pnitrophenol stock solution 1 2 3 4 S 6 7 8 9 10 ll 12 A Pt39 t39 P undil 112 14 18 116 undil 12 14 13 116 d1lut1on senes B C NaOH 110 120 140 130 1160 6 110 120 140 180 1160 6 D a ta 110 120 140 130 1160 110 120 1140 130 1160 quot buffer dye dye E Pquot 5Pha 110 120 140 130 1160 quot 110 120 1140 180 1160 buffer dye dye F T 110 120 140 130 1160 110 120 140 130 1160 buffer dye dye Preparation of the pnitrophenol diltuions series in NaOH and the buffers 1 2 3 Obtain 10 ml 01 M NaOH and pour into a trough Transfer 225 111 01 M NaOH into Row C Columns 1 12 using the multichannel pipette Using the multicharmel pipette and a trough Transfer 225 111 01 M acetate buffer into Row D Columns 1 12 Transfer 225 111 01 M phosphate buffer into Row E Columns 1 12 Transfer 225 111 01 M Tris buffer into Row F Columns 1 12 Transfer 25 111 dH2O into Columns 6 and 12 for Rows C D E and F wells with no dye Using the multichannel pipette transfer 25 111 of the pnitrophenol from Row A Columns 1 5 to Row C Columns 1 5 repeat the transfer of 25 111 of the pnitrophenol from Row A Columns 1 5 to each of the Rows D E and F Repeat step 5 above for Columns 7 ll Take microplate readings using the program ALKPHOSSEE Record the absorbances in Table C3 Table C3 Absorbance values from the microplate readings at 405 nm 1 2 3 4 5 6 7 10 ll 12 no dye I10 dye 1 1 60 110 120 U40 U80 1160 110 120 140 180 NaOH acetate buffer phosphate buffer Tris buffer 110 TABLE 11 PK A VALUES or SME BIOCHEMICAL SPECIES IN DILUTE SOLUTION PKa39 Species 2 00C 200C 370C EDTA 0 17 aspartic acid 1 213 202 195 H3PO4 0 206 213 221 glycine RCOOH 1 244 236 233 EDTA 1 26 citric acid 0 322 314 311 3alanine 1 365 357 352 formic acid 0 379 375 376 lactic acid 0 39 389 386 387 aspartic acid 0 401 392 388 7aminobutyric acid 1 409 404 403 succinic acid 0 428 422 419 acetic acid 0 478 476 477 citric acid 1 484 477 475 succinic acid 1 568 564 565 EDTA 2 63 carbonic acid 0 658 638 630 citric acid 2 639 639 643 imidazole 1 69 25oC H2pQ4 1 731 722 718 Tris 1 885 821 775 ammonia 1 1008 940 889 aspartic acid 1 1063 1012 975 Balanine 0 1100 1038 991 carbonic acid 1 1063 1038 1024 EDTA 3 106 y aminobutyric acid 0 1137 1071 1021 HPo42 2 123 250c EDTA ethylenediaminetetraacetic acid carbonic acid HZCO3 The Ka for this acid is formulated a C s X ahquot aC0H3903 Tris trishydroxymethylaminomethane Materials Equipment and Supplies pH meter Spectrophotometer pH 4 7 and 10 standardization buffers Reagents 050 mM pnitrophenol Dissolve 139 mg 1 mmole of pnitrophenol in a few ml of ethanol and dilute to 20 liters with distilled water 1 N HCI 01 N HCL 2 M acetic acid 1 M NaOH 01 M NaOH 01 M monobasic KH2PO4 1 M Tris base 01 M dibasic K2HPO4 005 M HCI and 005 M NaOH 111 Name Tum in these three pages with the write up attached DATA ANALYSIS AND POINTS FOR DISCUSSION Part A Preparation of Buffers 1 Table A1 instructs that 19 ml of monobasic phosphate mixed with 81 ml of dibasic phosphate is needed to prepare 100 ml phosphate buffer at pH 74 Beginning with 19 ml of monobasic phosphate and using the HendersonHasselbalch equation calculate the theoretical volume of the dibasic phosphate solution needed to reach a pH of 74 using activity coefficients for the monobasic and dibasic phosphate solutions Is the calculated amount of dibasic potassium phosphate and the empirically derived amount in Table A1 the same If not what else might be a contributing factor Activity Coef cients at three ionic concentrations Ion 0001 M 001 M 01 M HZPO41 0975 0928 0744 H130 42 0903 0740 0445 13043 0796 0505 0160 Part B Buffer Capacity 1 Provide a brief explanation for the trend in the measured pH values when the 01 M Tris buffer pH 90 was diluted as observed in Table B1 2a If the Tris buffer was exactly at pH 90 then calculate the expected pH values for tube 4 undiluted tube 5 12 dilution and tube 6 110 dilution after the addition of lml of 005 M HCl Test tube Buffer HCI Expected pH Measured pH 4 4mlof010MTrispH9 1m1005 M 5 4 ml of 0050 M Tris pH 9 1 ml 005 M 6 4m of0010MTrispH9 1011005 M b Does the expected trend follow the observed trend in Table B2 3a If the Tris buffer was exactly at pH 90 then calculate the expected pH values for tube 7 undiluted tube 8 12 dilution and tube 9 1 10 dilution after the addition of lml of 005 M NaOH Test tube Buffer NaOH Expected pH Measured pH 7 4m1of010MTrispH9 1m1005 M 3 4 ml of 0050 M Tris pH 9 1 ml 005 M 9 4ml of0010MTris pH9 1m1 05 M b Does the expected trend follow the observed trend in Table B3 4 For Tubes 4 9 which had the greatest affect on changing the pH of Tris from 9 the strong acid or the strong base Brie y explain Hint pH 90 is on what side of the pKa for Tris 112 Name Part C Spectrophotometric Determination of Buffer pH using the pH indicator dye pnitrophenol 1 Calculate the molar absorption coef cients of pnjtrophenolate at its absorbance maximum and pnitrophenol at its absorbance maximum 7L maximum Abs at 7L maximum 3 Mquot cm391 pni1ropheno1ate pnitrophenol 2 a Calculate the molar absorption coefficient of pnitrophenolate and pnitrophenol at 405 mn Abs at 7L 405nm 3 Mquot cmquot39 pnitrophenolate pnitrophenol b The determination of buffer pH using pnitrophenol is performed photometrically at 405 nm why is this wavelength chosen 3 a Using the data from Table C3 calculate the corrected average absorbance values of pnitrophenol at 405 nm also using the stock concentration of pnitrophenol 050 mM calculate the nal micromolar concentrations in Rows C D E and F columns 1amp7 2amp8 3amp9 4amp10 5amp11 6ampl2 dilutions 110 120 140 180 1 160 no dye nal dye concentration 000 MM Corrected average absorbances at 405 nm C NaOH 000 D acetate buffer 03900 E phosphate buffer 03900 F Tris buffer 03900 b Graph each result corrected average Abs 405nm versus concentration of dye There should be four lines on one graph if using MS Excel be sure to have the lines go through the 00 00 point and record the slopes of each line along with its corresponding R2 value Does each slope follow Beer s law Attach the graph 113 Name c Calculate the pH of the each bu er Recall that the pnitrophenol will be entirely in the pnitrophenolate form in the NaOH hence the concentration of the pnitrophenolate equals the total concentration of the pnitrophenol dye calculate the pH of each buffer using the slopes from the graph in 3b pH of phospahte buffer 715 log 81 buffer 31 N30 31 bu e How do these results compare with the pH value obtained with the pH meter Note the name of the dye is pnitrophenol which is also the name of the conjugate acid form of the pnitrophenol dye be sure to understand what is being discussed the dye or the conjugate acid form of the dye d Can the pH of the acetic buffer and the Tris buffer be determined accurately or reasonably Why or why not The answers may vary depending on the actual data obtained 21 Experiment 2 SPECTROPHOTOMETRIC METHODS PROTEIN DETERMINATION Objectives 1 Perform two spectrophotometric protein assays prepare standard curves and understand Beer39s Law 2 Compare spectrophotometric protein assays for sensitivity and potentially interfering substances Introduction Chemical analyses are part of almost every investigation in biochemistry The substances to be analyzed are present in milligram micrograrn or nanogram amounts often below the detection limits of classical gravimetric and volumetric procedures Since these compounds are usually in complex mixtures assays must be selective analyzing only the compound of interest There are several spectrophotometric assays for determining protein concentrations Each differs in sensitivity speci city and convenience This experiment will introduce three commonly used methods the absorbance of ultraviolet light A280 method the Bradford method and the bicinchoninic acid BCA method Spectrophotometric assays are designed to determine the concentration of a substance using Beer39s Law Often the concentration of a substance is determined by comparing an experimentally derived absorbance of the substance of unknown concentration against the absorbance of a set of solutions with known concentrations of the substance or a related substance measured under the same assay conditions The set of known concentrations of a substance is called the standards and its relationship to its measured absorbance is called the standard curve Figure 21 are examples of standard curves To construct a standard curve for protein concentration determinations a series of protein solutions with a range of known concentrations is prepared which are measured under identical conditions and the results are plotted as absorbance vs concentration Thus the absorbance of a sample of unknown protein concentration is located on the ordinate of the standard curve the y axis and its corresponding concentration is determined on the abscissa the xaxis The assays shown in Figure 21 either strictly obeys Beer39s Law right graph where the relationship between absorbance and protein concentration is linear or has a nonlinear relationship left graph This failure to obey Beer39s Law perfectly can be of chemical or physical origin However a nonlinear graph is just as useful By taking many points the standard gig is more precisely de ned Regardless of the nonlinearity and linearity of a particular standard curve either curve is rendered useless when i the absorbance exceeds the limits of the spectrophotometer Abs 2 20 and ii when the absorbance exceeds the limits of the system ie when increased concentrations of protein no longer leads to signi cant increases in absorbance the curve becomes atter or when lower concentrations of protein are below the level of detection and the absorbances do not change and or are near zero In every spectrophotometric analysis it is necessary to have a blank solution in addition to the standards and solutions of unknown concentrations The blank solution is used to account for the contribution of any absorbance due to the solution in which the lightabsorbing compound is dissolved in the absence of the lightabsorbance compound Be careful not to confuse the blank with the reference cuvette in the spectrophotometer The reference cuvette typically contains only water for biochemical systems The absorbance of the blank the reagent control is subtracted from the absorbance of each experimental sample This yields corrected absorbance values which are typically plotted as shown in Figure 21 where 000 mgml results in an Absorbance 000 These plots have units of Absorbance vs Concentration of 22 Protein The concentration of protein can be masssample volume volume of protein solution prior to the addition of reagent or massassay volume assay volume sample volume reagent volume absorbance absorbance protein concentration mgml protein concentration mgml Figure 21 Standard curves for hypothetical protein assays The results of an assay may give a curved line left graph or a straight line right graph Physical and Chemical Basis of Three Commonly Used Protein Assays A280 Method an example of a direct assay Direct spectrophotometry is used to measure protein and nucleic acid concentrations because it does not destroy the sample being analyzed and it is convenient The side chains of tryptophan and tyrosine are responsible for most of the ultraviolet UV absorption of proteins in the 260 to 290 nm region Figure 22 While phenylalanine also absorbs in the UV its molar extinction coef cient at 280 nm is much less than those for tryptophan and tyrosine Thus the absorbance of a protein solution at 280 nm depends on its relative tyrosine and tryptophan composition The absorbance at 280 nm A280 of 01 wv solutions ie 01 g100 ml or 1 mgml of different proteins varies from 00 to 15 For solutions containing a mixture of proteins an A280 value of 10 is typically observed for 1 mgml concentrations For a puri ed protein if either absorbance coefficient is known 0 or a at 280 nm then its concentration can determined from the A280 It is important to note that nucleic acids absorb strongly in the ultraviolet region When absorbing substances such as nucleic acids are present in protein solutions suitable corrections must be made for their contributions to the A280 or use another protein assay where nucleic acids do not interfere The absorption properties of the materials used in the construction of cuvettes determine their usefulness for spectrophotometric work ie the cuvettes themselves can absorb light Inexpensive plastic cuvettes are usable in the visible range of the electromagnetic spectrum However below about 330 mn these cuvettes will absorb light Therefore quartz cuvettes typically must be used for spectral work below about 330 run in the UV region Coomassie Blue Dye binding Method the Bradford Assay an example of an indirect assay A rapid and sensitive method for quantifying protein in solutions was developed by Bradford 1976 and was later improved by Read and Northcote 1981 This method depends upon the shift in absorbance maximum Om max of the dye Coomassie Brilliant Blue G250 from 465 nm to 595 mn which is caused by 2 4 the assay The Bradford BCA and A280 assays are based on different properties of the protein structure As shown in Table 21 proteins vary in amino acid composition It should not be surprising that an estimation of an unknown protein concentration using these three different assay methods can produce three different values However if the same protein assay method is used throughout an experiment while the protein concentration determined may be inaccurate the quantities of protein used for comparisons in an experiment will remain precise The development and choice of spectrophotometric assays includes the concept of sensitivity the measurable absorbance response corresponding to an amount of the chromophore present Sensitivity is most easily measured and compared using the absorption coef cients or molar absorption coef cients For example the coef cients of different standard proteins can be compared within one protein assay method or the coef cients of one standard protein within several protein assays can be compared New assays are always being developed and the sensitivity of response is a crucial consideration in comparison with currently available assays E SO3Na Cll3CH210ClLZOSO3 N mp NH C X CH3 Sodun Dodecylsufate sos rI1cH2 HSCHZCHZOH CHZCH3 Mercaptoethanol Figure 23 Structures of Coomassie Blue R250 SDS and mercaptoethanol Coomassie Blue G250 differs by the inclusion of two methyl groups positions noted by the arrows Coomassie Blue R250 Two BCA Molecules Complexed with Cu Figure 24 Compounds involved in color formation in the BCA assay Interfering Substances In a biochemistry lab choosing from several standard protein assays available largely depends on avoiding interfering substances During the isolation of an enzyme the protein content of complex solutions contains the multitude of substances present in the crude cell extract plus the buffers salts and other compounds 25 54 that are added during the isolation The ideal protein analysis would be selective only to total protein and not be in uenced or interfered with by other substances Interfering compounds are those that participate in the same chemical and physical properties as the protein assay Hence detergents are expected to interfere with the Bradford assay hydrophobicity The reducing agent 2mercaptoethanol is often included in cellular extracts or de ned solutions to improve protein stability thus the BCA assay must be avoided redox reactions Of course the disruption of cells releases nucleic acids nucleotides hemes etc many UV absorbing molecules so the A280 assay would be a poor choice for cell extracts Hence several protein assays have been developed each utilizing a different physical or chemical property of proteins that can be selected as needed Typically if a substance is present in the protein solution that will interfere with a speci c protein assay then that assay will be avoided and another suitable protein assay is chosen However often the presence of an unavoidable interfering substance in a protein assay can be compensated The ability to compensate for the presence of an interfering substance depends on its mode of action If the effect of the interfering substance is merely additive that is if the interfering substance contributes an absorbance under the assay conditions independently of the proteins then its contribution to the overall absorbance can be subtracted from an obtained absorbance of a solution Two conditions must be met to compensate for an additive interfering substance 1 the absorbance of the solution cannot of course exceed the limits of the spectrophotometer an absorbance greater than 20 or preferably 15 and 2 the concentration of the interfering substance or the volume of a solution containing the interfering substance added to the protein solution must be known in order to accurately account for the amount of absorbance due to a particular amount of interfering substance The protein assay if possible should contain a control with only the interfering substance included in the assay ie minus the protein solution Conversely if the absorbances of the proteins the interfering substances and the combination of proteins plus interfering substances are not additive then the interaction of the assay reagents with the different molecules is more complex than molecules merely acting independently of one another In these cases compensation ie correction for the presence of the interfering substance is impossible In research laboratories interfering substances can be removed from aliquots of samples being prepared for protein assays by several common methods such as protein precipitation sizeexclusion chromatography dialysis and filtration At times protein precipitation by denaturing agents such as trichloroacetic acid TCA perchloric acid HCIO4 or ethanol are used to concentrate the protein so that it can be more accurately measured in assays having low sensitivity and or to remove troublesome interfering substances References Segel lH 1976 Biochemical Calculations 2quot ed Chapter 5 John Wiley amp Sons New York Freifelder D 1982 Physical Biochemistry 2nd ed Chapter 14 pages 494500 504507 511512 W H Freeman and Co San Francisco Cooper T 1977 The Tools of Biochemistry Chapter 2 John Wiley amp Sons New York Long C ed 1961 Biochemistis Handbook pp 8182 D Van Nostrand Co Inc New York Kirschenbaum D M 1972 Atlas of Protein Spectra in the Ultraviolet Visible Regions lflPlenum New York Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding Analytical Biochemistry 1976 7224854 7 Read SM and Northcote DH Minimization of variation in the response to different proteins of the Coomassie blue G dye binding assay for protein Analytical Biochemistry 1981 1165364 8 Smith PK Krohn RI Hermanson GT Mallia AK Gartner FH Provenzano MDFujimoto EK Goeke NM Olson BJ Klenk DC Measurement of protein using bicinchoninic acid Analytical Biochemistry 1985 1507685 published erratum appears in Analytical Biochemistry 1987 1632279 9 PierceNetcom Coomassie Plus 10 PierceNetcom BCA Protein Assay Kit ExI ewew igt ON Table 21 Amino acid composition of selected proteins Moles Amino AcidMole of Protein Amino Acid BSA2 L ala A 46 23 asn 4 39 35 1 7 6 1 1 5 1 7 14 61 S9 4 26 28 28 thr 34 2 l 9 val 36 unknown IgtOLoJO10lJLoJlJOOOOv lJLaJOOOO5 Total 213 578 133 333 Bovine collagen 039l chain gelatin approximately 23540 daltons 2Bovine serum albumin 66296 daltons 3Chicken lysozyme 14314 daltons and 4Pig lactate dehydrogenase LDH H chain 36476 daltons Recall that 1 dalton is equivalent to 1 gmol daltons may also be expressed as kilodaltons 40000 daltons 40 kdal EXPERIMENTAL PART A QUANTITATIVE PROTEIN ANALYSIS BY ABSORBANCE or Uv LIGHT A280 Protein Assay Spectra proteins in the UV Run overlay spectra of these solutions in quartz cuvettes see page 013 for overlay directions there should be three curves on the same graph compound peak 7 nm Abs at peak 7 Abs at 280 nm 080 mgml BSA 040 mgml lysozyme 20 mgml gelatin When finished fmding the peaks and absorbances PRINT the overlay spectra A280 Protein Assay Refer to Table 22 for the A280 assay protocol to be used Tubes 114 represent the protein standard curve assayed in duplicate using bovine serum albumin as a protein standard while 1519 are the dilutions of the solution of unknown protein concentration tested in duplicate that fall within the standard curve 1 Obtain the stock solutions 6 ml 2mgml BSA and 15 ml unknown protein in labeled tubes 2 Tmn on the spectrophotometer select the Photometric mode and zero the instrument as usual 3 Prepare dilutions of the BSA standards For the unknown read 1 ml in the spectrophotometer then retrieve the 1 ml and use it to make the dilution series for the unknown in labeled tubes shown in Table 22 Record calculations and any notes made for preparing the dilutions in the lab notebook 4 Transfer the contents of each tube to a quartz cuvette push start to record the absorbance at 280 nm Table 22 Protocol for the A280 protein assay Tube Samples Water A230 ave A230 ave A330 Protein pl ave blank mgml 1 0 blank 1000 2 0 blank 1000 0 3 125 11120 mgml BSA 875 4 125 11120 mgml BSA 375 0 25 5 250 0120 mgml BSA 750 6 250 111 20 mgml BSA 750 05 7 375 0120 mgml BSA 625 8 375 11120 mgml BSA 625 03975 9 500 111 20 mgml BSA 500 10 500 01 20 mgml BSA 500 quot00 11 625 0120 mgml BSA 375 12 625 11120 mgml BSA 375 13925 13 750 pl 20 mgml BSA 250 14 750 11120 mgml BSA 250 15 15 1 ml unknown 0 undiluted l l dilution 16 12 dilution 500 500 111 unknown 17 12 dilution 500 500 pl unknown 18 14 dilution 750 250 pl unknown 19 14 dilution 750 250 11 unknown PART B QUANTITATIVE PROTEIN ANALYSIS BY THE BRADFORD PROTEIN ASSAY Spectra of the Bradford reagent and Bradford reagent plus protein 1 Prepare two solutions as follows a mix 500 pl of Bradford reagent 500 pl H20 b mix 500 pl of Bradford reagent 500 pl 50 pgml BSA 2 Run overlay spectra these solutions in PLASTIC cuvettes see overlay directions on page 013 There should be two curves on the same graph 3 When nished nding peaks and absorbances PRINT the overlay spectra peak A max nm Abs at peak A max Abs at 595 nm 05 ml Bradford reagent 05 ml H20 05 ml Bradford reagent 05 ml protein Microplate Bradford Protein Assay Background Refer to Table 24 below for the 96well microplate layout for the Bradford assay for determining the concentration of the unknown For colorimetric protein assays a standard curve is always prepared along with the sample of unknown protein concentration being determined The Bradford reagent is added last after all of the standards and samples are prepared and in the microplate The Bradford protein assay is considered a sensitive assay since low concentrations of proteins generate measurable changes in absorbance at 595 nm Since the concentration of the sample protein is unknown the dilution series of the sample will cover a broad range of protein concentration from 13dilution ie 13 to 1656l dilution ie 138 These dilutions are to be prepared in the microplate wells using the multicharmel pipette as directed below The dilution range used covers possible protein concentrations in biochemical work for example crude homogenates are often between 10 ugpl to 20 ugul while puri ed protein samples are often between 005 ugul to 05 ugul Recall that only one dilution of a sample needs to fall on the standard cIu39ve Note that the dilution series will be successive 13 dilutions of the previous rows Row A is simply a direct l3 dilution of a sample Row B is a 13 dilution of Row A thus creating a 19 dilution 13 x 13 19 The 13 dilution will continue successively to Row H Procedure Preparation of the Standard Curve and other Samples 1 Prepare 100 pgml working stock solutions of BSA from the 2 mgml stock BSA prepare 15 ml ml 2 mgml stock ml H20 2 Label 15 ml rnicrocentrifuge tubes from 1 to 16 3 Prepare the standard curve samples from the 100 pgml BSA working stock in 15 ml microcentrifuge tubes as shown in Table 23 tubes 1 16 4 Dispense 125 pl from each tube 1 16 into the microplate wells as shown in Table 24 29 Procedure Preparation of the Samples of Unknown Protein Concentration 1 Using the multichannel pipette dispense 125 pl of H20 into Columns 3 4 Rows A H draw the water from a plastic trough Refer to Tables 23 and 24 as needed Using the P200 pipette transfer 63 pl of the unknown sample into Row A Columns 3 4 mix by repeated pipette action being careful as much as possible to avoid introducing bubbles Using the multichannel pipette transfer 63 pl of the solutions from Row A Columns 3 4 to Row B Columns 3 4 simultaneously Mix by repeated pipette action be careful to avoid introducing bubbles Repeat 3 above by transferring 63 pl of the solutions from Row B Columns 3 4 to Row C Columns 3 4 and carefully mixing with the water continue transferring and mixing 63 pl from Row to Row until 63 pl has been transferred to Row H After mixing the solution in Row H pipette out and discard 63 pl of the solution Bach Well should then have 125 pl nal sample volume Procedure Dispensing the Bradford Reagent and Reading the Results 1 2 Obtain 10 ml Bradford reagent and pour into a plastic trough Using the multichannel pipette dispense 125 pl of the Bradford reagent successively to the columns beginning with Column 1 then Column 2 and through to Column 4 The nal volume of sample plus reagent is 250 pl which is close to lling the well without spilling from normal careful handling Take the microplate to the microplate reader place the microplate in the holder on instrument At the computer controlling the microplate reader select the program BRADFORDSEE from the Sessions tab found on the upper right corner or if the Bradford program is already open go to 5 Click on the START button found along the top tool bar If the START button is not visible then click on the Procedure tab found along the right edge of the program screen The plate will be shaken the absorbances will be read and the results will be printed all automatically If the program is already open it might ask if the previous results should be saved select CONTINUE Take away the microplate and the printout of the results DO NOT ATTEMPT TO HAVE THE COMPUTER PRINT ANOTHER COPY Partners should make a duplicate copy of the results at a photocopy machine Record the data onto Tables 23 Check that the standard curve is acceptable readable and reliable and check that corrected absorbance of at least one dilution A595 blank from the dilution series of the unknown sample is within the values of the standard curve If the unknown sample dilution series does not have at least one absorbance value on the standard curve then repeat the protein assay for the samples Wash the microplates ll they are reused a shake the contents of the assay out of the wells into a sink careful Coomassie will stain clothing b rinse with WARM tap water and shake out the water then rinse lightly with 50 ethanol a couple of times and vigorously shake out the ethanol Repeat waterethanolwater until no blue is visible 210 Table 23 Preparation of Samples for the Microplate Bradford Protein Assay and Data Table Sample Preparation Reading 125 pl sample 125 pl Bradford Reagent in microplate protein Tube Well BSA Standards pl H20 A595 ave A595 ave A595 ltgltl ave blank sample 3901 1 A1 0 pl blank reagent control 250 2 A2 0 pl blank reagent control 250 0 0 3 B1 10 pl of 100 pgml BSA 490 4 B2 10 pl of 100 pgml BSA 490 0002 5 C1 20 pl of 100 pgml BSA 430 6 c2 20 pl of 100 pgml BSA 430 0004 7 D1 40 pl of 100 pgml BSA 460 3 D2 40 pl of 100 pgml BSA 460 0003 9 E1 60 pl of 100 pgml BSA 440 10 E2 so pl of 100 pgml BSA 440 0012 11 F1 80 pl of 100 pgml BSA 420 12 F2 30 pl of 100 pgml BSA 420 0015 13 G1 100 pl of 100 pgml BSA 400 14 G2 100 pl of 100 pgml BSA 400 0020 15 H1 120 pl of 100 pgml BSA 380 16 H2 120 p1of100 pgml BSA 380 0024 Well Unknown Samples vol of dil A595 ave A595 ave A595 valid data sample ave blank yes or no A3 13 U3dilution 125 A4 13 U3dilution B3 13 19dilution B4 1x3 19dilution c3 133 127dilution c4 133 127dilution 1 D3 13 181 dilution D4 13 181dilution E3 135 1243dilution 1 E4 135 U243dilution 1 F3 13 1729 dllution F4 13 U729dilution 1 G3 13 12187dilution G4 13 12187dilution 1 H3 13 16561dilution 1 H4 13 16561dilution 1 2ll Table 24 Layout of the standards and samples in a 96well Microplate for the Bradford Assay BSA standard unknown Extra wells curve sample for repeating any and all of the Bradford Assay as needed 1 2 3 4 5 6 7 3 9 10 11 12 0000 0000 A ug111 us111 13 13 0002 0002 B us111 ugul 1x32 132 0004 0004 C ug111 ug111 13 133 0003 0003 D will us111 13 13 0012 0012 E us111 ugul 135 1x35 0016 0016 F us111 usI11 13 136 0020 0020 G ugiul usI11 13 13 0024 0024 H its111 ug111 13 13 DATA ANALYSIS AND POINTS FOR DISCUSSION DUE AT THE END or DAY 1 FOR EXPERIMENT 2 1 Construct a properly labeled Standard Curve for both protein assays A computer graphing program may be used to plot the data to obtain the equation for the straight line or the curve a secondorder polynomial for the Bradford plot If a computer program is used then report the equation of the trend line and the R2 value 2 Present the graphed Standard Curves to the TA the TA will evaluate the graph how good it is proper labeling etc PART C SENSITIVITY OBSERVATIONS REPEATING THE ASSAYS Sensitivity in absorbance measurements is how much of a change in the measured response in absorbance occurs per change in concentration of the chromophore Sensitivity of different chromophores are compared by their absorption coef cients or by their molar absorption coef cients When comparing sensitivities the greater the relative absorption coef cient the greater the relative sensitivity It is very important that corrected absorbance values be used when calculating an absorption coefficient or a molar absorption coefficient For protein assays the chromophore is the protein eg A280 assay or the protein complexed with a reagent eg Bradford assay or the results of the protein presence creating a change in the absorbance of a secondary reagent eg BCA assay To compare the relative sensitivities of protein assays the absorption coef cients 1 mgml391 cm l are used 212 Repeating either assay By now the standard curves for both the A280 assay and Bradford assay should have been constructed There is sufficient time to repeat either of these assays as judged by the data from Parts A and B If using MS Excel and the R2 value is lt 095 then the assay should be repeated If not using MS Excel then look at the standard curve and ask How con dent am I with the data if confidence is low then repeat the assay OR If the dilutions of the unknown solution appear irregular or no reliable dilution has an absorbance within the standard curve then repeat the assay In research labs the biotechnology industry and the health care professions judgment of reliability comes down to how con dent and willing one is to present data and results derived from the data to others Sensitivity with the Bradford Assay Refer to Table 25 for the preparation and reading of samples Prepare duplicate samples in microfuge tubes and transfer 125 pl of each sample to a microplate note the location of each sample in the microplatel Table 25 Sensitivity Observations with the Bradford reagent Sample Preparation Reading 125 pl sample 125 pl Bradford Reagent in microplate Protein Tube Samples pl H20 Ave A595 Ave A595 ave pg I pl blank assay vol 1 0 pl reagent control 250 00 0 2 3 100 pl of 01 mgml BSA 400 0010 4 5 100 pl 01 mgfml lysozyme 400 0010 6 7 100 pl 01 mglml gelatin 400 H 0010 8 9 200 pl of 01 mgml BSA 300 0020 10 11 200 pl 01 mgml lysozyme 300 G5 G5 0 l 12 13 200 pl 01 mgml gelatin 300 0020 14 Materials 2 mgml BSA 2 mgml lysozyme 2 mgml gelatin Bradford reagent unknown BSA solution 96 well microplates plastic troughs multichannel pipettes 2 13 Name Turn in this page with the writeup attached DATA ANALYSIS AND POINTS FOR DISCUSSION Overlay Spectra 1 Calculate the molar absorption coefficients 8 for tyrosine and tryptophan at 280 nm using the overlay spectra data in Figure 22 on page 23 2 Calculate the absorption coefficients a for BSA lysozyme and gelatin at 280 nm using the overlay spectra data obtained in Part A Arrange the absorption coef cients from the most sensitive greatest a value to the least sensitive lowest a value in the table 27 below Provide a brief explanation based on the amino acid compositions shown in Table 21 3 Using the overlay spectra data obtained in Part B for the Bradford reagent and the Bradford reagent plus protein provide a brief explanation for performing the Bradford protein assay at 595 nm Bradford and A280 Protein Assays 1 Attach the standard curves for both assays A computer graphing program may be used to plot the data to obtain the equation for the straight line or the curve a secondorder polynomial for the Bradford plot If a computer program is used then report the equation of the trendline and the R2 value 2 Calculate the protein concentration of the unknown protein solution as determined in both assays Arrange these results in a single properly labeled Table Sensitivity 3 Calculate the absorption coef cients a for lysozyme gelatin and BSA from the corrected absorbances in the Bradford Assay from Table 25 Use the estimated pathlength of 055 cm for the 250 pl assay volumes in the microplates Absorption coef cients hence relative sensitivities must be calculated from concentrations in the assay volumes Arrange the absorption coefficients from the most sensitive greatest a value to the least sensitive lowest a value in the table 27 below Table 27 Comparative absorption coefficients A280 assay Bradford assay Protein Absorp coef Protein Absorp coef Protein Absorp coef mgmlquot cmquot mgml39l crn l mgmlquot cmquot 0010 pgpl 0020 pgul Experiment 3 Enzymes Assays and Kinetics Objectives 1 Determine the enzymatic rate of alkaline phosphatase as a function o a variable pH constant enzyme and substrate concentrations b variable enzyme concentration constant pH and substrate concentration c variable substrate concentration constant pH and enzyme concentration i competitive inhibitor 2 Calculate the speci c activity and turnover number for alkaline phosphatase 3 Calculate the Km for the substrate the Vmax for the enzyme and the Ki for a competitive inhibitor 4 Determine the speci c activity of lactate dehydrogenase Introduction Enzymes are biological catalysts that lower the activation energy of a reaction so that the reaction proceeds at a rate much greater than the uncatalyzed rate Thus enzymes allow ef cient synthesis and degradation of biological molecules Biochemistry focuses much research on characterizing the structure cellular function and kinetic properties of enzymes Biotechnological methods allow for the alteration of protein structure and function for such purposes as basic understanding industrial applications or development of therapetics Also the areas of bioinforrnatics known as proteomics and metabolomics have an ambitious goal of describing the structures and mctions of all enzymes in all cells under any condition Thus the puri cation of proteins Experiment 4 and subsequent analyses eg enzyme kinetics Experiment 3 are crucial for examining natural and altered proteins and feeding information into bioinformatics driven analyses and hypotheses formation and biotechnological applications in medicine and industry Enzyme Activity The rate of most biological reactions in the presence of an enzyme is greater than the rate in its absence Because uncatalyzed rates of most biologically important reactions are effectively zero at biological temperatures the mere demonstration of a reaction in a biological extract is taken as evidence of the presence of an enzyme However a nonenzyme control must always be measured for any rate contributed by a mixture For a puri ed enzyme nonenzyme controls are performed in the absence of the enzyme ie buffer and substrates only Since most biological catalysts are proteins a few RNAS act as enzymes Ribozymes an enzyme derived rate is eliminated by treatments that destroy proteins such as heat or addition of proteases However there are exceptions For example Taq Polymerase used in PCR reactions Experiment 4 is stable at temperatures near 100 C whereas Nitrate Reductase that xes inorganic nitrogen is extremely labile in the presence of very low concentrations of oxygen Or if the enzymatic reaction uses two or more substrates then omitting one substrate serves as a nonenzyme control the substrate omitted must not be the one that is being quanti ed or whose derived product is not being quanti ed The observation of enzyme activity in a crude cell or tissue extracts can be complicated The detection or an enzyme or an accurate measure of an enzyme catalyzed rate is complicated by other enzymes that compete for substrates or utilize products by the absence of a necessary activator or by the presence of an inhibitor Thus fractionation separate proteins chemical additions to achieve appropriate concentrations of reactants cofactors and or activators or dialysis to remove inhibitors may be required to measure enzymatic rates reliably and accurately 3 Enzyme Assays Reaction rates are expressed as micromoles of substrate converted to product per minute which is de ned in Intemational Units IU 10 IU 10 umole Pmin The measurement of a reaction rate entails determining the amount of substrate that has disappeared dSdt or the amount of product that has appeared dPdt during a timed interval Measurement of product formed is usually more accurate than substrate utilized since there is less error in subtracting a small nmnber the initial product concentration is zero from a relatively large number the nal concentration of product Actual experimentally observed enzyme reaction rates depends upon many factors including 1 concentration of enzyme 2 concentration of the substrates 3 buffering species 4 temperature 5 species of ions 6 pH 7 regulatory molecules 8 contaminants 9 ionic strength Spectrophotometry Absorption spectroscopy is the most frequently employed method of detection in enzyme assays For many enzymes their substrates and products do not absorb light In such cases arti cial nonphysiological substrates or products are synthesized that do absorb light Spectrophotometric principles apply to kinetic analyses but with time as an additional parameter Thus the change in concentration of a reactant or product with time dcdt is proportional to the change in absorbance with time dAdt The time parameter is applied to Beer s Law so that gg dAdz di Si 1 Other common detection methods for measuring reaction rates include uorescence radioactivity and chemiluminescence Initial Velocity Speci c experimental assay conditions are set so that the initial velocity of the reaction can be measured conveniently and reproducibly rates must not be too fast to observe nor too slow to be practical Substrates are at appropriate concentrations and the enzyme concentration is adjusted so that the rate of change in absorbance is linear with time A linear rate is important as it is used to estimate initial velocities Vo velocity at time zero A linear rate allows for the extrapolation of the rate to time zero which is the initial rate when the concentration of product is zero and there is no back reaction Since the rate of the reaction is measured for a de ned substrate concentration the concentration of substrate must not change appreciably during the time of measurement In practice this usually means that no more than 5 of the substrate should be converted to product over the time period of the assay If there are additional substrates or cofactors they must be present in nonlimiting concentrations In order to ful ll the assumption of the MichaelisMenten model the concentration of substrate must be much greater than the concentration of enzyme so that the rate is dependent on enzyme concentration Also enzymes reactions are reversible so if the equilibrium strongly favors one side of the reaction the kinetics of the reaction can be studied most easily by assaying it in the favored direction This avoids appreciable back reactions The conditions of nearly constant substrate concentration and no appreciable back reaction defme initial velocity F ixedtime and Continuous Enzyme Assays Enzyme assays are usually carried out in one of two ways xed time and continuous A continuous assay typically monitors the appearance of product or disappearance of substrate in realtime in a spectrophotometer The advantage is to monitor the linearity of the assay In a xed time assay the enzyme and substrate are incubated for a xed period of time and then the reaction is terminated by the addition of a solution that completely prohibits the enzyme 3 3 reaction from occurring known as the stop solution The advantage is to measure many assays simultaneously since the terminated reactions are often stable for extended periods Enzyme Kinetics According to MichaelisMenten analysis the initial velocity Vo of an enzyme reaction is given by 0 Km S where S is the substrate concentration Vmax is the velocity achieved at saturating substrate concentrations and Km is a constant characteristic of the substrateenzyme interaction From Equation 2 if S gtgt Km ie at high saturating substrate concentrations the Km term becomes insigni cant and the denominator of the MichaelisMenten equation becomes simply S va Vina kcatiE S 3 which gives zeroorder kinetics with respect to substrate concentration and rstorder kinetics with respect to the concentration of enzyme This is the condition used for assays in which determination of enzyme concentration is desired since the velocity is dependent on enzyme concentration E and independent of substrate concentration S The substrate dependence of an enzyme must be measured in order to determine the Vmax and Km values for an enzyme This is done by assaying the enzyme with varying initial concentrations of substrate A MichaelisMenton graph of enzyme activity velocity versus substrate concentration results in a curved graphed line as shown in Figure 31 which only allows for very close estimations of Km and Vmax 70 IL quot 60 1350 E E 3 392Vmax 92 o E 5 3939gt 0 012345678910 S Km 1mMgt Figure 31 MichaelisMenten plot for a hypothetical enzyme catalyzed reaction 3 4 The preferred method is to replot the data in a linear form using the LineweaverBurk equation K 1 M 1 j j Vmax n3 Imax 1 v 4 On a LineweaverBurk plot the y axis intercept gives 1V max and the xaxis intercept is 1Km as shown in Figure 32 One disadvantage of the LineweaverBurk plot is that the points at low substrate concentrations which are the smallest and frequently the least accurate values may lend undue weight in determining the best t line Values obtained for kinetic constants Km and Vmax were used in the early days of biochemistry in the determination of metabolic pathways ie the biosynthesis and biodegradation of molecules Many enzymes have had their kinetic properties thoroughly examined and values for kinetic constants are known For biochemical and biotechnological research today kinetic constants are useful in understanding enzyme action for example in the bioengenering of cellulases for enhancing the bioconversion of waste products Hence understanding experimental kinetics is relevant and applicable 007 006 005 004 003 E 1 nmoles per min391 002 00 j393939 L 0 V quotaquot 4 0 I 1 2 3 1 quot36 No57 I81 quot quotquotquot1 m Figure 32 LineweaverBurk plot of the data in Figure 31 The Vmax and Km values are both obtained by a linear extrapolation of the data When enzyme velocity is measured at substrate concentrations below levels giving Vmax a velocity v is obtained having the same units as maximum velocity and v changes constantly with time as substrate is being depleted v is dependent on S It is also important to maintain initial rates the velocity always must be proportional to the concentration of enzyme whether substrate is saturating or not At very low substrate concentrations where S ltlt Km the S in the denominator can be ignored and the rate of the reaction is directly proportional to the substrate concentration Eq 5 V Kn1S N Km In practice the velocity is expressed in a variety of ways depending on the information sought such as umoles P per min IU IU per ml of assay mixture IU per ml of original stock enzyme solution or IU 3 5 per mg of protein speci c activity The Appendix to Experiment 3 contains a detailed description of the typical equations used in kinetic analyses Turnover number catalytic center activity and specific activity Additional de nitions of activity are useful particularly when purifying an enzyme As described above enzyme activity is commonly expressed as International Units IU If an enzyme is puri ed and its molecular weight known the enzyme activity can be stated in terms of umoles of product per minute per umole of enzyme an expression also known as molecular activity or turnover number units minquot Knowing the turnover number at saturating substrate concentration allows for the direct comparison of enzymatic rates determined for different enzymes and for kcat to be determined The international unit for kcat is moles of product per second per mole of enzyme units secquot39 If the number of catalytic sites per molecule of enzyme is known the catalytic center activity is further expressed as moles of product per minute per mole catalytic center Another useful defmition of enzyme activity is the speci c activity de ned as IUmg protein when the mass of protein in an assay is known The mass of protein is determined independently by a protein assay such as the Bradford or BCA protein assays Enzyme Inhibitors Enzyme inhibitors are classi ed as reversible noncovalent interactions and irreversible covalent interaction The three most common types of reversible inhibition for MichaelisMenten kinetics are competitive noncompetitive and uncompetitive The use of LineweaverBurk plots reveals the type of inhibition also re plots of data derived from LineweaverBurk plots are useful for determining Ki values for the inhibitor One aspect of experimentation with reversible inhibitors which should be emphasized is that the order of addition of substrate inhibitor and enzyme is usually not crucial as long as either the substrate or enzyme is added last to initiate the reaction This is true because a steadystate initial reaction rate is reached soon after all three components are mixed pH Dependence of Enzyme Activity The maximum velocity of an enzymecatalyzed reaction is determined under a set of conditions chosen to optimize some aspect of the enzyme s performance Frequently the enzyme activity assay is carried out at a pH ionic strength temperature etc giving the highest velocity achievable For example the temperature may be set at the physiological temperature of the tissue from which the enzyme is derived 37 C for most mammalian tissues or at room temperature for convenience Enzyme activities are measured as a function of one of these parameters with all other parameters held constant and a bell shaped curve is ideally obtained The peak of the curve is noted as the optimum condition for that parameter Relatively small changes in many of these parameters have little effect on enzyme activity However small changes in pH of the reaction mixture often have large effects on enzyme activity The pH of the solution affects the ionization states of the substrates and or products and the amino acid side chains and any ligands of the enzyme Also the pH can affect the stability of enzymes Enzyme activities are often surveyed for a pH optimum to help ensure the stability and function of the enzyme and to achieve maximum velocities for kinetic analysis Thus the proper choice and preparation of buffers is critical Alkaline Phosphatase EC 3131 protein database accession number P191ll Alkaline phosphatase is a metalloprotein with a broad speci city for the hydrolysis of phosphate esters The metal cofactors are two zinc atoms and one magnesitun atom which are coordinated by highly conserved amino acids across many species Alkaline phosphatase consequently can be irreversibly inhibited by chelators that bind to the metal ions The hydrolysis of a phosphomonoester results in an alcohol and inorganic phosphate the reverse reaction is so unfavorable that it is not appreciable hence 3 6 inorganic phosphate produced is essentially only a competitive inhibitor and not a substrate for phosphorylation by alkaline phosphatase cellular phosphorylation reactions are accomplished by kinases and phosphorylases This lack of a signi cant back reaction allows for initial rates linear rates to be obtained in enzyme assays relatively easier than for most other enzymes Since neither the alcohol nor the inorganic phosphate are chromophores an arti cial substrate p nitrophenylphosphate is widely used for alkaline phosphatase assays Alkaline phosphatase converts pnitrophenylphosphate to p nitrophenol and inorganic phosphate The product pnitrophenol equilibrates between the conjugate acid form pnitrophenol and the conjugate base form pnitrophenolate only the pnitrophenolate absorbs light at 410 nm The total amount of product formed in the alkaline phosphatase catalyzed reaction is determined by measuring the amount of the pnitrophenolate formed and then calculating the amount of acid form also produced using Beer s Law and then HendersonHasslebalch equations Lactate Dehydrogenasc EC 11127 protein database accession number P19858 Under conditions of low oxygen levels such as in exercising muscle or submerged plants NADH produced by glycolysis accumulates in cells as it is not reconverted to NAD by the TCA cycle quickly enough However most organisms have retained the anaerobic metabolic capability to regenerate NAD from NADH by transferring electrons from pyruvate to an end product such as lactate or ethanol For muscle cells this activity is Lactate Dehydrogenase LDH The reaction catalyzed by LDH is shown in Figure 33 The apparent equilibrium constant Keq for this reaction is 28 x 10395 at pH 70 AG0 25 kJmol which means the reaction with NADH and pyruvate as substrates is favored over the reaction with NAD and lactate The physiological direction is indeed the favored rate of NADH and pyruvate as substrates resulting in lactate and NAD as products G 9 O 0 0 0 0 ea 9 9 HQ c H NAD C20 NADH H I CH3 CH3 Llactate pyruvate Figure 33 The reaction catalyzed by LDH References 1 Stryer L 1988 Biochemistry 3rd ed chapter 8 W H Freeman and Co SF 2 Lehninger A A 1975 Biochemistry 2nd ed chapters 8 and 9 Worth Publishers Inc NY 3 Segel I H 1976 Biochemical Calculations 2nd ed Chapter 4 John Wiley amp Sons NY 4 Segel l H 1975 Enzyme Kinetics John Wiley amp Sons NY 5 Dixon M and Webb B C 1964 Enzymes 2nd ed Academic Press Inc NY 6 Le Du MH er al 2001 Crystal Structure of Alkaline Phosphatase om Human Placenta at 18l Resolution JBC 27612 91589165 7 Ghosh and Fishman 1966 On the Mechanism of Inhibition of Intestinal A lkaline Phosphatase by LPheylalanine JBC 24111 25162522 8 Holbrook JJ Liljas A Steindel SJ and Rossman MG 1975 quotLactate Dehydrogenasequot in The Enzymes P D Boyer ed Vol XI part A pp 191292 Academic Press Inc New York A general review of lactate dehydrogenase 9 Alberts et al 2002 Molecular Biology of the Cell Fourth Ed Garland Science New York 10Ne1son and Cox 2005 Lehninger Principles of Biochemistry Fourth Ed Freeman New York EXPERIMENTAL A ALKALINE PHOSPHATASE REACTION RATE As A FUNCTION or PH In this experiment the pH dependence of alkaline phosphatase a ubiquitous enzyme that catalyzes the hydrolysis of phosphate monoesters is examined A convenient assay for alkaline phosphatase follows the hydrolysis of pnitrophenylphosphate substrate by measuring the absorbance of pnitrophenolate product at 410 nm as a mction of time 0 OH II O N OPO H20 0 N O op 0 H 2 I 2 I pKa 715 Irnitrophenvlphosphate pnitrophenolate The product is in equilibrium between the pnitrophenol and pnitrophenolate forms but only the anion form absorbs light at 410 run this equilibrium is pH dependent The HendersonHasselbalch equation is used to determine the total concentration of product formed at a given pH by calculating the acid form of the product pnitrophenol Total product is the sum of protonated and unprotonated forms Preparation of Tris buffers To determine the pH dependence of alkaline phosphatase a series of different pH values is needed A series of 010 M Tris buffers varying in pH between pH 7 and pH 10 are prepared for this experiment Use large test tubes and make 20 ml of each buffer that will be needed to determine the pH dependence of alkaline phosphatase 1 Calibrate the pH meter between 7 and 10 2 Mix the Trisbase and Trisacid solutions as described in Table 31 below 3 Measure and record the pH of each buffer by pouring a 5 ml into a clean plastic cup Table 31 Tris buffers to be prepared for the alkaline phosphatase assay Tube 010 M Tris base ml 010 M TrisHCl ml Measured pH 1 20 18 2 50 15 3 10 10 4 15 50 5 18 20 6 19 10 Continuous alkaline phosphatase assay The continuous assay is performed by continuous measurement of the rate of appearance of product monitored at 410 nm using the Shimadzu spectrophotometer The alkaline phosphatase reaction mix contains a buffer at a chosen pH enzyme and its substrate pnitrophenylphosphate High pH solutions will slowly hydrolyze the phosphate from the substrate resulting in nonenzymatic formation of product pnitrophenol To minimize the nonenzymatic production of pnitrophenol the reactions are initiated by the addition of substrate after the buffer and enzyme are mixed Also nonenzyme controls are performed complimenting each enzymatic reaction which is performed by adding the substrate to 38 only buffer and monitoring the rate as with the enzyme catalyzed reactions The six enzymecatalyzed reactions and the six nonenzyme control reactions are to be recorded for 1 minute each 1 8 Obtain 500 pl of 0125 mgml alkaline phosphatase enzyme keep this stock sample on ice Also obtain 3 ml of 10 mM pnitrophenylphosphate keep this stock at room temperature The concentrations of the alkaline phosphatase reaction mixture in a 1 ml nal volume are 0075 M buffer and 20 mM pnitrophenylphosphate 50 ul of alkaline phosphatase alkaline phosphatase stock concentration is 0125 mgml the reaction is started by adding the appropriate volume of 10 mM pnitrophenylphosphate Calculate the volume of the bu er require per 1 ml continuous assay volume Calculate the volume of the substrate required per 1 ml continuous assay volume Set parameters for kinetics on the spectrophotometer as follows Select 8 Optional Program Pack it 410 nm Measured time 60 sec Lag 0 sec Rate 60 sec and Interval 10 sec Obtain two 15 ml plastic cuvettes Auto zero with H20 then discard the H10 in the sample cuvette Just prior to performing the assay transfer the appropriate volume of the 01 M Tris buffer to the sample cuvette then add 50 ul of alkaline phosphatase mix gently The reaction is started by adding the appropriate volume of the 10 mM pnitrophenylphosphate to the cuvette Immediately and quickly mix by inverting the cuvette covered with Para lm only once or twice and insert it into the spectrophotometer and press Start The spectrophotometer will record the reaction in real time For proper controls all six reactions are repeated as above in the absence of enzyme Mix 800 pl of buffer and 200 pl substrate in the sample cuvette quickly insert it into the spectrophotometer and press Start Any observed rate of absorbance is must be subtracted from the observed enzymatic derived rate of absorbance obtained above which is the corrected rate Keep all raw data generated from the spectrophotometer and record pertinent data in Table 32 Table 32 Continuous assay of Alkaline Phosphatase Assay nonenz Enz Corrected pnitrophenolate pnitrophenol total product Assay pH AA410 AA41O AA410 umol min pm01min pmolminquot buffer minquot mar minquot IU IU IU 1 2 3 4 5 6 note pnitrophenolate at 410 nm 8 17600 M391 cmquot 3 9 B ALKALINE PHOSPHATASE REACTION RATE As A FUNCTION OF ENZYME CONCENTRATION The xedtime kinetic assay method is used in Part B and Part C Fixedtime assays are run for a speci ed length of time then the reactions are terminated ie stopped at the speci ed time and the samples are read spectrophotometrically The key issue for xedtime assays is to choose data that represent valid assays ie determining Vo As observed in Part A alkaline phosphatase showed greatest activity at the highest pH values Therefore a buffer having a high pH pH 95 is used for Part B and Part C of this Experiment F ixedtime Alkaline Phosphatase Assays The reactions are terminated at speci ed time intervals by adding a high concentration of a K2HPO4 which inhibits enzyme activity by competitive inhibition Furthermore the pH values of the terminated reactions are greater than 915 which will ionize nearly all the product to pnitrophenolate After all of the reactions are stopped then the absorbance of the product is read directly by the microplate reader The microplate protocol for this assay is shown in Table 33 The microplate reader measures absorbance for the assay at 405 nm Enzyme assays are run in Rows E amp F for 10 minutes and Rows G amp H for 2 minutes A serial dilution of the enzyme is prepared in Row D and transferred to the enzyme assays in Rows E H as required A standard curve for the product pnitrophenol must be derived from reading the plate as discussed in the introduction of Experiment 2 thus is included The serial dilution of the pnitrophenol standards is prepared in Rows A amp B 1 Obtain 10 ml 01 M Tris pH 95 2 ml 10 mM pnitrophenylphosphate 10 ml 1M K2HPO4 500 pl 05 mM pnitrophenol 250 pl 0125 mgml alkaline phosphatase Two groups share one trough with the 01 M Tris one trough with 10 mM pnitrophenylphosphate and one trough with 1 M KgHPO4 This ensures sufficient volumes for accurate pipetting Table 33 Microplate Protocol for FixedTime Alkaline Phosphatase Assay 1 2 3 4 5 6 7 s 9 10 11 12 A pnitcrlroplhenol I2 m1 122 123 M A95 blank stan ar s B pnitctlroplhenol 12quot 12 12 12 12 12quot blank stan at s C D elelzstwgree serial 02 W 02quot 12 11110115 E enzyme 10 39 120 121 2 123 4 no assays mm enz F enzyme 10 quot 2 2 3 2 110 assays mm enz enzyme 2 12 02 02 02 02 no assays mm enz H enzyme 2quot 12 22 2 2 H0 assays 2 mm enz 3 10 Preparation of the pnitrophenol standards 2 In Rows A amp B Columns 1 7 dispense 125 pl 01 M Tris pH 95 using a multichannel pipette 3 In Rows A amp B Column 1 dispense 125 pl 050 mM pnitrophenol to the two wells using a P200 4 Using a multichannel pipette transfer 125 pl from Rows A amp B Column 1 to Column 2 and mix with pipette action 5 Continue the 125 pl transfers and mixing through Column 6 After the nal mixing in Column 6 the last 125 pl is removed and discarded 6 In Rows A amp B Columns 1 7 dispense 125 pl 1 M K2HPO4 using a multichannel pipette for nal volumes of 250 pl The concentration of pnitrophenol in Rows A amp B Column 1 is 0125 mM To be technically precise 125 pl 064 M K2HPO4 would be added which results from the sum of 45 pl H20 and 80 pl 1 M K2HPO4 as is done for the stopped enzyme reactions below Preparation of the Alkaline Phosphatase dilutions 7 In Row D Columns 2 5 dispense 100 pl of 01 M Tris pH 95 using the P200 pipette 8 In Row D Column 1 dispense 200 pl of alkaline phosphatase enzyme using a P200 pipette 9 Using the P200 pipette transfer 100 pl of alkaline phosphatase enzyme from Column 1 to Column 2 mix well with pipette action being careful not to introduce bubbles 10 Continue the 100 pl transfers and mixing successively through to Column 5 Preparation and running the fxedtime enzyme assays 11 In Rows E H Columns 1 5 using a multichannel pipette dispense 125 pl of 01 M Tris pH 95 to each well In Column 6 dispense 135 pl of 01 M Tris pH 95 for the nonenzyme control 12 Using a multichannel pipette transfer 10 pl of the enzyme dilution series from Row D Columns 1 5 to Row B Columns 1 5 repeat by transferring 10 pl of the enzyme dilution series from Row D to Row F then Row G and then Row H Columns 1 5 13 Assays are started with pnitrophenylphosphate and stopped with K2HPO4 as follows 10 minute reactions START the reactions by dispensing 35 pl of 10 mM pnitrophenylphosphate to Row B Columns 1 6 using the multichannel pipette and then for Row P Columns 1 6 STOP the reactions at exactly 10 minutes by dispensing 80 pl 1 M K2HPO4 to Row B Columns 1 6 using the multichannel pipette The nal volume is 250 pl and then for Row F Columns 1 6 2 minute reactions START the reactions by dispensing 35 pl of 10 mM pnitrophenylphosphate to Row G Columns 1 6 using the multichannel pipette and then for Row H Columns 1 6 311 STOP the reactions at exactly 2 minutes by dispensing 80 ul 1 M KZHPO4 to Row G Columns 1 6 using the multichannel pipette The nal volume is 250 pl and then for Row H Columns 1 6 Reading the microplate 14 Take the microplate to the microplate reader place the microplate in the holder on instrument 15 At the computer controlling the microplate reader select the program ALKPHOSSEE from the Sessions tab found on the upper right comer or if the ALKPHOS program is already open go to 16 16 Click on the START button found along the top tool bar If the START button is not visible then click on the Procedure tab found along the right edge of the program screen If the program is already open it might ask if the previous results should be saved select CONTINUE Take the microplate and the printout of the results 17 Calculate the average corrected absorbances for Tables 34 and 35 also enter these corrected values onto the class data EXCEL spread sheet at the front of lab Table 34A 10 minute Alkaline Phosphatase reaction rates as a function of enzyme concentration Average Corrected Corr Ave sample mg Abs4o5 Abs4o5 Average Corrected Absms minquot V0 IU enzyme after 10 after 10 min Abs4o5 after Average from class from class in reaction min 10 min Abs4o5 minquot data data 12 12 12 12I 1 22 122 123 123 12 1x24 no enz 000 000 000 000 000 no enz 000 3 12 Table 34B 2 minute Alkaline Phosphatase reaction rates as a function of enzyme concentration sample mg enzyme in reaction AbS4o5 after 2 min Average AbS4o5 after 2 min Corrected Average AbS405 after 2 min Corrected Average Abs4o5 minquot Corr Ave AbS4o5 lllill l from class data Vo ID from class data 12quot 12 12 12 122 122 123 12 12 12 no enz 000 no enz 000 000 000 000 000 Table 35 Absorbances of the pNitrophenol Dilution Series at 405 nm columns gt 1 2 3 5 pnitrophenol pM 125 625 312 156 781 391 000 Row A Row B Average AbS4o5 Corrected Average AbS4o5 000 Corrected Average Ab405 from class data 000 3 13 C REACTION RATE As A FUNCTION OF SUBSTRATE CONCENTRATION By varying the substrate concentration the Km for pnitrophenylphosphate and the Vmax for alkaline phosphatase are determined 39om a LineweaverBurk plot Also the inhibition constant Ki for the competitive inhibitor inorganic phosphate is determined A xedtime assay protocol is used and is outlined in Table 36 Reaction mix cocktails are prepared for the assays containing buffer and enzyme in the presence or absence of potential effectors and the reactions are initiated with substrate and stopped with K2HP04 1 Obtain 12 mls 02 M Tris pH 95 750 pl 0125 mgml alkaline phosphatase AP 750 pl 10 mM pnitrophenylphosphate pNPP 500 pl 25 mM phosphate Pi and 4 mls 1 M K2HP04 Preparation of the substrate dilution series in RowA 2 In Row A Columns 1 and 7 dispense 280 pl 10 mM pNPP 3 In Row A Colunrms 2 5 and 8 11 dispense 140 pl H20 4 Transfer 140 pl pNPP from Column 1 to Column 2 mix well then transfer 140 pl from Column 2 to Column 3 then Column 3 to Column 4 then Column 4 to Column 5 mixing well each time 5 Repeat the serial dilution of step 4 for Row A Columns 7 through 1 1 Preparation of the reaction mix cocktails use 5 ml test tubes 6 No inhibitor mix well 1530 pl Tris 1938 pl H20 and 204 pl AP With a P200 dispense 153 pl of the reaction mix into Rows B and C Columns 1 5 7 In the presence of 50 JM Pi mix well 765 pl Tris 928 pl H20 408 pl 25 mM Pi and 102 pl AP Dispense 153 pl of the reaction mix into Rows D and E Columns 1 5 8 In the presence of 100 pM Pi mix well 765 pl Tris 887 pl H20 816 pl 25 mM Pi and 102 pl AP Dispense 153 pl of the reaction mix into Rows B and C Columns 7 11 9 In the presence of 1 50 uM Pi mix we1l765 pl Tris 847 pl H20 1224 pl 25 mM Pi and 102 pl AP Dispense 153 pl of the reaction mix into Rows D and E Columns 7 11 10 Nonenzyme Controls mix well 765 pl Tris and 1071 pl H20 Dispense 153 pl of the reaction mix into Row F Columns 1 5 and Columns 7 11 1 1 LENGTH of FIXEDTIME REACTION The length of time chosen for the xedtime assay 2 minutes see Part B Starting and Stopping the Fixedtime Reactions 12 Pour the 1 M K2HP04 into a plastic trough START the reactions with the 530 pl multichannel pipette transfer 17 pl of pNPP from Row A Columns 15 to Row B Columns 15 and then transfer 17 pl of pNPP from Row A Columns 1 S to Row C Columns 15 Run the reactions for 2 minutes 314 STOP the reactions with the 30300 ul multichannel pipette after 2 minutes dispense 80 pl of 1 M KHP04 to Row B Columns 15 and then 80 ul of 1 M K2HPO4 into Row C Columns 15 13 Repeat steps 11 and 12 for all rows D and E then F until all reactions are nished 14 Repeat steps 11 12 for Columns 7 ll 15 Read the stopped reactions in the microplate reader at 405 nm ALKPHOSSEE program 16 Record absorbance data obtained from the microplate reader on Table 37 17 Calculate the average corrected absorbances per minute in Tables 37 also enter these corrected values onto the class data EXCEL spread sheet at the front of lab Table 36 Protocol Alkaline Phosphatase reactions as a function of substrate concentration 1 2 3 4 5 6 7 8 9 10 11 10 mM 5 mM 25 mM 125 mM 625 pM 10 mM 5 mM 25 mM 125 mM 625 JM A pNPP pNPP pNPP pNPP pNPP pNPP pNPP pNPP pNPP pNPP 100 uM 100 pM 100 uM 100 uM 100 uM 13 no Pi no Pi no Pi no Pi no Pi Pi Pi Pi Pi Pi 100 uM 100 pM 100 pM 100 uM 100 uM C no Pi no Pi no Pi no Pi no Pi Pi Pi Pi Pi Pi 50 pM 50 pM 50 uM 50 pM 50 1M 150 uM 150 pM 150 pM 150 uM 150 uM 1 Pi Pi Pi Pi Pi Pi Pi Pi Pi Pi 50 uM 50 uM 50 nM 50 uM 50 uM 150 pM 150 pM 150 uM 150 t1M 150 pM E Pi Pi Pi Pi Pi Pi Pi Pi Pi Pi non non non non non non non non non non F enz enz enz enz enz enz enz enz enz enz Table 37 Alkaline Phosphatase Activity as a function of substrate concentration 315 AbS4o5 after 2 minutes Average Corrected AbS405 min 39 1 00 mM pNPP 050 mM pNPP 025 mM pNPP 0125 mM pNPP 00625 mM pNPP 100 mM pNPP 050 mM pNPP 025 mM pNPP 0125 mM pNPP 00625 mM pN PP Columns 1 5 no Pi no P1 50 11M 50 uM non enzyme 000 000 000 000 000 Columns 7 11 100 pM 100 pM 150 uM Pi 150 uM Pi non enzyme 000 000 000 000 000 316 Table 38 Rates and Inverse Rates from Class Data May be generated on MS Excel VoIU 1lVo an 100 0500 0250 0125 00625 10 20 40 80 16 mM mM mM mM mM nMquot mMquot mMquot mMquot mMquot pNPP pNPP pNPP pNPP pNPP p NPP pNPP pNPP pNPP pNPP no Pi S0 uM Pi 100 uM P1 150 11M Pi D LDH ENZYME ASSAY The absorption of NADH at 340 nm is not typically found for other biologically important compounds Thus 340 nm serves as a highly selective wavelength to follow the changes in NADH concentration It is for this reason that dehydrogenases were once the most studied enzymes The molar extinction coefficient for NADH 8340 6220 M4 cmquot while for NAD it is 8340 00 M4 cm In this experiment the disappearance of NADH is followed by spectrophotometry dAbs 340 umdt dSdt using a continuous assay Valid assays will be established empirically this is accomplished by experimentally nding the dilutions resulting in linear rates under the given conditions The validity of a linear rate is then con rmed by performing the enzyme assay at another dilution and determining if the two rates are directly proportional ie the rates are dependent on the enzyme concentration Overlay Spectra ofNAD and NADH Run overlay spectra of NAD393939 and NADH from 400 nm to 220 nm in quartz cuvettes basic operation for overlay spectra is on pg 013 Solutions of 004 mM NAD and 004 mM NADH are available Table 39 Results from the overlay spectra of NAD and NADH compound peak A nm Abs at peak 9L Abs at 340 nm 004 mM NAD 004 mM NADH 317 LDH Enzyme Assay The continuous assay is performed by following the disappearance of the NADH substrate monitored at 340 nm using the Shimadzu spectrophotometer The LDH assay is performed in a final volume of 10 ml by adding 30 ul of solution containing the LDH enzyme to 970 pl of the reaction mix that contains pyruvate and NADH in a Tris buffer To minimize the reverse reaction enzyme is added last Also two nonenzyme controls are perfonned i with the substrates and buffer only and ii with enzyme and only the NADH substrate 1 Label a 15 ml microfuge tube LDH then obtain 200 l1 of the available 010 mgml solution of LDH from the front table and keep this on ice 2 To save time and to increase the reproducibility of the LDH assays prepare a sufficient volume of an LDH assay cocktail containing the components of the reaction mixture except the enzyme The cocktail mix suitable for conducting 10 assays is Component Vol per Assay Vol per 10X Cocktail Final Cone in 10 ml assay 200 mM Tris pH 73 900 41 90 ml 30 mM pyruvate 35 pl 350 pl 66 mM NADH 35 pl 350 pl 3 Set parameters for kinetics on the spectrophotometer Select 8 Optional Kinetics Package 7L 340 mn Measured time 60 sec Lag 0 sec Rate 60 sec and Interval 10 sec 4 Prepare the 10X cocktail mix keep at room temperature 5 Prepare a set of serial dilutions of LDH 200 pl each using cold Tris buffer keep dilutions on ice It is recommended that 200 pl of a 1 10 dilution be prepared then prepare a 140 dilution and a l100 dilution by serial dilutions using the 1 10 dilution Test these three dilutions by running the assays steps 6 and 7 to assess what dilutions may be needed to achieve linear rates 6 For each assay remove 970 pl of the assay cocktail and place it in a 10 ml plastic cuvette 7 Add 30 pl of appropriatelydiluted LDH enzyme solution to start the reaction Immediately after adding the enzyme rapidly cover the cuvette with Para lm invert it once place it in the spectrophotometer and record the absorbance at 340 nm for 60 sec at 10 sec intervals 8 Evaluate the results for linearity A linear assay allows for an estimation of initial velocity Vo by extrapolation Ask are there at least two rates from different dilution that are i linear and ii proportional to each other according to their dilutions For example if a 18dilution resulted in a linear rate of 024min then a 1 16dilution should result in a rate of approximately 012min If the rate is not linear then the enzyme is either too concentrated a de nite slowing in the change of absorbance per 10 sec intervals or too dilute to achieve a constant measurable rate Record the data in Table 310 Valid assays display linear rates that are proportional to their dilutions 318 9 Run the nonenzyme controls i 970 pl reaction mix 30 pl dilution buffer ii 970 pl reaction mix without pyruvate 30 pl of the diluted enzyme that gave a linear rate Record the data in Table 310 if more trials are needed then record data next to Table 310 or on a separate sheet Table 310 LDH assays dilution AA 340 Valid Tnal assayed min Yes or No 1 110 2 140 3 1100 9 10 nonenzyme 1 nonenzyme ii Materials commercial alkaline phosphatase 1 M K2HPO4 96 well microplates 25 mM potassium phosphate 10 mM pnitrophenylphosphate 01 M Trisbase 01 M Tris pH 95 05 mM cysteine make just prior to use 15 ml plastic cuvettes 01 M TrisICl 02 M Tris pH 95 050 mM pnitrophenol in 1120 004 mM NAD 004 mM NADH commercial lactate dehydrogenase 200 mM Tris pH 73 66 mM NADH 30 mM pyruvate 3 19 Name Turn in these pages with the writeup attached DATA ANALYSIS AND POINTS FOR DISCUSSION Perform Exp 8A Attach the printed page asked for at the end of the exercise 1 Molecular weight Theoretical pl 2 Bovine calf intestinal alkaline phosphatase is a homodimer so what is the molecular weight of the mctional holoprotein ie of the homodimer holo complete Part A Reaction rate as a function of pH 1 Using the data from Table 32 plot the initial velocities IU vs assay pH for the continuous assays Are these results expected Brie y discuss your answer hint what is the name of the enzyme Attach the graph Part B Reaction rate as a function of enzyme concentration 1 Access the class data spread sheet email and the class website for Table 35 Calculate the corrected average absorbances for the pnitrophenolate standards from the class data Table 35 then plot the corrected average absorbances vs concentration of pnitrophenolate Determine the el AA Ac for the pnitrophenolate standards in 250 pl Attach the graph 2 Access the class data spread sheet email and the class website for Table 34 i Calculate the corrected average absorbances per minute and standard deviation from the class data Any data that falls outside onestandard deviation from the mean may be eliminated and then a new average and standard deviation are calculated ii Using the 31 for pnitrophenolate determined above calculate the initial velocities from the class data For valid data from both time intervals plot one graph Vo IU vs protein mass mg of alkaline phosphatase Determine the speci c activity of alkaline phosphatase A IU A mg from the graph Attach the graph iii Calculate the turnover number lU umol enzyme minquot for alkaline phosphatase from the speci c activity Use the MW of the Bovine calf intestinal alklaine phosphatase homodimer calculated above Then calculate the catalytic center activity of alkaline phosphatase Turnover number Catalytic center activity 3 20 Part C Reaction rate as a mction of substrate and inhibitor concentrations Access the class data spread sheet email and the class website for Table 38 Calculate the corrected average absorbances per minute and standard deviation from the class data Any data that falls outside onestandard deviation from the mean may be eliminated and then a new average and standard deviation are calculated Using the 1 for pnitrophenolate determined in Part B calculate the initial velocities Vo IU Enter these values in Table 38 then calculate the inverse values for Table 38 Class Data 1 From the class data in Table 38 plot 1Vo vs 1 p NPP for all conditions on the same graph in the presence and absence of phosphate Attach the graph 2 Determine the Km for pnitrophenylphosphate and Vmax for alkaline phosphatase from the LineweaverBurk plot 3 Determine the Ki for phosphate using the graphical method slopes of LineweaverBurk lines vs Pi See Biochemical Calculations pg 251 Attach the graph Your Group s Data 4 From your group s data in Table 37 plot 1V o vs 1 p NPP for all conditions on the same graph in the presence and absence of phosphate Attach the graph 5 Determine the Km for pnitrophenylphosphate and Vmax for alkaline phosphatase from the LineweaverBurk plot 6 Determine the Ki for phosphate using the graphical method slopes of LineweaverBurk lines vs Pi See Biochemical Calculations pg 251 Attach the graph Part D Speci c Activity of Lactate Dehydrogenase 1 For the Valid Assays Only see Table 310 complete the table below Show all work Corrected Trial dilution 330 AA 340 IU mg LDH Speci c activity assayed min min1 per assay per assay nonenzyme i 00 2 Why under what circumstances or conditions would the nonenzyme ii control leaving out pyruvate be used Experiment 3 Appendix Analytical Treatment of Kinetic Data Experiment 3 requires a thorough understanding of how to perform a kinetic analysis for an enzyme and the mathematical calculations that convert the raw spectral data into standard units Later Experiments also will make use of kinetic analysis It is strongly suggested that you read and work through this appendix with the goal of being able to understand the derivations of the equations used in kinetic analysis A IU umol product per minute For a 11 mole ratio of Substrate converted to Product S lt gt P A umol P mini 5 A umol S minquot The importance of this relationship is apparent in a kinetic analysis that is measuring the rate of the disappearance of substrate but the results are to be expressed in terms of rate of product formed Converting the negative rate of substrate disappearance to the positive rate of product produced is just a simple substitution of equivalents The rate of a product formed should never be expressed as a negative value B Spectrophotometric determination of reaction rates 1 cm path length has been omitted The Shimadzu spectrophotometer used in MCBl20L provides a visual display of the kinetic results by showing the C1l139VCd obtained graphed as Abs vs time printing out the change in Abs over speci c time intervals and the AAmin It is important to remember that you must decide whether or not the given AAmin number represents a valid assay by assessing the linearity of the resulting rate The steps from obtaining the AAmin ie the raw data and converting it to units of IU are as follows i AAmin recorded absorbance change over time observed on a spectrophotometer ii E g M min391 converts the raw data to molarity units a Mquot it s the rate per liter of assay iii AAmin 106 umolmolj uM min converts molarity to micromolarity since the 3 M4 intemational unit IU uses micromoles iv AAfmin 106 umolfmol vol of assay L IU converts micromolarity into micromoles 8 M391 ie converts concentration to a quantity IU is the international unit IU umol P minquot this is IU in the assay v AAmin 106 umolmol vol assay L 3 Mquot IUml vol of added enzyme ml dilution of added enzyme converts IU in the assay to lUml in the original undiluted enzyme solution Exp 3 Appendix 1 vi AAmin 106 umolmol vol assavLL total vol of original sample ml I 9 M4 total IU vol of added enzyme ml dilution of added enzyme converts IUml of original undiluted enzyme solution to total IU in the entire volume of original undiluted enzyme solution An Example calculation NADH Hquot pyruvate lt gt NAD lactate The disappearance of the substrate NADH is measured at 340 nm a N ADH 340nm 6220 M4 30 ul enzyme is used to start the reaction in 1 ml assays as in Experiment 4 Puri cation table sample vol of sol n AAmin dilution tested IU total IU crude extract 25 ml 0125 l500 11 mole ratio of NADH converted to NAD so AAmin of substrate AAmin of product IU in the assay 0l25min 6220 Mquot106 umolmol0001L 00201 umol minquot 00201 IU Total IU in original enzyme solution 00201 umol minquot 25 m1 0030ml 1500 8375 total IU Complete the table sample vol of sol n AAmin dilution tested IU total IU crude extract 25 ml 0125 1500 00201 8375 C Speci c Activity IUI mg protein First of all the mg protein represents all of the protein present not merely the enzyme of interest In the above example calculation the crude extract is a homogenate prepared from lysed cells that contains nearly all soluble proteins within the cells The activity assay is of course designed to measure only amount in IU of the enzyme of interest There are three methods for obtaining the speci c activity i graphical IU in assay versus mg protein in assay slope of the graphed line AIUAmg is the speci c activity This is the preferred method when an accurate speci c activity is desired especially when the enzyme is puri ed rates from several dilutions at least 3 of the enzyme solution are required to construct the graph The slope is generally derived from the best t line that compensates for the variation in the measured data Exp 3 Appendix 2 If a speci c activity is needed just for comparative purposes as in assessing the steps for a protein puri cation then methods i and ii below are suf cient ii IU in assay mg protein in assay mg protein in assay mgml protein sol n x ml used in assay x dilution iii total IU total mg protein total mg protein mgml protein so1 n x total vol of original sample D Turnover Number of an Enzyme IU umol enzyme pmol minquot umolquot391 minquot If an enzyme is puri ed then the mg of protein in the speci c activity equation is only the enzyme of interest The mg of enzyme is converted to micromoles of enzyme to arrive at the tumover number by umol enzyme mg protein x 10393 2 mg106 umol mol MW enzyme g mol Molecular weight notations MW Mr Md 1 gmol of protein 1 dalton 1 dal 1000 dal 1 kdal The Turnover Number can be found by either one of two methods i calculating IU umol enzyme convert mg to from umol the speci c activity IUmg of a puri ed enzyme ii graphically IU vs umol enzyme slope of the line is IU umol enzyme min391 Exp 3 Appendix 3 Experiment 4 AMPLIFICATION OF A GAF4 DoMAIN SEQUENCE BY THE POLYMERASE CHAIN REACTION Objectives 1 Primer design for the i PCR ampli cation of a GAF4 domain from the cyanobacterium Nostoc punctiforme ii directional cloning of the ampli ed GAF domain into an expression plasmid 2 Amplify the GAF4 domain DNA using PCR methodology 3 Perform a restriction enzyme digest and agarose gel analysis on the PCR generated DNA fragment to con rm the identity of the ampli ed DNA 4 Quantify DNA spectrophotometrically Introduction To dissect speci c functions of proteins and determine tertiary structures it is critical to isolate a protein of interest away from other proteins But a protein isolated from a natural source may be a homogeneous population that is expressed from a single gene or a heterogeneous population that is expressed 39om multiple genes within a gene family that complicates isolation procedures Also the de novo puri cation of some proteins are very dif cult such as integral membrane proteins transient proteins or low abundant proteins In the postgenomic era experiments on protein structure and function are accomplished in large part on proteins whose genes have been cloned by molecular biological techniques into an expression vector usually a plasmid and expressed in an organism The organism may be its natural home or a host organism Also these molecular biological cloning techniques allows for the cloning of portions of proteins ie mctional domains from within a larger protein to study the domain or utilize a functional attribute of a domain fused to another protein sequence Cloning is usually a straight forward process by using the polymerase chain reaction PCR to isolate a speci c gene sequence facilitated by the known genomic sequence information available from the databases and easily accessed online Many proteins are composed of structural building blocks known as domains Domains are identi ed by their tertiary structure in addition to a simple primary structural determination Protein domains are peptide sequences conferring speci c particular structural and or mctional roles within a larger protein and a speci c domain may be found in many proteins of different functions Structural motifs are short conserved stretches of sequence found within domains for enzymes a motif may identify the active site However a particular motif found within a domain may or may not possess the function implied by the motif in such cases the motif may be retained to maintain the tertiary structure of the functional domain These domains or more precisely the stretches of DNA coding for domains can be switched between different DNA sequences coding for other proteins hence creating new proteins with modi ed functions The molecular mechanisms placing one domain next to another are well established events in the genome transposition rearrangements inversions translocations deletions and duplications and homologous recombination There are many families of protein domains found in all kingdoms of life The phytochrome family of photoreceptors is an example of a protein family constructed of domains Figure 41 As research interests biochemists study the structures and possible functions of proteins protein families and their domain substructures The goals of such inquiries are to understand the origin and evolution of acquired mctions to discover altered or new 42 functions of proteins and to engineer proteins toward speci c mctions for improvements in health and agriculture 8 3 ho a 391mr 39 v1 Cquotcrn1 nquot kR 1m ant rnr39quot lb ilquot39quotT HaKo39t H Kv39 i39 p P P lt quot i to l l n fl n I 3 quot OO l39quotIE P5amp3 32 G39 F P I 39 953 39 3 H 39quotl39J 391 I ur1 quot ii E l39 r lt39 1 limit 7quot quotTquot H 39ltA l vlt ATa 39 393r39393939 1 J i i itM3 QIi Figure 41 Phytochrome family of photoreceptors a The photoreceptors have two functional portions a photosensing region and a dimerizationsensing region and exist as homodimers in association with the dimerizationsignaling region b Each of the general regions in a are made of distinct domains A bilin chromophore is covalently bound to a GAF domain and it is the chromophore that detects light quality and quantity light absorption triggers a conformational change in the receptor structure that is transmitted to other signal transduction pathways via the dimerizationsignaling region Note that the signaling region is constructed of histidine kinase related domains HisKA and HisKATPase and the photosensing regions are composed of three primary domains PAS GAF and PHY This is a highly simpli ed comparative model for example cyanobacterial phytochromes may contain multiple GAF domains with or without attached chromophores Polymerase Chain Reaction The biochemical approach to assessing the function for example of a protein of interest is to clone the protein s gene into an expression plasmid The resulting recombinant plasmid is placed in a host organism typically E coli or yeast which allows transcription and translation to occur in the host The cloned protein gene is usually expressed at high concentrations within the host this expressed protein can be analyzed om host cells that are illy or partially lysed or on preparations that are enriched for the expressed protein after partial puri cation or full puri cation The most direct means of cloning a protein coding gene is to make a DNA copy of the gene by the polymerase chain reaction method PCR and then incorporate this copied DNA segment into a plasmid Arguably one of the most signi cant breakthroughs in biochemical and molecular research efforts was the advent of the polymerase chain reaction PCR methodology In brief the method is similar to the DNA sequencing technique but without the ddNTPs and repeated multiple times PCR is quick and reliable for genes and genomes for which the sequences are known underscoring the power of the genome projects to determine the primary DNA sequences of many genomes from many organisms A prokaryotic gene can be copied directly from the genome since there are no introns Introns are non protein coding segments of DNA interspersed in the gene along with exons that are the protein coding segments Eukaryotic genes typically contain introns while prokaryotic genes do not Hence PCR reactions designed to create a copy of a contiguous protein coding segment must be performed with mRNA for most Eukaryotic genes which had introns removed by RNA processing 44 30 cycles are performed by a therrnocycler machine that very quickly raises and lowers the temperature required for each step Hence between 225 33554432 and 23 l073741824 copies of a segment of DNA is made Also notice that with each cycle the length of the template DNAs shorten to just that of the segment being copied the original parent strands and the early extended strands that were copied still remain but they are unobservable Agarose Gel Analysis The identity of the resulting DNA segment from a PCR reaction needs to be con rmed to prevent the perpetuation of an artifact ie copying the wrong segment of DNA The analysis of the DNA segment is initially performed by assessing its size in basepairs bp by agarose gel electrophoresis Electrophoresis is the separation of molecules in solution passing through a solid matrix under an electric force Agarose is a polysaccharide of galactose units and galactose derivatives crosslinked by hydrogen bonding create a gel An agarose gel matrix of 07 to 12 wv separates DNA fragments of about 20000 to 200 bp At physiological pH nucleic acids are polyanions due to the ionized phosphate groups in their phosphodiester backbone As such nucleic acids migrate towards the anode in an electric eld and therefore can be separated from one another by electrophoresis Also each DNA strand has the same chargetomass ratio due to the regular intervals of phosphate groups along the DNA molecule hence the relative electrophoretic mobility of linear DNA is determined by mass size alone The DNA in an agarose gel is visualized as bands by staining with any number of dyes that intercalates between the bases of DNA molecules and uoresces when excited with ultraviolet light some common dyes are ethidium bromide SYBR Safe and GelRed Like most uorescence detection methods it is very sensitive The mass of a linear piece of DNA eg a PCR reaction product is estimated by its mobility compared to the mobilities of a set of linear DNAs of known sizes aka markers A typical standard curve created from the known size standards Figure 43 shows mobility of linearized DNA versus size the mobility of DNA is nonlinear and the size mass is typically referred to as basepairs bp or kilobase pairs kbp hence the units are log bp or log kbp to achieve a linear standard curve In addition to size EtBrstained gels can be used to estimate the amount of DNA in a given band by visually comparing its uorescence intensity to that of a known amount of marker DNA often to one or more of the size standards mobility mm or cm log bp Figure 43 A standard curve for the electrophoresis of DNA Restriction Enzyme Mapping The size alone of a piece of DNA does not verify that it is the correct sequence eg is the PCR amplified DNA product the correct gene or is it an artifact such as a copy of another member of a gene family A quick analytical veri cation of sequence performed in the lab is restriction enzyme mapping Restriction enzymes catalyze the cleavage of double stranded DNA molecules generating smaller DNA fragments These enzymes are found in a wide variety of organisms and hundreds of puri ed restriction enzymes are available from a several biotechnological supply companies Restriction enzyme names typically consist of an abbreviation for the source organism eg Eco for E coli followed by a Roman numeral or number to indicate the enzyme Table 41 Table 41 A short list of restriction endonucleases Arrows indicate speci c cleavage sites Name Recognition Site Organism BamHI GlrGATCC Bacillus amyloliquefaciens II EcoRI Gl AATTC Escherichia colz39RY13 Hindm Al39AGCTT Haemophilus in uenzaeR Ncol Cl CATGG Nocardia corallz39naATCC 19070 Ndel CAlrTATG Neisseria denitri cans NRCC 31009 Pstl CTGCALG Providenica stuarti 164 S811 Gl TCGAC Streptomyces albus G Smal GGG lCCC Serratia marcescens Sstl GAGCTl C Streptomyces Stanford Xbal TJICTAGA Xanthomonas campestris pv badrii Restriction enzymes recognize and cleave DNA at speci c sequences that are typically palindromes The importance of this enzymatic speci city is that the digestion of a DNA molecule produces a set of discrete speci c sized fragments The pattern of restriction enzyme sites in a DNA segment is a ngerprint of that molecule and is referred to as a restriction map Hence if a PCR reaction has ampli ed the correct gene then digesting the PCR product by an enzyme whose recognition sequence is present will generate smaller sized fragments of known sizes Figure 44 shows the result of a restriction enzyme mapping of a hypothetical PCR generated DNA fragment If a 1000 bp fragment is expected the agarose gel con rms a 1000 bp band in Lane 1 is produced The sizes of the smaller fragments from restriction enzyme digests with PstI and Sstl also con rm the identity of the PCR product A 1000 bp PCR fragment with the predicted restriction enzyme recognition 0 sites Lanes Agarose Gel Lane assignments 1 PCR fragment uncut 2 Pstl digest 3 Sstl digest 4 size standards I Sstl 200 l Pstl 400 600 bp distance 2 3 I 3 Sstl 800 1000 4 he l000 4 800 4 600 1 400 2 200 100 Figure 44 Restriction enzyme mapping of a DNA fragment analyzed by agarose gel electrophoresis 46 Another important consequence of restriction enzyme digestion is the formation of cohesive ends DNA agments from different sources have the same sticky ends cohesive ends if they have been generated by digestion with same restriction enzyme eg Figure 45 shows the result of digestion with EcoRI Thus two different DNA fragments can be armealed together by complementary base pairing of the cohesive ends under appropriate conditions of temperature and ionic strength DNA ligase catalyzes the formation of a phosphodiester bond between the 339 hydroxyl and 539phosphoryl groups of adjacent nucleotides held in position by hydrogen bonding of the complimentary base pairing The reaction is called ligation Blunt end fragments such as those generated by Smal are much more di cult to ligate since they do not have complimentary cohesive ends however blunt ends do allow the ligation of any two fragments of DNA generated by different bluntend forming restriction enzymes This is recombinant DNA the recombining of DNA segments not naturally found together V Restriction enzyme reaction 5 GAATTC3 gt 5 G AATTC3 3 CTTAAG5 439quotquot 3 CTTAA G5 A Ligation reaction Figure 45 Cohesive ends sticky ends produced by Eco RI cleavage of double stranded DNA top Combining DNA fragments with Eco RI ends by Ligation bottom reformation of the Eco RI recognition site Nostoc GAF4 domain Nostoc punctiforme is a cyanobacterium which is a photosynthetic organism hence sensing changes in light intensity and wavelength and making appropriate metabolic and physiologic responses is critical In cyanobacteria there are many light sensing molecules that collectively span the visible spectrum called cyanobacteriochromes Cyanobacteriochromes sense light by a linear tetrapyrrole that is attached onto a cysteine belonging to a protein domain known as GAF The GAF domain units are universally found in many proteins in all kingdoms which contain the structural motifs for cyclic MP regulated nucleotide phosphodiesterase Adenylyl cyclase and the bacterial transcription factor lj hlA The precise mechanistic details of the GAF sensing and signaling events in the cyanobacteriochromes remain unknown One Nostoc cyanobacteriochrome sensory protein from the NpR6012 gene contains four GAF domains in tandem The fourth domain Nostoc GAF4 domain is cloned in this experiment Figures 46 and 47 The GAF1 domain of NpR6012 does not contain an attached bilin dimerizationsignaling J GAF4 09 ea lt NpR6012 gene from Nostoc punctiforme Figure 46 The NpR6012 gene from Nostoc punctiforme The fourth GAF domain is designated NpR60l2g4 phycocyanobilin is attached via a Cysteine 47 The GAF 4 domain peptide sequence is shown in Figure 47 The peptide is 176 amino acids The designation for the beginning and end of the domain is based on the tertiary structure not shown EKAVTK ISNRIRQSSD 600 VEEIFKTTTQ EVRQLLRCDR VAVYRFNPNW TGEFVAESVA HTWVKLVGPD IKTVWEDTHL 660 QETQGGRYAQ GENFVVNDIY QVGHSPEHIE ILEQFEVKAY VIVPVFAGEQ LWGLLAAYQN 720 SGTRDWDESE VTLLARIGNQ LGLALQQTEY LQQVQGQSAK 760 Figure 47 The sequence for amino acids 585 760 of the Nostoc GAF4 domain NpR6012g4 The underlined C687 is the bilin attachment site UniProtB accession number B2IU14 The attached linear tetrapyrrole is a bilin called phycocyanobilin which appears blue in color to the eye and has a molar absorption coefficient of 132000 M4 cm391 at 652 nm Figure 48 COOH COOH HC J HC IX 3939 3 quotF 0 N AN 39 N 39 N O H H H Figure 48 The structure of phycocyanobilin In order to dissect the biochemical molecular and cellular functions of the GAF domains these domains are cloned and expressed apart from their native biological environments The molecular structure of a cloned GAF domain can be altered for example by sitedirected mutagenesis The resulting recombinant mutated GAF gene then can be returned to its native organism Nostoc in this example to examine the effect such mutations may have on the sensory and signaling pathways Directional Cloning The isolation of a protein coding gene is accomplished primarily by PCR followed by the directional insertion of the PCR product into a plasmid Plasmids are extra chromosomal circular double stranded DNA molecules found in numerous prokaryotes and yeasts and is one type of DNA molecule known as vectors that are used for cloning DNA sequences of interest Cloning vectors are bio engineered from plasmids or viruses that are autonomously replicated in a host cell thus also replicating the inserted DNA fragment They range in size from two to several hundred kilobase pairs kbp Plasmids and other cloning vectors have been engineered to a ne point to where many plasmids are available and a speci c plasmid is chosen for a speci c purpose Expression plasmids are chosen to express a functional protein in host cells General Plasmid Functions In this experiment the vector pBADInteinCBD Figure 49 was chosen to express the cloned Nostoc GAF4 domain Plasmids have common features that allow them to persist in host cells and for experimental use The plasmid pBADIntein CBD is an example of a bioengineered plasmid meaning the functional portions of the plasmid are derived from multiple sources and pieced together 48 Origin of replication It is essential for a vector to have a sequence that pennits its autonomous replication and retention in host cells For bacterial hosts the ori sequence lls this function shown as the pBR322 ori Antibiotic resistance gene The presence of an antibiotic resistance permits only those host cells that have taken up a plasmid to grow in media containing an antibiotic The ampicillin resistance gene encodes Blactamase an enzyme conferring resistance to the antibiotic ampicillin Multiple cloning site A multiple cloning site MCS is typically included which contains several restriction enzyme recognition sites not found anywhere else within the plasmid sequence ie unique single sites The collection of restriction enzyme sites in the MCS gives several possibilities for the insertion of foreign DNA segments into the plasmid The pBADInteinCBD MCS contains only three restriction sites NcoIXholSmaI it was constructed using the pBAD MCS and the pTYB2 MCS for the specific purpose of cloning the Nostoc GAF protein Expression plasmids Expression plasmids must contain four essential DNA elements to transcribe and translate an inserted protein coding gene These elements might be included with an inserted gene or provided by the plasmid for convenience These elements are located in or near the MCS to reconstruct a functional recombinant gene 1 Promoter A promoter is required for RNA Polymerase to bind This is the pBAD promoter 5 to the MCS on pBADInteinCBD so the inserted gene is under its control 2 A ribosome binding RBS A RBS site located between the promoter and the initiator methionine codon allowing the translation machinery to bind 3 An initiator methionine codon All nearly all organisms begin translation of mRNAs with a start methionine codon AUG Often a plasmid will provide a start methionine followed by a short set of other codons within the MCS if the start methionine of an inserted gene is not present Of course the codons of an inserted gene must be inframe with the plasmid initiator methionine codon and accompanying codons 4 A stop codon A translation stop codon is included with an inserted gene Plasmids might provide a stop codon along with a preceding short set of other codons Again the codons of an inserted gene must be inframe with the plasmid provided stop codon if used The pBADInteinCBD provides a stop codon at the end of the InteinCBD peptide sequence the InteinCBD peptide sequence begins at the Cysteine Plasmids are often engineered to contain other features included for experimental convenience later protein isolation controlling expression etc The pBADInteinCBD has two such additional elements 5 An epitope tag An epitope tag is an amino acid sequence recognized by antibodies or other high af nity binding proteins or molecular complexes that will detect the presence of the translated cloned protein This is important for nonenzyme proteins or where enzymatic activity may be compromised or poor The pBADInteinCBD contains the InteinCBD sequence adjacent to the MCS A cloned gene whose protein is translated in frame with InteinCBD sequence will create a fusion protein with the InteinCBD sequence located the Cterminal end of the translated protein The actual affmity tag is the CBD portion of the InteinCBD peptide CBD means Chitin Binding Domain The fusion protein is 49 detected ie reports its existence by binding to the CBD which is used to af nity purify the recombinant ision protein Intein is a selfcleaving peptide under high concentrations of a thiol reducing agent such as dithiothreitol the InteinCBD fusion is removed from the expressed cloned protein fusion leaving a puri ed protein see Experiment 5 MCS Ncol Smal ATG CCA TGG other sites CCC GGG TGC TTT MET CYS InteinTBD RBS s f 5 U pBADlInte1nCBD E 42 kbp j 3 3 033322 or Figure 49 The pBADInteinCBD plasmid pBADTM is from Invitrogen the Smal site and Intein CBD sequence is derived from pTYB2TM from NEB 6 Transcriptional regulatory protein Genes cloned into the MCS of pBADInteinCBD are under control of the pBAD promoter Transcription is repressed in the presence of the araC regulator protein encoded by the araC gene on pBADInteinCBD itself Transcription is induced in the presence of arabinose which binds to the araC regulator protein altering the promoter region allowing RNA polymerase to bind and begin transcription In brief arabinose induces protein expression Such a system prevents the overexpression of a cloned protein that may be harmful to the host cells until expression is induced for an experiment Primer Design A recombinant protein coding gene must be inserted into the MCS of a plasmid properly so that the elements of an expressed gene are in the correct orientation 5 promoter gt RBS gt initiator methionine codon gt protein coding region gt translation stop codon3 Inserting a protein coding gene in the correct orientation is known as directional cloning Directional cloning is accomplished by adding different restriction enzyme recognition sequences on the 5 ends of the PCR primers The first choice of recognition sites depends on i the availability of the same sites 410 in a plasmid MCS and ii the chosen sites are not present within the gene to be cloned to avoid the destruction of the inserted gene when digested with the restriction enzymes to generate compatible sticky ends The sequences of the primers used in this experiment are shown in Figure 410 These sites are chosen in conjunction with the engineered NcoI and SmaI sites in the pBADInteinCBD MCS and these sites are not present in the Nostoc GAF4 domain gene sequence GAGAAAGCTGTCACCAAGATCAGTAACCGCATCCGGCAATCTTCAGATGTAGAAGAAATCTTCAAA 66 ACAACCACTCAAGAAGTACGACAATTACTGCGATGCGATCGCGTCGCAGTCTATCGCTTCAACCCT 132 AATTGGACTGGTGAATTTGTTGCAGAATCAGTAGCTCACACTTGGGTAAAACTGGTAGGCCCCGAT 198 ATCAAGACTGTCTGGGAAGATACCCACTTACAAGAAACTCAAGGAGGTCGATATGCCCAAGGGGAG 264 AACTTTGTTGTAAATGACATTTATCAGGTAGGTCATTCTCCTEgCACATTGAAATTTTAGAGCAA 330 TTTGAAGTCAAAGCTTATGTAATTGTTCCTGTATTTGCCGGGGAACAATTGTGGGGATTGCTAGCA 396 GCTTATCAAAATTCTGGAACTCGTGATTGGGATGAATCGGAAGTCACCTTGTTAGCACGCATTGGC 462 AACCAGTTAGGGCTAGCATTACAACAGACTGAATATTTGCAGCAAGTACEAGGGCAGTCAGCCAAA 528 Figure 410 The nucleotide sequence for the Nostoc GAF4 domain N pR60l2g4 Forward primer 5 CCCCATGGGAGAAAGCTGTCACCAAG Ecol Reverse Primer 5 CCCCCGGGTT T GGCT GACT GCCCTTGT SmaI Boxed sequence TGT is the Cysteine codon that is the bilin attachment site The Forward primer sense primer Since the GAF4 domain peptide sequence is internal to the larger cyanobacteriochrome protein it lacks a start Methionine The NcoI site allows the inclusion of a start Methionine inframe to the 5 end of the of the inserted Nostoc GAF4 domain sequence This adds three amino acids to the Nterminus of the Nostoc GAF4 domain Met ATG Pro CCA and Trp TGG Figure 411 The Reverse primer antisense primer The Smal site allows the in ame C terminal addition of the InteinCBD peptide to the 3 end of the of the inserted Nostoc GAF 4 domain sequence This adds Pro CCC and Gly GGG to the Cterrninus of the Nostoc GAF 4 domain Figure 411 For both primers the 5 addition of two cytosine nucleotides improves ability of the restriction enzymes to bind to their recognition sites and each has 18 nucleotides complementary to the GAF4 sequence Ncol Smal ATGCCATGGGAGAAAGCTGTCACCAAGM mACAAGGGCAGTCAGCCAAACCCGGGTGC GGTACCCTCTTTCGACAGTGGTTCN WTGTTCCCGTCAGTCGGTTTGGGCCC M P W E K A V T K Q G Q S A K G P C Figure 411 The insertion of the GAF4 domain PCR product into the Ncol and Smal sites of the pBADInteinCBD The Italicized nucleotide and amino acid sequences are plasmid derived whereas the nonItalicized sequences are GAF4 derived Ligation Transformation and Selection Ligation After the PCR ampli cation of the Nostoc GAF4 domain gene the resulting DNA fragment is inserted into the pBADInteinCBD plasmid where it can be stored in host E coli cells and used for later expression The PCR ampli ed Nostoc GAF4 domain gene and the pBADInteinCBD plasmid are both digested with Ncol cohesive ends and Smal blunt ends These two DNAs are joined together by the binding of the Ncol cohesive ends the enzyme ligase covalently joins the Smal blunt 411 ends and the Ncol cohesive ends forming a recombinant plasmid pBADGAFInteinCBD Figure 412 illustrates the construction of the recombinant plasmid PCR ampli ed Nostoc GAF4 domain 528 bp pBADInteinCBD 42 kbp Ncol GAF Smal pBAD promoter Ncol Smal Intein CBD I Q 1 Restriction enzyme digests with Ncol and Smal 2 Ligation with T4 Ligase reforms the phosphodiester linkages Recombinant plasmid pBADGAFInteinCBD 47 kbp Figure 412 The construction of the recombinant plasmid pBADGAFlnteinCBD Transformation and Selection The transfer of plasmids into E coli host cells is called transformation The host E coli cells are transformed by either the CaClheatshock method or the electroporation method The CaCl2heat shock method requires placing approximately 50 picomoles of plasmid into a dense solution of E coli prepared in a high concentration of CaCl2 known as competent cells this mix is incubated at 42 C for 30 seconds to 2 minutes and the plasmid is takenup by the E coli The transformed E coli cells are typically selected on an agar plate with an antibiotic only those E coli cells containing a plasmid with the antibiotic resistance gene transforrnants will survive The electroporation method again requires placing approximately 50 picomoles of plasmid into a dense solution of E coli but prepared in 10 glycerol known as electrocompetent cells the mix is place in a electroporation cuvette and the plasmid is transferred into the E coli with a burst of electric current in an instrument called an electroporator Again the transformed E coli cells are selected on agar plates containing the appropriate antibiotic The electroporation method is superior but requires the purchase of the electroporator and electroporation cuvettes Figure 413 A concluding remark on PCR and cloning The ability to sequence entire genomes and PCR technology allowed for the experimentation on many previously intractable proteins such as transient signal transduction proteins proteins difficult to purify etc The primers used in PCR are ordered from several companies that will synthesize the submitted sequences and have them delivered within a couple of days for little expense eg two primers each 21 nucleotides in length with delivery costs approximately 3000 in 2012 Cloning genes by PCR requires the presence of DNA containing the target sequence to be ampli ed The acquisition of DNA samples is often an inconvenience However many research labs are bypassing the de novo amplification of genes by having entire genes commercially synthesized from sequence information 412 available from the online accessible data banks The synthetic genes are then inserted into plasmids or other vectors as described above PCR ampli cation or PCRbased sitedirected mutagenesis may be performed on such a gene to study the function of the expressed protein etc Plasmid 0 N ansformation g selection on an agar plate antibiotic gtLr1 W 5535ac 4 mv m39iEm2 fa1Tft c 39 Lvwm 3 9 K 1 1 g Q m L 5 n 3 ll 5 n n n o n n n n n u n c o o u o o a n o u o o o o u n n n n n o o n n n o u u 1 A 7 a 1 an 31 5 n n n o n o n n n u n u a o a c o o I n u I o o u o o u o o n o n a o o n u o n o n I a I 3 5 n n n o q u a c o o a o o o u u o u n n u u n 3 n o n n n n o o n o n u n n a 4 35 U E n n o a a o o u o o o o n n n o 1 g E c a a a n 9 a o n n a o o o a o u u c o s 1 as a a n u o o o 1f lr B wDou r39 1oL1 lt39 39W39 mC W1 5 quot E n M L 3 ws Hi 4 mue o ww 5139 3939V f E coli cells transformed no uptake E coli host of plasmid Lives Dies Figure 413 Transformation and selection of host E coli cells References Lynch M 2002 Gene Duplication and Evolution Science 297 945 947 Nembaware V Crum K Kelso J Seoighe C 2002 Impact of the Presence of Paralogs on Sequence Divergence in a Set of MouseHuman Orthologs Genome Research 12 1370 1376 Hardison R 1998 Hemoglobins from Bacteria to Man J Exp Biol 201 1099 1117 Clark A 1994 Invasion and maintenance of a gene duplication PNAS 91 2950 2954 Li WH Gojobori T 1983 Rapid evolution of goat and sheep globin genes following gene duplication Mol Biol amp Evol 1 94 108 6 Holsinger K 2001 2004 Evolution in multigene families httpldarwineebuconnedueeb3 48 lecture notesmolevolmultigenemolevolmulti genehtml 7 Hartwell L et al Genetics From Genes to Genomes 2quot Edition 2004 McGraw Hill 8 Hart D Jones E Genetics Analysis of Genes and Genomes 6quot Edition 2005 Jones amp Bartlett 9 Gogarten P 2000 Evolutionary Theory An Esalen Invitational Conference httpfwwwesalenctrorg 10 Jain R Rivera M Lake J 1999 Horizontal gene transfer among genomes The complexity hypothesis PNAS 967 38013 806 11 Horizontal Gen Transfer httpenwikipediaorgwikiHorizontalgenetransfer 12 Ochman H Lawrence J Groisman E 2000 Lateral Gene Transfer and the Nature of Bacterial Innovation Nature 405 299304 13 Brown J 2003 Ancient Horizontal Gene Transfer Nature Reviews 4 121132 14 Stanhope M Lupas A Italia M Koretke K Volker C Brown J 2001 Phylogenetic analyses do not support horizontal gene transfers from bacteria to vertebrates Nature 41 1 940944 15 Baldo A McClure 1999 Evolution and Horizontal Transfer of dUTPaseEncoding Genes in Viruses and Their Hosts Joumal of Virology 739 77107721 16 Parkash R 1998 How Genes Evolve Resonance Feb 1998 2834 17 Scientists Uncover Transfer of Genetic Material Between BloodSucking Insect and Mammals httpwwwsciencedailycomreleases20100410043015 5856htm 18 Yoshida S Maruyama S Nozaki H Shirasu K 2010 Horizontal Gene Transfer by the Parasitic Plant Striga hermonthica 0 Science 28 328 1128 19 HosttoParasite Gene Transfer in Flowering Plants Phylogenetic Evidence from Malpighiales Davis C Wurdack K wwwsciencexpressorg 15 July 2004 Page 2 101126science1100671 20 PCR optimization primer design 1997 httpwwwqiagencon11iteratureqiagennews0597975pcropdf 21 Jacob F 1977 Evolution and Tinkering Science 1964285 11611166 22 Campbell I Downing A 1994 Building Protein Structure and Function from Module Units Trends in UhJDJ lJv 413 Biotechnol 125 168172 23 Bork P Doolittle R 1992 Proposed Acqusition of and Animal Protein Domain by Bacteria PNAS 8919 89908994 24 Wikipedia Protein domains Experimental Day 1 Part A PCR ampli cation of a Nostoc GAF4 domain sequence 1 Obtain and label the top of the lid of a 05 ml PCR tube 2 Transfer 5 pl of the target DNA solution that contains the Nostoc GAF4 sequence of interest into the labeled PCR tube the target DNA is approximately 10 ng to 04 ng per reaction 3 Transfer 45 pl of the PCR reaction mix cocktail into the PCR tube and mix well The reaction mix contains the following reagents standard concentrations per reaction 345 pl H20 5 pl 10X PCR buffer solution IX PCR buffer solution 10 mM TrisHCl pH 90 50 mM KCl and 15 mM MgCl2 4 pl stock dNTPs stock 25mM each dNTP nal concentration 200 pM each dNTP 05 pl Forward GAF primer nal 50 pmol stock 100 pmolpl 05 pl Reverse GAF primer nal 50 pmol stock 100 pmolpl 05 pl Taq DNA Polymerase nal 125 units The fmal reaction volume is 50 pl 4 Add one drop of mineral oil then place the tube in a rack at the front of the lab The instructor or TA will start the PCR therrnocycler when every group is ready Programmed PCR reaction cycles Step 1 9500 for 30 seconds Step 2 55 C for 30 seconds Step 3 72 C 1 minute Step 4 Repeat steps 1 3 for 24 additional cycles Step 5 72 C for 10 minutes Step 6 RT 5 The PCR reaction will take approximately 15 hours Part B GeneJetTM PCR Puri cation Kit cleanup of the PCR product The following protocol is designed to remove primers nucleotides polymerases and salts from the PCR reaction mixture thereby purifying DNA ie PCR product ranging from 100100000 bp using a GeneJetTM PCR Puri cation Kit Fermentes 1 Retrieve the tube with the PCR reaction mixture 2 Transfer the aqueous layer below the mineral oil using a P200 into a 15ml microcentrifuge tube 3 Add an equal volume of BindingBuffer ie 50 pl to the aqueous layer and mix well 4 l4 4 Place a GeneJet column onto the Vacuum manifold load the entire PCR sample in BindingBuffer onto the column and leave the columncap open 5 Turn on the vacuum It may be necessary to press down on the lid of the manifold to achieve a good seal After the sample has passed through the column turn off the vacuum 6 Add 750 pl of WashBuffer to the column and restart the vacuum to wash the bound DNA and leave the vacuum on for about two minutes to ensure the removal of the BindingBuffer 7 Remove the column from the manifold and insert it into a 15ml microcentrifuge tube labeled quotCLEAN PCRquot 8 Add 30 pl of 10 mM TrisHCl pH 85 elution buffer directly onto the center of the column and centrifuge the column in the 15 ml tube for 1 min at maximum speed The DNA PCR product is now in the liquid that has eluted from column 30 pl Discard the column 9 SAVE this sample the Clean PCR product in the refrigerator for Day 2 Day 2 Part C Restriction enzyme digest and agarose gel electrophoresis of the PCR reaction product 1 Add 20 pl dH2O to the Clean PCR product to bring the volume to 50 pl 2 Label a 05 ml microfuge tube for the Hind III restriction enzyme reaction 3 Transfer 5 pl of the Clean PCR product to the labeled tube SAVE the remaining 45 pl Clean PCR product 4 Transfer 15 pl of the restriction enzyme reaction cocktail to the labeled tube and mix well The reaction mix contains the following reagents per reaction 125 pl H20 2 pl 10X Restriction enzyme buffer solution compatible with Hind III eg NEB Buffer 2 IX NEB Buffer 2 10 mM TrisHCl 50 mM NaCl 10 mM MgCl2 1 mM Dithiothreitol pH 79 at 25 C 05 pl Hind III nal approx 20 units The nal reaction volume is 20 pl 5 Incubate at 37 C for 60 minutes While the reaction is incubating pour the agarose gel Part D Part D Agarose gel analysis of the PCR product and the restriction enzyme digests 3 groups will share one agarose gel 1 The agarose gel casting tray is atbottomed walled on two sides and open at both ends Place the tray in the apparatus so that the open ends are closed by the walls of the apparatus 8 9 415 Dissolve 12 g of agarose in 80 ml of 05 X TBE by heating the mixture in a microwave oven for approximately 12 minutes The gel will be 15 agarose that is dense enough to see DNA bands below 1000 bp Allow to cool to a comfortable temperature before pouring Just before pouring the agarose add 4 ul of the GelRedTM stain and swirl to mix the stain then immediately pour the gel Insert the thicker side of one comb and let sit until the agarose has solidi ed GelRedTM stock is l0000X 4 ul gives 05X in 80 ml Once the agarose has solidi ed remove tray with the gel and reorient it 900 so that the top of the open end of the gel ie the end with the combwells is near the cathode negative electrode cathode and the bottom of the gel is near the anode positive electrode anode Add 400 ml 05 X TBE buffer just enough to cover the gel completely Then remove the comb the buffer helps the comb slip out easier without tearing the gel Retrieve the restriction enzyme reaction when nished and add 4 pl 6X loading dye Transfer 5 ul of the Clean PCR product into a 05 ml microfuge tube add 5 u1dH2O and then add 2 ul 6X loading dye Obtain 5 ul of the DNA mass ruler standard premixed with dye see Table 42 below Load the agarose gel according to Table 43 below 10 Connect the electrodes with the lid of the apparatus and run the electrophoresis at 125 volts While the agarose gel is running quantify the Cleaned PCR product using the nanodrop Part E 11 When the leading purpledark blue dye has migrated half the distance of the gel turn the power supply off and photograph the gel Table 42 DNA Mass Ruler Standards DNA Length Amount of DNA base pairs loaded on the gel 1000 100 ng 700 70 ng 500 50 ng 200 20 ng 100 10 ng 416 Table 43 Sample Loading for the 15 Agarose Gel Lane Sample 1 2 3 uncut Cleaned PCR 12 pl first group 4 Hind III digested entire 24 pl 5 Standards 5 pl 6 7 uncut Cleaned PCR 12 pl second group 8 Hind III digested entire 24 pl 9 Standards 5 pl 10 11 uncut Cleaned PCR 12 pl third group 12 Hind III digested entire 24 pl 39 13 Standards 5 pl 14 Part E Quantifying the PCR product Nanodrop Spectrophotometer To quantify the amount of the PCR product the Nanodrop Spectrophotometer is used The Nanodrop spectrophotometer is a highsensitivity instrument that measures UV and visible absorbances in as little as 2 pl sample volume The sample is loaded onto a ber optic cable and the absorbance is measured across 1 mm gap distance ie this instrument reads both a 1 mm and a 01 mm path length This spectrophotometer is fast and obviously saves sample volumes 1 On the Nanodrop startup menu select Nucleic Acid The rst user will initialize the instrument by cleaning the pedestals and loading a water sample Initialization should be necessary only at the beginning of the lab period 2 In the upper left hand corner of the screen note the display Nucleic Acid Before measuring a sample scrub the pedestals of the Nanodrop briskly with a Kimwipe Please treat the Nanodrop gently DO NOT lift the arm by the exible ber optic cable DO NOT bang the arm down onto the bottom pedestal Lower the arm gently 3 Measure the absorbances of the Clean PCR product a Load 4 pl of dH2O onto the bottom pedestal Lower the ber optic cable arm gently b Find the button labeled Blank on the screen and click it The machine will create a meniscus between the two arms and display a blank screen c Wipe the water off the pedestals with a Kimwipe Load 4 pl of a sample the PCR product onto the lower pedestal Click the button labeled Measure When the machine displays the spectrum record Absorbances in Table 44 below and print the data d After measuring the sample clean the pedestal with a Kimwipe Table 44 Nanodrop measurements Sample A260 A280 A260A280 PCR product Materials agarose 6X DNA loading dye 2 mgml ethidium bromide 05X TBE DNA Mass Ruler 100 bp to 1000 bp Ferrnentes GeneJetTM PCR Puri cation Kit Columns and accompanying solutions Binding Buffer WashBuffer and 10 mM Tris pH 85 elution buffer PCR ingredients 10X PCR buffermix nal 1X concentration 10mM TrisHC1 pH 90 50mM KCI 15mM MgC12 Stock dNTPs 25mM each dNTP nal concentration 200 pM each dNTP DNA primers speci c to Nostoc GAF gene nal concentration 50 pmolpl each primer Taq DNA Polymerase nal 125 units Restriction enzyme and accompanying buffer Hind III Restriction enzyme reaction mix cocktail for one reaction 15 pl cocktail volume 20 pl nal volume 125 pl H20 2 pl 10X buffer speci ed for the enzyme by manufacturer 05 pl restriction enzyme Name Turn in this page with the writeup attached DATA ANALYSIS AND POINTS FOR DISCUSSION Experiment 8B 1 Attach the requested print out 2 a What restriction enzyme recognition sites are available in the MCS of pBADInteinCBD b Are any of these sites present in the Nostoc GAF4 DNA sequence 3 a What is the expected size in bp of the PCR product in Experiment 4 b Locate the Hind III recognition site in Figure 410 when the PCR product is digested with Hind III what are the expected sizes of the bands DNA Quanti cation 1 When DNA is sufficiently isolated from other cellular components the concentration of DNA is easily determined by spectrophotometry An absorbance value of 10 corresponds to 50 pgml of double stranded DNA at 260 nm in a 1 cm path length or using the Beer s Law expression a 260mn 0020 pgml 1 cm a Calculate the concentration of DNA in the Clean PCR sample b Pure double stranded DNA has an A250A230 ratio of approximately 18 What is the A260A230 ratio for the PCR product from the nanodrop results Agarose Gel Analysis Verifying that the correct gene was ampli ed 1 Is the expected size of the PCR product observed on the agarose gel 2 Are the expected sizes of the Hind III digested PCR product observed on the agarose gel 3 Based on the answers to questions 1 and 2 above is the identity of the Nostoc GAF4 sequence veri ed 4 What method will most accurately determine that the identity of the PCR band is the Nostoc GAF4 gene sequence NpR6012g4 Li I Experiment 5 PURIFICATION OF A CLONED GAF DOMAIN PROTEIN EXPRESSED IN AN E COLI HOST Objectives 1 Expression of a cloned GAF 4 domain from Nostoc punctiforme in an E coli host 2 Af nity puri cation of the GAF4 domain using the Intein Chitin Binding Domain epitope tag Introduction Life science research is an integrated enterprise connecting molecular biochemical cellular developmental genetic and evolutionary biology These disciplines have generated enormous amounts of information on protein structures and activities on genetic loci coding for proteins and on complex patterns of regulation of expression and cellular functions that are available from databases Protein puri cation and characterization is a vitally active arena for broadening the informational databases and for increasing the understanding of proteins and their cellular activities a short list of examples include native protein structure versus bioengineered protein structure gene expression cytoskeletal structure cell division metabolism enzyme mechanisms proteinprotein interaction signal transduction receptor function immune responses and energy conversion The classical approach to protein puri cation de novo ie orn a natural source has been replaced by a knowledgebased approach incumbent with genome sequence infonnation The knowledge based approach is to begin with the genomic sequence or a cDNA sequence of a protein and then to devise a cloning strategy once cloned expression is induced and the protein is puri ed from its host organism The GAF domain of Nostoc punctiforme Nostoc punctiforrne is a cyanobacterium which is a photosynthetic organism hence sensing changes in light intensity and wavelength and signaling the appropriate metabolic and physiologic responses is critical One well known group of light sensing molecules is the phytochrome family of photoreceptors see Figure 41 Phytochromes are photoreceptors that sense light via a covalently attached linear tetrapyrrole a bilin to a cysteine In higher plants the attached bilin exists in two states a ground state known as the red light absorbing form Pr and a far red light absorbing form P The Pr form senses light by absorbing light in the red spectrum which converts the bilin to the Pfr form The Pfr form will naturally return to the Pr ground state or exposing the Pfr form to farred light will convert it to the Pr form Figure 51 The P form is believed to stimulate signal transduction pathways The interconversion of Pr and Pfr is also called RFR photocycling hv red photon Pr Tquotgt P T9 signal transduction hv farred photon natural conversion Figure 51 The photo conversion between the red and far red forms of phytochrome The bilin chromophore in these photoreceptors is attached to a cysteine of a GAF domain see Figure 41 within the photosensing portion of the protein The GAF domain is an universal structural unit found in the 52 phytochrome photoreceptor family and many other signaling and sensory proteins from all kingdoms of life GAF cyclic QMP regulated nucleotide phosphodiesterase gdenylyl cyclase the bacterial transcription factor FhlA In cyanobacteria there are a series of light sensing molecules in the phytochrome family that collectively span the visible spectrum called cyanobacteriochromes Cph The bilins of the Cph are attached to GAF domains via a conserved cysteine however some are RFR photocycling as the higher plant phytochrome while others exhibit blue and green light BG photocycling or green and red light GR photocycling or orange and green light OG photocycling hence this collection of sensory molecules span the entire visible range of light The Nostoc GAF4 domain cloned in Experiment 4 see Figure 46 the rst GAF domain does not contain a bilin and the other three GAF domains each have an attached bilin phycocyanobilin Figure 48 The phycocyanobilin of GAF 4 domain exhibits a GR photocycling Figure 52 hv red photon CphR 39 gt CphG hv green photon K natural conversion Figure 52 The GR photocycling observed from the Nostoc GAF4 domain NpR60l2g4 The precise mechanistic details of sensing and signaling events via the phycocyanobilin containing GAF domains remain unknown In order to dissect the biochemical molecular and cellular functions of these light sensory GAF domains these domains are cloned and expressed apart from their native biological environments The molecular structure of a cloned light sensory GAF domain then can be examined for example by altering its structure using site directed mutagenesis The resulting recombinant mutated GAF gene then can be returned to its native organism Nostoc in this example to examine the effect such mutations may have on the sensory and signaling pathways Puri cation of Expressed Recombinant Proteins in E coli To con dently establish the properties of any protein the protein must be puri ed This means it must be separated from all other types of proteins found within cells and also from a variety of other molecules such as nucleic acids lipids carbohydrates amino acids and salts etc Due to the use of molecular biological tools in this post genomic era the de novo puri cation of proteins is typically not the rst nor primary approach to studying proteins The de novo puri cation of proteins often requires large amounts of material with variable quality And much of the earlier biochemical structurefunction analysis of proteins relied on the discovery of natural mutants for example sickle cell anemia is caused by a single amino acid mutation By contrast cloning the genes for proteins into a plasmid and expressing those genes in a host organism allows for the control of expression and simpli ed puri cation steps Plus the cloned genes are amenable to creating speci c mutations for direct experiments on protein structurefunction which bypasses the need to nd naturally occurring mutants this experimental process is known as directed evolution The genes for proteins of interest are often ampli ed by PCR and then directionally cloned into plasmids to form functionally expressed genes that are hosted in E coli or yeast These proteins can be puri ed by conventional puri cation methods However a cloned protein gene is often inserted into a plasmid with the purpose to fonn a fl1SlOl l protein with an epitope sequence available on the plasmid known as an epitope 53 tag an epitope fusion permits the direct detection of the protein for example by Westem blot analysis Experiment 7 and or puri cation of the protein by af nity chromatography this Experiment Expression and Puri cation of the Nostoc GAF4 domain Expression To induce protein expression for the pBADGAFInteinCBD plasmid meaning to allow RNA polymerase to bind to the promoter and begin transcription the sugar arabinose is added to growth media The arabinose binds to the araC regulator protein altering the promoter site to accept RNA polymerase binding This requires use of an E coli host strain de cient in arabinose metabolism In this experiment the E coli strain XJb DETM is used which is de cient in one of the genes in the arabinose operon araB Puri cation The basic steps of purifying a soluble protein whether from a de novo source or from a host organism involve homogenization and fractionation Homogenization is the lysis of cells releasing the cellular contents and then the creation of a crude extract which is the removal of insoluble material from the homogenate Crude extracts are typically prepared by ltration or centrifugation If purifying a membrane protein the insoluble membranes would be kept while the soluble portion would be discarded After crude extracts are prepared several steps are usually required to purify a protein The number and variety of possible puri cation steps can be many and the actual procedure for purifying a protein is determined empirically However since so many proteins have been successfully puri ed there are generalized strategies to follow The initial steps of these strategies in large part depend on the cellular location of the protein to be puri ed for example membrane proteins often require the addition of detergents that are non ionic and nondenaturing to release them from the membranes and to keep them soluble in an aqueous solution whereas proteins located in organs such pancreas or liver often requires the addition of protease inhibitors as these tissues contain high levels of proteases Also some proteins are located in several types of tissues and cells often a tissue is chosen if it is known to contain the greatest cellular concentrations of the target protein The earliest proteins puri ed were mostly soluble proteins located in the cytosol of cells and organelles that are simpler to extract in a homogenate The cloning of a protein gene into a plasmid can allow for the elimination of many puri cation steps in a protocol by employing an epitope tag An epitope is an amino acid sequence that binds to another molecule or molecular complex with high speci city By creating a fusion protein between a protein of interest and an epitope tag the protein of interest can be puri ed by af nity chromatography As described in Experiment 4 the Nostoc GAF4 domain is cloned inframe with the InteinCBD sequence to create just such a fusion protein Given the wide variety of detailed strategies to purify proteins from host cells only a brief introduction to the basic strategies used to purify the Nostoc GAF4 domain from E coli host cells by the InteinChitin Binding Domain af nity chromatography is presented Homogenization Tissues and cells must be disrupted to release proteins in a soluble form Both the disruption technique grinding freezing and thawing treatment with organic solvent or mild detergents etc and the disruption buffer are chosen with the stability of the particular protein in mind The use of high ionic strength solutions detergents stabilizing agents or chelating agents are often employed to release enzymes bound to membranes or protein complexes Homogenization is usually performed with cold buffer at a volume between 5 to 10 times the mass of the tissue ie 10 ml buffer per 1 g of tissue This helps ensure that the tissue will become suf ciently dispersed homogenized and diluted which reduces the destruction of proteins by protease activity Removal of unbroken cells large cell fragments and unwanted subcellular particles and organelles from eukaryote sources is accomplished by velocity centrifugation at relatively low to moderate speeds The resulting supernatant yields a crude extract composed of everything solubilized from the cell while the pellet contains the heavier particles such as unbroken cells 54 In this experiment the XJbDETM host E coli cells are a strain engineered to autolyse This means the usual techniques of using a French press or tissue shearing are bypassed which is not necessarily superior yet allows many groups of students in MCB l20L the ability to break open the host E coli without lining up to use special equipment The XJbDETM strain carries a 7 endolysin gene which codes for a protein that breaks the peptidoglycan complex of the cell wall this gene is induced by arabinose and after the cells are frozen they lyse easily upon thawing The problem that occurs with these cells is the release of the genomic DNA creating a highly viscous lysate hence the addition of DNaseI is used to digest the DNA and reduce viscosity Fractionation Fractionation is the separation of components from one another The choice of fractionation procedures used is based on the physical and chemical properties of the protein in question Under speci c conditions these properties include the protein mass net charge solubility binding characteristics kinetic properties hydrophobic interactions or a combination of two properties such as the chargemass ratio PhysicalChemical Property Common Fractionation Techniques mass sizeexclusion chromatography SDS gel electrophoresis ltration net charge ionexchange chromatography isoelectric focusing electrophoresis solubility salt precipitation organic extraction binding characteristics af nity chromatography hydrophobic interaction chromatography kinetic properties af nity chromatography Native gel electrophoresis hydrophobic interaction reverse phase chromatography hydrophobic interaction chromatography Protein puri cation typically involves several steps with each step applying a different procedure since proteins have different combinations of properties Procedures used to fractionate proteins include concentration by the addition of salt organic solvent or ltration column chromatography ie size exclusion ionexchange affmity differential centrifugation and electrophoresis Earlier steps in a scheme use methods of relatively high capacity but relatively low speci city for example ammonium sulfate precipitation or ultra ltraion Large quantities of crude or partially fractionated proteins are quickly and ef ciently processed Conversely later steps use methods of high speci city but of low capacity for example affinity chromatography Because of its high solubility in water and very low heat of solution ammonium sulfate NH42SO4 is a highly effective and widely used salt for protein fractionation and concentration Each protein has its own degree of solubility in ammonium sulfate so fractionation occurs when some proteins precipitate and others remain soluble at particular concentrations of ammonium sulfate The relationship between grams of ammonium sulfate added and resulting percent saturation is not a linear one Tables listing the mass of ammonium sulfate per volume of solution or volume of saturated ammonium sulfate required to take a solution at a given percent saturation to a higher one are found in many references for protein puri cation The puri cation steps using ammonium sulfate is referred to as taking ammonium sulfate cuts and in biochemistry jargon the cut refers to the pellet In this experiment ammonium sulfate is used at a relatively high concentration nominally 60 saturated ammonium sulfate solution The purpose is to precipitate and concentrate the proteins from the host E coli lysate which contains the expressed recombinant GAF4InteinCBD fusion protein The concentrated proteins in the pellet are resuspended with a buffered solution suitable for applying it to the Chitinaf nity column used in the next step Hence the ammonium sulfate step used does not result in the substantial fraction of proteins Fractionation by Column Chromatography Chromatography is the movement of molecules in a solution called the mobile phase that passes over a solid stationary phase also referred to as the matrix The 55 molecules in the mobile phase interact differently with the matrix The differential mobility of the molecules is what affects their separation For column chromatography a cylindrical column is packed with a solid matrix and the mobile phase is run through the column by gravity or using a pump The solid matrix is usually beads made of silica polystyrene or sugar polymers that can be chemically modi ed to possess speci c physical properties to interact with the molecules of the mobile phase The choice of matrix determines the principle of separation actionation Four common types of column chromatography used for the separation of proteins and peptides are sizeexclusion ionexchange af nity and reversephase The amino acid composition of proteins and peptides and or the tertiary structures affecting the degree of availability of speci c amino acids over the surface of proteins determines their interaction with the different types of matrices Affinity Chromatography In af nity chromatography action is based on the ability to bind to a speci c molecule referred to as a ligand attached to the matrix beads When a protein solution is run through an af nity column a protein that binds to the ligand is retained in the colurrm while all other proteins are found in the owthrough fraction Then the bound protein is eluted with another molecule in solution having a higher af nity for the protein than that of the ligand or free ligand in molar excess For enzymes using a substrate as a ligand is not usually productive since the enzyme may catalytically convert the ligand and render the af nity property useless Af nity chromatography typically yields very pure preparations of speci c proteins in a single column passage There are many variations of the af nity process described above In this experiment for the InteinCBD af nity chromatography the CBD domain binds to chitin beads and the elution of the protein of interest is achieved by incubation with a reducing agent such as dithiothreitol DTT The reducing agent induces the selfcleavage and removal of the InteinCBD through the cysteine of the Intein see Figures 53 and 54 the protein of interest the Nostoc GAF4 domain in this experiment is eluted while the InteinCBD remains bound to the chitin beads Bound fusion protein GAF4InteinCBD W95quot can Chitin Bead PCB DTT 4 C quot39 16 hours Free GAF Intein can Chitin Bead A PCB PCB phycocyanobilfn Figure 53 InteinCBD binding to chitin beads elution of the Nostoc GAF4 domain by selfcleavage of the Intein sequence in the presence of a reducing agent 56 Following and Quantt ring the Isolation Process The progress of protein puri cation is followed for each the fractionation step used For example after centrifugation is the target protein in the pellet or the supernatant Also how much of the target enzyme is recovered after each fractionation step is employed Hence there needs to be an assay speci c for the target protein Enzymes For enzymes the total activity of the target protein and the total mass of all protein are both measured The ultimate goal is to isolate the target protein from all other cellular proteins Thus the total TU is compared to the total amount of protein all proteins present in the fraction determined by a protein assay to calculate the speci c activity Activity IUassay see Experiment 3 Total IU IUassaytotal fraction volume volume of the fractionassaydilution Speci c Activity total IU total mg As the progress of the puri cation proceeds the speci c activity increases until the enzyme is homogeneous at which time the speci c activity is constant Nonenzyme proteins For nonenzyme proteins other methods such as SDSPAGE can be used to follow the enrichment of the target protein through the various steps of a puri cation scheme or if an antibody is available an ELISA assay can be used Or some intrinsic property of the target protein can be measured In this Experiment the presence and quantity of the Nostoc GAF4 domain is assessed by the intrinsic absorption properties of the phycocyanobilin chromophore The concept of the speci c activity is employed without an enzyme activity by de ning the absorption properties of the chromophore as the activity For example using the molar absorption coefficient the nnol of phycocyanobilin PCB is calculated Activity umol PCB assay Total PCB unol PCB assaytotal fraction vol vol of the fractionassaydilution Speci c Activity total PCB total mg Nostoc GAF domain Cterminus Sma I Intein GCCAAA CCCGGG TGCTTTmmm D 9 ltgt4uot7 Aa Lys Pro CV5 Phe D I DTT 4 C quot 16 hours Nostoc GAF domain Cterminus Sma l Intein I ass j l s I i quotGCCAAA CCCGGG lTGCTTTmmm Ala Lvs Pro U 3 E El The Cysteine of the lntein sequence selfcleaves in the presence of a reducing agent note under these circumstances the Nostoc GAF domain has two additional amino acids fused to the Cterminus although the InteinCBD is removed Figure 54 The SelfCleavage of the Intein sequence Assessing Purity The homogeneity ie the purity of a protein preparation can be dif cult to judge The demonstration of purity depends on a negative result which is the inability to demonstrate heterogeneity Most often the nal judgment of purity rests on the results of polyacrylamide electrophoresis under 57 denaturing conditions ie SDS PAGE Experiment 6 showing a single protein band or only those bands that correspond to the subunits of a multisubunit protein complex Concentration Dialysis and Storage At the end of a puri cation procedure a puri ed protein requires storage and may need to be dialyzed and or concentrated Three standard concentrating methods are salt precipitation lyophilization 39eezedrying and ltration Concentration and dialysis are often desirable since higher protein concentrations in selected buffered solutions will maximize protein stability Storage may require addition of stabilizing agents such as glycerol antimicrobial agents or antioxidants such as 2 mercaptethanol or dithiothreitol Daily storage may only require placing samples in a refrigerator approx 4 C to 10 C while longer term storage may require storage at 20 C or 80 C General Care and Handling of Proteins Extremes of pH or temperature might irreversibly denature any enzyme Many compounds reactive towards amino acids also can cause inactivation of an enzyme Other less obvious experimental conditions such as the forces at interfaces between solution and air or solution and solid can disrupt protein structure Thus keep most protein solutions on ice avoid abrupt changes in pH or temperature avoid reactive or adverse chemicals and avoid foaming when stirring When an enzyme concentration is very low it may be necessary to stabilize it typically by adding glycerol or a noninterfering protein eg BSA Commercially available enzymes such as Taq polymerase or restriction enzymes are supplied in solutions containing glycerol at concentrations between 20 to 50 percent References 1 Scopes R 1987 Protein Puri cation Principles and Practice 2nd Edition SpringerVerlag New York 2 Whitaker John R 1994 Principles of Enzymology for the Food Sciences 2nd Edition Marcel Dekker New York 3 Alberts et al 2002 Molecular Biology of the Cell Fourth Ed Garland Science New York 4 Nelson and Cox 2005 Lehninger Principles of Biochemistry Fourth Ed Freeman New York 5 httpwwwornlgovscitechresourcesIIumanGenomeposterschromosomehbbshtm sickle cell anemia 6 httpwwwnebcomnebecommproductsproductn6702asp pTYB2 plasmid 7 httptoolsinvitrogencomcontentsfsmanualspbadmanpdf pBAD plasmid 8 Shleif R 2010 AraC protein regulation of the L arabinose operon in Escherichia coli and the light switch mechanism of AraC action httpgenebiojhuedu0urspdf127pdf 9 Chitin Bead affinity chromatography NEB Chitin Beads manufacturer s protocol 10 Ho YS et al 2000 Structure of the GAP domain a ubiquitous signaling motif and a new class of cyclic GMP receptor The EMBO Joumal 19 5288 5299 11 Gambetta GA Lagarias JC 2001 Genetic engineering of phytochrome biosynthesis in bacteria PNAS 98 19 1056610571 12 lkeuchi M lshizuka T 2008 Photochem Photobiol Sci 7 11591167 13 Rockwell NC et al 2008 Biochemistry 472773047316 14 Narikawa R et al 2008 J Mol Biol 388 844855 15 Hirose Y Narikawa R Katayama M 2010 PNAS 10719 88548859 16 Rockwell NC Lagarias JC 2010 Eur J ChemPhysChem 116 11721180 17 Kehoe D 2010 PNAS 10720 90299030 18 Rockwell NC et al 2011 PNAS 10829 1185411859 19 lshizuka T et al 2011 Biochemistry 506 953961 20 Kim PW et al 2012 Femtosecond Photodynamics of the RedlGreen Cyanobacteriochrome NpR6012g4 from Nostoc punctiforme Biochemistry 512 608618 21 Rockwell NC Martin SS Lagarias JC 2012 Biochemistry 51 35763 585 22 Rockwell NC Martin SS Lagarias JC 2012 Biochemistry 51 96679677 Experimental Prelab Part A Starter culture inoculate 8 x 2 ml LB plus 50 ugml Ampicillin 25 ugml Kanarnycin with XJbDETM transformants containing pBADGAFInteinCBD Shake overnight at 37 C 58 Pre Iab Part B Inoculate 8 x 90 ml growth media LB 50 ugml Ampicillin 25 ugml Kanamycin 30 mM arabinose 10 mM IPTG 10 mM MgCl2 in 500 ml asks with the 2 ml overnight starter culture Shake at room temperature for 16 to 20 hours There is a second plasmid present that contains the operon for the biosynthesis of the phycocyanobilin chromophore the plasmid carries a Kanamycin antibiotic resistance gene and this operon is induced with IPTG This is enough cells for two lab rooms 22 groups Day 1 Part C Cell collection and lysis three groups will use the contents of one ask l Distribute 30 ml of the overnight induced cultures into 40 ml Oak Ridge centrifuge tubes centrifuge tubes with caps 2 Collect the cells by centrifugation at 7000 rpm 7K rpm for 10 min 3 Each group obtains one centrifuge tube of pelleted cells 4 Carefully pour off the supernatant and keep the cell pellet 5 Resuspend the cell pellet in 5 ml H20 in the centrifuge tube Recap and label the tube with a sharpie 6 Give the capped labeled centrifuge tube to a TA or the instructor after all samples are collected the sample will be frozen at 80 C for 30 min 7 During the 30 min freeze prepare a 95 saturated solution of ammonium sulfate Add 13 g ammonium sulfate to 20 ml of H20 in a 50 ml beaker stir until dissolved Set aside until step 12 8 After the 30 min freeze swirl the centrifuge tube of frozen cells in a 250 ml beaker half full with warm tap H20 until the pellet is fully thawed vortex frequently the process takes approximately 5 min 9 As soon as the cells have thawed and lysed add 60 ul 100X DNase I buffer 100 mM Tris pH 75 500 mM MgCl2 130 mM CaCl2 into the cell lysate recap and vortex it is important to distribute the buffer evenly throughout the viscous mixture 10 Add 80 pl of DNase I 10 units ul recap and mix by inverting the tube 10 times Transfer the contents into 4 labeled 15 ml microcentrifuge tubes and incubate in the 37 C H20 bath for 30 min DNase 1 decreases the viscosity of the solution by partially degrading the genomic DNA 11 The total volume of cell lysate is approximately 6 ml Transfer the DNase I treated cell lysate into a new 40 ml centri ige tube Add 40 ml 20 mM Hepes pH 80 and vortex Centrifugation at 10000 rpm for 10 min which removes unbroken cells and cell debris the autolysis is approximately 70 ef cient Transfer the supernatant post DNase I fraction into a new centrifuge tube Measure the volume of the post DNase I fraction ml Use the P1000 SAVE 200 ll of the post DNase I fraction in a labeled microfuge tube Perform the spectral analysis as follows i Set the Shimadzu spectrophotometer on Overlay Spectrum Mode Parameters 800 nm 400 mn Absorbance range 00 02 after running the spectrum adjust the range so that the A652 mn peak is maximized in the selected range ii Place plastic cuvettes containing H20 in the spectrophotometer press the F1 key for baseline correction this autozeroes the cuvettes across the spectrum of wavelengths iii Retrieve the sample cuvette remove the water add 10 ml of the fraction to the sample cuvette iv Shine GREEN light through the solution for 1 minute v Place the cuvette in the spectrophotometer and press start to run the spectrum Adjust the absorbance range as needed vi Use the cursor arrows record the Abs at 652 nm and Abs at 750 nm Part D Ammonium sulfate precipitation 12 Add 16 ml 95 saturated ammonium sulfate to the 10 ml of Post DNase I fraction recap and mix by inverting the tube 10 times to ensure the complete mixing of the solutions the solution is nominally 60 saturated ammonium sulfate 13 Let sit on ice for 30 min 14 Collect the protein pellet by centrifugation at 10000 rpm for 10 min Make sure the tube is labeled 15 Carefully pour off the supernatant Keep the pellet which should be blue in color 16 Resuspend the pellet with 500 pl of 20 mM Hepes pH 80 repeatedly collect and dispense the 500 pl of solution until the blue precipitate is dissolved try to avoid the whitish occulent precipitate higher up on the tube Transfer the resuspended pellet to a 20 ml test tube then add 5 ml chitin Column Buffer 20 mM Hepes pH 80 01 mM EDTA 500 mM NaCl 01 TritonXl00 this is the Resuspended NH42SO4 fraction Measure the volume of the Resuspended NH42SO4 action ml Use the P1000 SAVE 200 pl of the Resuspended N H42S04 fraction in a labeled micro ige tube Perform the spectral analysis as described in Step 11 above Abs 652 and Abs 750 Part E Chitin affinity column the column is already equilibrated with Column Buffer and ready to use 17 Load the chitin affmity column with the resuspended protein pellet in colurrm buffer by pipetting the solution into the column cylinder above the bed of chitin beads pipette the solution against the inside of the glass column so the solution ows gently down the sides Place a beaker below the column Then remove the bottom cap to open the column and let the solution ow through the column Let the meniscus of the solution reach the top of the chitin beads then recap the bottom of the column to stop the ow DON T LET THE COLUMN RUN DRY After loading the resuspended protein pellet there should be a blue colored band on top of the beads this is the GAF4IIntein CBD fusion protein hold a sheet of blank white paper behind the column to help visualize the blue color 510 18 Add 5 ml of Column Buffer to wash the beads the bed of chitin beads is 05 ml hence 10 column volumes of wash is 5 ml which ensures a complete washing Uncap the column and let the wash ow through When the meniscus reaches the top of the bed of chitin beads recap the column 19 Immediately add 15 ml 3 column volumes of eshly prepared Cleavage Buffer 30 mM DTT in 20 mM Hepes pH 80 01 mM EDTA 500 mM NaCl When the meniscus of the solution reaches approximately 05 cm from the top of the chitin beads recap the bottom of the coltunn 20 Cover the top of the column with para lrn label the column with tape attached to the column 21 Place the column in the beaker at the front of lab they will be placed the refrigerator until the next lab period Day 2 Part E continued 22 Retrieve the column 23 Label a 5 ml test tube to collect the eluted Nostoc GAF4 domain 24 Add 15 ml 3 column volumes of Elution Buffer 20 mM Hepes pH 80 01 mM EDTA 500 mM NaCl to the column then remove the cap from the colunm and collect the eluate in the 5 ml test tube until the column stops owing approximately 2 ml Measure the volume of the Post DTT elution fraction ml Use the P1000 SAVE all of the Post DTT elution fraction in the 5 ml test tube labeled Part F Preparation of samples for SDSPA GE Analysis 25 i Transfer a 25 pl aliquot of each of the SAVED actions to labeled microfuge tubes ii Add 25 ul of 2X SDSPAGE sample buffer to each of the 25 ul fraction aliquots iii Heat at 100 C for 2 min iv Store in refrigerator until Experiment 6 Part G Final Spectral Analysis RedGreen Photocyclin g 26 Set the Shimadzu spectrophotometer on Overlay Spectrum Mode Parameters 800 mn 400 nm Absorbance range 00 04 27 Place plastic cuvettes containing H20 in the spectrophotometer press the F1 key for baseline correction this autozeroes the cuvettes across the spectrum of wavelengths 28 Retrieve the sample cuvette remove the water add 10 ml of the eluted GAF4 domain 29 Shine GREEN light through the solution for 1 minute 30 Return the cuvette to spectrophotometer and press start to run the spectrum after rurming the spectrum adjust the range so that the A652 nm peak is maximized in the selected range after Green light exposure 31 Use the cursor arrows record the Absorbances at 750nm 652 nm 538 mn 51 1 32 Remove the cuvette then shine RED light through the solution for 1 minute 33 Return the cuvette to spectrophotometer and press start to run the spectrum 34 Use the cursor arrows record the Absorbances at 750 nm 652 nm 538 nm 35 Print the nal overlay spectra should display two curves Part H Braafford Protein Assay 36 Determine the concentration of protein in each of the SAVED fractions Follow the procedure for the Bradford Protein Assay in Experiment 2 Materials XJbDETM cells transformed with pBADGAPIntein CBD ampR and kanR LB Amp Kan LB 50 pgml amplicillin 25 pgml kanamycin 65 ml sterile growth media in 500 ml LB 50 pgml amplicillin 25 pgml kanamycin 1 mM MgClg asks 3 mM Larabinose 1 mM IPTG Larabinose amp MgCl2 15 M arabinose amp 05 M MgCl2 500X stock IPTG 100 mM l00X stock ampicillin 100 mgml 2000X stock kanamycin 50 mgml 2000X stock DNasel 10 units ml in DNase 1 storage buffer DNase reaction buffer 100X stock 100 mM Tris pH 75 500 mM MgCl2 130 mM CaCl2 DNase I storage buffer 50 glycerol 1 mM Tris pH75 5 mM MgCl2 13 mM CaCl2 Hepes pH 80 200 mM Hepes pH 80 10X stock Column Buffer 20 mM Hepes pH 80 500 mM NaCl 01 mM EDTA 01 TritonX l 00 Cleavage Buffer 20 mM Hepes pH 80 500 mM NaCl 01 mM EDTA 30 mM Dithiothreitol Elution Buffer 20 mM Hepes pH 80 500 mM NaCl 01 mM EDTA Chitin beads New England Biolabs S6651S 20 mM Hepes pH 80 columns 50 ml beakers stir bars 100 C heat block 2X SDSPAGE sample buffer solid ammonium sulfate 37 C H20 bath 40 ml centrifuge tubes Bradford Protein Reagent plastic cuvettes Centrifuges with Sorval small rotors 20 mgml BSA Shimadzu spectrophotometer 96well microplates 30300 multichannel pipettes balances 512 Name Turn in these two pages with the writeup attached DATA ANALYSIS AND POINTS FOR DISCUSSION Experiment 8C 1 Attach the two printed alignments from the exercise I Local alignment of the GAF4 sequence with the parent cyanobacteriochrome and II Sequence alignment of four bacteriochromes one being the Nostoc B2IU14 Sequence 2 a Using the information from the rst exercise 8C 1 Locate the four GAF sequences on the multiple alignments of the full length cyanobacteriochromes produced in the second exercise 8C II and draw a bracket around the four local GAF alignments b Cysteine 687 is the attachment site for the phycocyanobilin chromophore Locate Cysteine 687 in the GAF4 Sequence in the Nostoc punctiforme cyanobacteriochrome note or highlight the location Write the CONSERVED six amino acids prior to and after the GAF4 Cys687 among the four proteins for a nonconserved amino acid write an X Sequence corresponding to Cys Nostoc punctiforme GAF4 687 c Locate the similar sequence within the three other GAF regions bracketed in step a and identify the bracketed sequences corresponding to the Nostoc punctiforme GAFS ie GAF1 GAF2 GAF3 Sequence corresponding to Cys Nostoc punctifonne GAF3 Sequence corresponding to Cys Nostoc punctifonne GAF2 Sequence corresponding to Nostoc punctifonne GAF1 there is no Cys to align with Cys 687of GAF4 d What do these results infer about the function of the three Stretches of sequence for the other three proteins what is already known about the GAF 1 domain compared to the other three GAF domains in the Nostoc punctiforme cyanobacteriochrome protein 513 Purification and Spectral Analyses 1 Complete the Puri cation Table El phycocyanonbilin at 652 nm 132000 M4 cm391 1 cm Volume Total umol yield mgml Total mg gmol PCB Fraction ml PCB protein mg Post DNase I treatment 100 Resuspended NH42SO4 pellet Post DTI elution Absorbance after exposure to 1 minute of Green light Total umol PCB Abs 652 nm Abs 750 nm El 106 umol mol fraction volume L 2 Did the Nostoc GAF 4 preparation exhibit GR photocycling Attach a copy of the nal overlay spectrum 3 If photocycling was observed then what conc1usions can be reached regarding the expression and puri cation of the Nostoc GAF 4 domain 6 Experiment 6 ELECTROPHORESIS OF PROTEINS IN POLYACRYLAMIDE GELS Objectives 1 Learn the techniques of discontinuous SDS polyacrylamide gel electrophoresis 2 Demonstrate the relationship between mobility and protein subunit molecular weight in SDSPAGE 3 Analyze the purity of the Nostoc GAF domain puri cation 39om Experiment 5 by SDSPAGE Introduction Proteins have pH dependent net charge in solution due to charged amino acid side chains on their surfaces The net charge affects the physical properties of a protein such as migration in an electric eld during electrophoresis or separation by ion exchange chromatography Also coupled with their mass proteins have a particular charge to mass ratio at a given pH Polyacrylamide gel electrophoresis PAGE is a powerful method for separating proteins i by their mass when denatured with the detergent SDS or ii by their net charge and mass under nondenaturing conditions The theoretical net charge and isoelectric point of a protein is obtained for a speci c pH from the amino acid composition using the pKa values of the ionizable groups However a theoretically calculated net charge or pl may not re ect that of the native protein For example some amino acids such as an uncharged lysine may be buried in the interior of a protein molecule and therefore does not contribute to the surface charge of a native protein or only one of two tandem lysines are likely ionized a positively charged amine can be stabilized by the unpaired electrons of the adjacent uncharged amine plus two similarly charged groups may lead to signi cant charge repulsion possibly distorting the secondary structure of the peptide chain The pKa values of the ionizable amino acid side chains and those of the carboxyl and amino groups on the alphacarbon are found in Table 61 Table 61 pKa values of amino acid side chains and the a carboxyl amp oamino groups amino acid pKa a groups pKa range average side chain pKa arginine R 1248 o carboxyl 182 238 216 lysine K 1053 onamino 880 1096 947 tyrosine Y 1007 cysteine C 808 histidine H 600 glutamate E 425 asparate D 365 Adapted from Lehninger Principle of Biochemistry 4quot Ed 2004 Nelson and Cox Table 31 Polyacrylamide gels Electrophoresis is the migration of charged molecules by an applied electric force Polyacrylamide gel electrophoresis is a method for separating proteins from one another in a buffered solution by their migration through pores of a solid polyacrylamide matrix under an electric current A polyacrylamide gel is prepared by a freeradical chain reaction Figure 61 The freeradical initiator is the persulfate anion which activates the 39eeradical propagator tetramethylethylenediamine TEMED The 62 TEMED in turn activates acrylamide molecules which forms an acrylamide polymer by successive addition of acrylamide units The molecule bisacrylamide which consists of two acrylamide units joined through their CONHg groups permits the crosslinking of growing polyacrylamide chains The result is a matrix of covalently linked acrylamide and bisacrylamide units 9 9 9 H3C CH3 H3C O OO 0 INCCN gt NCCN 0 0 H30 H2 H2 CH3 H3C H2 H CH3 persulfate TEMED free radical H2 0 NH2 I 0 ZCquotC I 2 acrylamide CH 0C 2 H H H30 390 CH3 quotj INCCN H2 H3 H2 H CH3 I TEMED I d p 39quot39 B bisacrylamide V N C l C H2 N H2 01 C 0 quot21 O O 7 H2C free radical with potential crosslink between two growing chains Figure 61 Reactions involved in crosslinking acrylamide chains Mobility The rate at which proteins move through in an electric eld is de ned as its electrophoretic mobility p which is equal to the ratio of the terminal velocity of the molecule V to the electric eld force E which is also equal to the ratio of the net charge of the molecule q to the ictional coef cient inherent to an electrophoretic system f u D Ef 2 I The units of electrophoretic mobility are cmsecvoltscm or cm volt391 sec Another way to conceptualize the rate of migration in a PAGE system is when the terminal velocity V is equal to the product of the net charge and the electric eld force qE divided by the ictional coef cient f v m The velocity of a molecule increases with an increase in either the net charge of the molecule and or the applied voltage ie an increase in the electric eld force The velocity of a molecule is also opposed by the frictional forces countering the net charge and voltage The ictional coef cient is a complex variable 63 frictional forces are created by such factors as the density of acrylamide affecting the porosity of the matrix the viscosity of the solution and the size and shape of the molecules in the solution moving through the matrix For example the larger the size of a molecule the slower its velocity or the lower the percentage of acrylamide ie larger porosity used to prepare the matrix the higher the velocity of a molecule For a given PAGE system with a de ned buffer percent polyacrylamide and applied voltage proteins are separated by their differential velocities which are proportional to their charge to mass ratios V or qm 3 PA GE apparatus Positively charged proteins will migrate towards the cathode negative electrode while negatively charged proteins will move towards the anode positive electrode Approximately 80 of cellular proteins have a net negative charge at physiological pH values A simpli ed PAGE system for the analysis of negatively charged proteins is shown in Figure 62 The upper and lower reservoirs contain the same buffer that was incorporated into the gel and the pH typically is between values of 83 to 89 to maintain the net negative charges of the proteins The buffer serves not only to maintain a relatively constant pH but also as a conducting electrolyte I cathode samples POW T SUPP polyacrylamide gel between two plates buffer quotIquot anode Figure 62 A simpli ed polyacrylamide gel electrophoresis system The electrical current is conducted through the buffer and gel by moving ions and through electrodes and external wire circuits by electrons The reactions occurring at the electrodes the cathode and the anode in the buffered solution participate in the electrical circuit These reactions at the cathode are Electrons in the circuit 2 e 2 H20 lt gt 2 0H H2 4 The bufferinthe system HA 0H lt gt A H20 5 Conversely the reactions at the anode are Electrons in the circuit H20 lt gt 2 H 12 02 2 e 6 The buffer in the system H A lt gt HA 7 As shown in Figure 62 protein solutions are applied to the sample wells formed at the top of the gel To keep the protein solution om diffusing out of the well glycerol or sucrose ie nonionic molecules is typically added to the protein solution increasing its density The higher density of the protein solutions allows them to remain at the bottom of the sample wells under the reservoir buffer When the power supply is applied a voltage gradient is established in the gel and the negatively charged proteins migrate into the gel Cations in the system of course move towards the cathode thus net positively charged proteins will migrate into the upper reservoir and be lost The simpli ed polyacrylamide system presents a problem when analyzing the migration of proteins in the gel Analysis of polyacrylamide gel results usually include measuring the distances the proteins have migrated in the gel and the sharper the observed positions of the migrated proteins ie the distinction between proteins also called the resolution the greater the accuracy of the measured distances The resolution is in part a function of the volumes of the protein solutions loaded in the sample wells The lower the sample volume loaded ie the thinner the sample loaded the greater the resolution because the positions of the migrated proteins are better de ned The sharpness or broadness of the positions of the migrated proteins re ect how thin or how thick the sample volumes in the wells appear upon loading The volume required for loading depends upon the concentration of total protein in the sample and or the concentration of a speci c protein to be analyzed Discontinuous PAGE system Ornstein and Davis see references developed a differential buffer and gel system for concentrating the proteins within a loaded sample volume which improves the resolution of the proteins after running the gel This system is referred to as discontinuous disc gel electrophoresis an upper stacking gel of 4 to 5 acrrylarnide and buffer pH of 68 and a lower resolving gel of higher percent acrylamide and buffer pH of 89 see Figure 63 Thus there is a distinct discontinuity in both the pH and the percent acrylamide between the stacking gel and the resolving gel The stacking gel The stacking gel is typically between 4 to 5 acrylamide which results in a very large porosity in the matrix Thus when the voltage is applied proteins of all sizes migrate through the large pores easily and are not separated by their different sizes The reservoir buffer at pH 83 contains high concentrations of glycine which is a zwitterion When voltage is applied the net charge of glycine in the reservoir buffer is negative and runs towards the anode as do the net negatively charged proteins in the wells When the glycine encounters the stacking gel at pH 68 the glycine becomes only slightly negatively charged and moves slowly This creates a narrow zone of electrical resistance where the chloride ion a counter ion from HCl when titrating the Tris buffers to their pH values runs at the leading edge of this zone referred to as the ion front followed by other smaller negatively charged molecules such as the bromphenol blue dye added to the loading buffer to visually follow the mobility of the molecules in the electric eld so the ionfront is more commonly referred to as the dyefront Behind the zone of electrical resistance ie the dyefront anions in the system namely the net negatively charged proteins and trailing glycine molecules are moving faster than and catch up to the dyefront Once caught up to the dyefront these anions carmot pass the leading chloride ions of the dyefront Thus the anions ie the proteins stack up at the dyefront and are concentrated behind the zone of electrical resistance This concentration effect improves the resolution of the proteins separated in the resolving gel 65 939YCine PH 3393 glycine p H8quot 3 Qlycine PH 3 3 F j stmkirg ml j T pH639B 0 cl T j P cl Cl j running gel pH89 0 CI txornp lend blue j m quot D II nu p wwJ cl 39 cl 39 or H b c N equott39 T dim rent Current ison Proteins stack Trailing ion glycine overtook protein samples are I oade d inthe stacking 93 L the proteinsifebasing them fro m the stacking condition Brom ph enol blue however is stiilstacked Figure 63 Discontinuous polyacrylamide gel electrophoresis system The resolving get When the dyefront reaches the resolving gel the proteins begin to separate by their size and charge according to the relationship V or qm The percent acrylamide of the resolving gel is much greater than that of the stacking gel decreasing the porosity of the matrix Also the higher pH value of the resolving gel pH values approximately 88 to 90 allows glycine to have a net negative charge which eliminates the zone of electrical resistance Behind the dyefront proteins are separated by their different masses and net charges Protein Staining General staining Proteins do not absorb light in the visible region of the spectrum therefore staining methods are used to visualize the proteins in a gel after electrophoretic separation After electrophoresis the proteins are rst xed in the gel with an acetic acid solution or a mix of ethanol and acetic acid which denatures and precipitates the proteins within the gel The most common general staining method uses Coomassie Blue R250 structure in Figure 23 which allows approximately one to ten micrograms of a single protein to be visualized as a blue band in the gel The Coomassie blue staining works similarly to the Bradford protein assay that uses Coomassie blue G250 the dye binds hydrophobically to the proteins in the gel and binds in proportion to the mass of protein Thus the visual intensity of staining by Coomassie is relative to the mass of protein present 66 Quantitative Analysis of Protein Mobility in a PAGE system Mobility Mobility of a protein is the distance it migrated in the gel under the conditions used When the dyefront ie the leading anions reaches the bottom of the resolving gel the voltage is switched off and the proteins in the gel are stained The distance that an observable protein band traveled is measured and compared A ruler with millimeter divisions is most commonly used The distance from the top of the resolving gel to the middle of a stained protein band in the gel is recorded The mobility obviously is a mction of the length and the percent acrylamide of the gel For example a longer gel results in a greater measured mobility whereas a greater percent acrylamide results in a lower measured mobility SDSPA GE Arguably one of the most widely used methods in any life science laboratory is SDSPAGE as described by Laermnli see references Sodium dodecylsulfate SDS is an anionic detergent Due to the amphipathic property of SDS it is very useful in solubilizing proteins in an aqueous solution The hydrophobic hydrocarbon tail of SDS buries itself into proteins to escape water while the hydrophilic negatively charged sulfate head interacts with the aqueous solution When excess SDS is present it binds to most proteins with a near constant ratio of 14g SDS10g protein There are two major consequences of SDS binding First proteins are fully denatured to an extended chain This means that monomeric proteins are simply denatured whereas native multisubunit protein complexes are dissolved into their component subunits by breaking the noncovalent associations within the complex Second a large net negative charge for the SDSprotein complex is introduced which renders the charge contributions of the ionizable amino acid side chains insigni cant Hence the SDSprotein complexes essentially have the same charge to mass ratio ie qm constant For this reason SDSPAGE separates proteins almost entirely by their molecular size velocity is inversely proportional to molecular size V on 1 molecular size 8 The mobility of smaller proteins are generally faster than those of larger proteins due to less frictional resistance moving through the polyacrylamide matrix ie sieving effects For a de ned SDSPAGE system ie percent acrylamide etc a reproducible measure of protein mobility is the relative mobility The relative mobility Rm is the ratio of distance the protein has moved into the resolving gel divided by the distance that the bromphenol blue dyefront has moved For analytical purposes a standard curve is created when mobility or Rm values are plotted versus the logarithm of known protein molecular size Figure 64 Several proteins of known molecular size are used to construct such a standard curve Thus the molecular size most commonly referred to as molecular weight MW of a single protein and the individual subunits of protein complexes can be determined by SDSPAGE and a corresponding standard curve Additional analyses derived by SDSPAGE are determining the subunit composition of multimeric complexes and assessing the purity of protein isolation procedures For example the tetrameric protein lactate dehydrogenase LDH is composed of subunits of equivalent molecular weights In SDSPAGE the enzymatically active tetramer MW 140000 Daltons is dissolved into inactive monomers MW 35000 Daltons Only one stained band of 35000 Daltons is observed in the gel However the enzyme Ribulose 15bisphosphate carboxylaseoxygenase Rubisco from higher plants is a hexadecamer ie 16 subunits with a molecular weight of 540000 Daltons which is composed of eight subunits of 56000 Daltons and eight subunits of 12000 Daltons Analysis of Rubisco by SDSPAGE results in two stained bands at 56000 Daltons and 12000 Daltons Of course structural information available for thousands of proteins from bioinformatic databases greatly aids in proposing expected results and analysis of actual results For protein puri cation procedures the observation of a single stained band or the minimum number of stained bands of a multisubunit protein is generally suf cient to assume the protein is puri ed 67 10 06 quot 04 02 J l I I I 00 40 42 44 46 48 50 52 log Daltons Figure 64 SDSPAGE Standard Curve There are limitations to keep in mind when interpreting SDSPAGE results For example aberrant molecular weights are often obtained by SDSPAGE for glycoproteins with the attached complex carbohydrates Another situation is for subunits linked by disul de bonds that distort subunit composition analysis it is for this reason that the reducing agent 2mercaptoethanol is usually included with SDS in preparing the proteins for SDSPAGE Also very hydrophobic integral membrane proteins or highly basic proteins having a large net positive charge might not migrate in a gel according to their actual molecular weights References 1 Omstein L 1964 Ann N Y Acad Sci 121 321349 2 Davis B J 1964 Ann N Y Acad Sci 121 404427 3 Laemmli U K 1970 Nature London 227 680685 4 Spiker S and Isenberg I 1983 in quotMethods in Enzymologyquot Vol 91pp 236247 5 Wilson C M 1983 in quotMethods in Enzymologyquot Vol 91 Staining proteins in gels 6 Blackshear P J 1984 in quotMethods in Enzymologyquot Vol 104 pp 237255 7 Merril C R Goldman D and Van Keuren M L 1984 in quotMethods in Enzymologyquot Vol 104 pp 439446 8 Nelson D and Cox M 2004 Lehninger Principles of Biochemistry 4th Ed Freeman amp Co NY Chap 3 9 YJ Han et al 2010 Plant Cell Physiol 524 596609 Experimental A SDS POLYACRYLAMIDE GEL ELECTROPHORESIS SDSPAGE 1 Three groups will share one gel 2 Setup the BioRad Electrophoresis Apparatus with the precast 12 Ready GelTM as shown by the instructor 3 Fill the upper reservoir with reservoir buffer until the level is above the upper gel Check to be sure that there is no leakage of buffer from the upper reservoir to the lower Slowly and gently remove the comb If there is no leakage om the upper reservoir then ll the lower reservoir with reservoir buffer 4 The lane assignments for loading the gel is shown in Table 62 Retrieve the samples prepared for the SDSPAGE from Experiment 5 reheat these samples in the heating block at 100 C for 2 min then cool 68 to RT Refer to Table 62 One groups loads lanes 13 a second group loads lanes 46 and a third group loads lanes 79 The molecular weight standards for lane 10 is ready to load take this sample only when ready to load the gel 5 Connect the electrodes to the apparatus Start the electrophoresis at 200 V constant voltage Bubbles that begin to form indicate the apparatus is functioning properly The bromphenol blue dye present in the sample will become narrow and condense into a thin band as it migrates through the stacking gel Table 62 Lane assignments for the SDS polyacrylamide gel Lanes Groups Samples prepared in SDS sample buffer uglolzgglln 1 20 pl post DNase I extract 2 1 20 pl resuspended ammonium sulfate fraction 3 20 pl affinity column puri ed GAF 4 domain 4 20 pl post DNase I extract 5 2 20 pl resuspended ammonium sulfate fraction 6 20 pl af nity column puri ed GAF4 domain 7 20 pl post DNase I extract 8 3 20 pl resuspended ammonium sulfate fraction 9 20 pl affinity column puri ed GAF 4 domain 10 10 pl Nonstained SDS MW Standards see Table 63 Table 63 Nonstained SDSprotein molecular weight standards Sigma Chemical Co SDS7B Nonstained SDS Protein Standards MW daltons bovine serum albumin 66000 ovalbumin 45000 carbonic anhydrase 29000 Blacto globulin 1 8400 lysozyme 14300 6 Stop the electrophoresis when the dyefront reaches 05 cm from the bottom of the gel approximately 45 min Turn off the power supply pour out the buffer and remove the plates Rinse the electrophoresis apparatus thoroughly Be very careful in handling the apparatus the electrode wires break easily 7 Using the thin edge of the green spatula gently pry the plates apart Squirting a stream of water from the water bottle will help to separate the plates The gel should stick to one of the plates Cut the stacking gel away from the resolving gel using the green spatula and cut away the bottom of the gel at the dye ont properly discard the removed acrylamide 8 Transfer the resolving gel to a plastic petri dish add a suf cient volume of 20 mM of the Zinc staining solution 20 mM zinc acetate in 150 mM Tris pH 70 to cover the gel let sit at RT for 10 min 9 Photograph the Zinc stained gel using the shortest UV wavelength 302 nm 10 Transfer the resolving gel to a plastic dish containing su icient Coomassie Blue stain to cover the gel Cover and let stain until the next lab period 69 Next Laboratory Period B DESTAINING THE SDSPAGE Recycle the staining solution by pouring the stain into the Used Stain container Rinse the gel with tap water to remove excess dye then soak it in 10 vv acetic acid ie destain solution adding two sheets of Kimwipes to help remove the Coommassie Blue until stain is eliminated from gel place the container on the orbital shaker After destaining photograph the gel using the image processor Materials Reagent Recipe Reservoir buffer 06 g Tris 288 g glycine to 1 liter nal pH should be 83 Small pore gel acrylamide 40 g acrylamide 107 g bis acrylamide to 100 ml Ammonium persulfate 10 l g ammonium persulfate to 10 ml Prepare just before use 50 glycerol and bromphenol blue 50 mg bromphenol blue 500 ml glycerol 500 ml water Sample dilution buffer 125 ml solution B 50 mg bromphenol blue 500 ml glycerol to 1 liter pH should be 6769 Destaining and Storing solution 10 acetic acid 10 SDS 10 Ammonium persulfate Prestained SDSprotein markers Sigma SDS7B Solution A separating gel buffer 15 M Tris pH 88 brought up to pH 89 with cone HCl Solution B stacking SDS gel buffer 05 M Tris pH 68 brought up to pH 68 with cone HCI Coomassie Stain 01 Coomassie Blue R250 45 methanol 10 acetic acid 10X SDS reservoir buffer 303 g Tris base 1507 g glycine up to 1 liter with water Dilute to 1X on day of use 2X sample dilution buffer for SDS gel 0125 M Tris pH 68 4 SDS 20 glycerol 10 mercaptoethanol 0004 bromphenol blue Zinc staining solution 20 mM zinc acetate in 150 mM Tris pH 70 Name Turn in this page with the writeup attached DATA ANALYSIS AND POINTS FOR DISCUSSION Exp 8D 1 Attach the print out requested from Exp 8D 2 What is the estimated molecular weight of the Nostoc GAF4 domain SDSPAGE 1 Attach a copy of the photo of the destained SDSPAGE gel with proper labeling of lanes 2a Determine the mobility or Rm for each of the protein molecular weight standards and the Nostoc GAF4 domain prep b Plot a standard curve mobility vs log MW of the molecular weight standards c Determine the MW value of the Nostoc GAF 4 domain prep from the standard curve using the linear portion of the graph and d Prepare a Table with the names of the standards and the Nostoc GAF4 domain along with their mobility values log MW and MW e Does the GAF4 MW determined by the SDSPAGE correspond with the predictedexpected value 3 Brie y assess the purity of the Nostoc GAF4 domain 4 Does the zinc stained band correspond with the Coomassie stained band If yes then what does this mean The MW of the InteinCBD peptide is 55 kD does the MW of the zinc stained band in the Post DNase I extract correspond to the MW of the GAF4 domain 55 kD InteinCBD Integrate the Data from Experiment 5 with Experiment 6 5 Using the MW of the GAF4 peptide determined 39om Experiment 6 calculate the total mass of the Nostoc GAF4 domain based on the molar concentration of the chromophore present determined in Experiment 5 6 Calculate the total mass of the Nostoc GAF4 domain determined by the Bradford Protein Assay in Experiment 5 7 How do the two masses compare with one another as determined by the two methods If a large discrepancy is observed comparing the results of the two calculations of concentration and total mass then what may be a biochemical reason for any large discrepancy 55 kD InteinCBD derived from the NEB Irnpactm plasmid constructs Experiment 7 WESTERN BLOT ANALYSIS OF A CYANOBACTERIAL BILIVERDIN REDUCTASE EXPRESSION IN E COLI Objectives 1 Examine the integrity of the pASK75 plasmid containing the BvdR gene by plasmid minipreps and restriction enzyme analysis 2 Examine the full expression of the BvdR protein ie transcribed and translated by Western Blot analysis use of a control plasmid E coli host cells with the pASK75 plasmid without the BvdR gene Introduction In 1997 an open reading frame ORF was found at locus sir 784 in cyanobacterium Synechocystis PCC 6803 upon completion of its genomic sequence Due to sequence similarities with other proteins listed in the databanks the ORF was shown to be similar to biliverdin reductase from mammals and was armotated as a putative biliverdin reductase BVR The ORF encodes a 328 amino acid polypeptide which is 23 identical to mammalian biliverdin IXa reductase A multiple sequence alignment of the protein encoded by locus sir 784 with BVR sequences from rat and human is shown in Figure 71 RatBVR MDAEPKRKFG VVGV R SV LRDLKDPR AFLNLIG 40 HumanBVR MNTEPERKFG VVGKI R GSV MRDLRNPHP AFLNLIG 41 Slr1784 MSENFAVATPVR IVGT Y QR EVFRGDRR QL S 39 RatBVR F SRR LGSLDEVR I LEDALRSQ IDVAY CSESS 77 HumanBVR F SRR LGSIDGVQ I LEDALSSQ EVAY CSESS 78 Slr1784 F GNS NTAKFADTFGVRPQ QALINDP IDLVL TINQ 82 RatBVR S EDYIRQFLQAGKHVLVEYPMTLSF AQEL ELAAQKGRVL 120 HumanBVRS EDYIRQFLNAGKHVLVEYPMTLSL AQEL ELAEQKGKVL 121 L LTY M L L Sr1784 L GAIAEAA AGKHV LEYPLA GKK QQLAREKGKL 125 RatBVR HEEHVELLMEEFEFLRREVLGKELLKGSLRFTASP 155 HumanBVR HEEHVELLMEEFAFLKKEVVGKDLLKGSLLFTAGP 156 Slr1784 H EHIELLGGVHQAIRQNLGKIGEVFYARYSTIMGQNPLAPQRW 168 HumanBVR LEEERFGFPAFSGISRLTWLVSLFGELSLVS TLEERKED 196 Slr1784 TYHHQQFGFPLVAALSRISRFTDLFGTVQQVD QCRFWDQPNP 211 RatBVR QYMK MT QLETQNKGLLSW EEKGPGLKRN YVNFQFTS 234 HumanBVR QYMKMT CLETEKKSPLSW EEKGPGLKRN YLSFHFKS 235 Slr1784 EYFRACLATYLQFNNGLKAEV YGKGEVFHQNE IFTLHGDR 254 RatBVR S EVPSVGVNKNIFLK QDIFVQKLLD 263 HumanBVR GSL NVPNVGVNKNIFLK QNIFVQKLLG 264 Slr1784 TLIFVGETGRLIQGQT TEITVGSRRGLFRQ TEAVLDYL 295 RatBVR QVSAEDLAEKKRIMHCLGL SDIQKLCHQKK 295 HumanBVRQFSEKELAEKKRILHCL L EEIQKY CSRK 296 SIr1784 TTGKPLYVDLEASLYALEV DLCAQACGYKVEN 328 Figure 71 Multiple Sequence Alignment of Cyanobacterial and Mammalian Biliverdin Reductases RatBVR LEEERFGFPAFSG ISRLTWLVSLFGELSL ISE TLEERKED 195 72 BVR catalyzes the reduction of biliverdin IXu BV to bilirubin BR comprising the nal step of heme catabolism in mammals and some species of sh as seen in Figure 72 Hence fmding an enzyme of typically vertebrate function in a cyanobacterium was a surprise which also underscores the importance of the genome projects NADH NADPH O2 Heme Bulwerdnn NA0 oxygenase reduclase NADF OH H0 0 O Heme Figure 72 Heme catabolism to biliverdin and reduction of biliverdin to bilirubin Recently in 2009 recent in biological research so yesterday in processor development bilirubin was discovered in a plant Strelitzia nicolai the White Bird of Paradise Tree While hemes and similar structures such as chlorophylls are abundant and well characterized molecules in photosynthetic organisms bilirubin was unknown in higher plants Subsequent investigations showed bilirubin is present in other species of Strelitzia Strelitzia regime the Bird of Paradise Flower and Strelitziacaeae Phenakospermum guyanense the South American Travelers Palm aka Red Travelers Palm Bilirubin is responsible for the bright orange red colorations observed in Strelitzia Today bilirubin s presence has been veri ed in diverse angiosperms in eight species from the orders of Zingiberales Arecales and Myrtales but only contributes color in Strelitziaceae Given the presence of bilirubin the BVR enzyme is likely found in many organisms In marrnnals the reduction of heme is a detoxi cation process in the placenta whereas plants it results in pigment accumulation Bilirubin accumulation in Strelitzia is pleasing to the human eye but the functions of the pigment accumulation is uncertain The wide biological occurrence of bilirubin illustrates how various cells types through evolution have adapted unique purposes for similar metabolic reactions The pASK75 Expression Plasmid Functions In this experiment the vector pASK7S Figure 73 was chosen to express a cloned putative BVR protein Plasmids have common features that allow them to persist in host cells and for experimental use These features on the pAS 75 are described as follows Origin of replication It is essential for a vector to have a sequence that permits its autonomous replication and retention in host cells For bacterial hosts the On sequence lls this function 73 Antibiotic resistance gene The presence of an antibiotic resistance permits only those host cells that have taken up a plasmid to grow in media containing an antibiotic The bla gene encodes 3lactamase an enzyme conferring resistance to the antibiotic ampicillin by the pASK75 resistance is noted amp lllultiple cloning site The collection of unique restriction enzyme sites in the MCS contains the restriction enzyme recognition sites for Xbal StuI EcoRI Kpnl BamHI Xhol Sall PstI and HindIII Promoter A promoter is required for RNA Polymerase to bind This is the tetA promoter just 5 to the MCS on pASK75 so the inserted gene is under its control Ribosome binding RBS A RBS site located between the promoter and the initiator methionine codon allowing the translation machinery to bind The RBS in pASK75 is just 3 to the Xbal site Initiator methionine codon The pASK75 provides two initiator methionine codons and inframe amino acids if needed starting with the tetA gene and the OmpA gene The cloned BvdR gene included its own start methionine thus neither of the plasmid start methionines were used Stop codon The pASK75 provides a stop codon at the end of the streptag peptide sequence A Streptag epitope sequence The pASK75 contains a streptag sequence adjacent to the MCS A cloned gene whose protein is translated in ame with streptag sequence will create a fusion protein with the strep tag sequence located the Cterrninal end of the translated protein Streptag is a peptide that binds the protein streptavidin with high affinity The fusion protein is detected ie reports its existence by using a streptavidinenzyme conjugate similar to the conjugated antibody system used for the ELISA The streptag sequence can also be used to affinity purify the recombinant fusion protein The T etR Repressor Protein Genes cloned into the MCS of pASK75 are under control of the tetA promoter but transcription is reduced in the presence of the tetR repressor protein encoded by the tetR gene on pASK75 The tetR repressor protein binds to the tetA promoter preventing access by RNA polymerase Transcription is increased in the presence of anhydrotetracycline which binds to the tetR repressor protein prohibiting the tetR repressor protein from binding to the tetA promoter In brief anhydrotetracycline induces protein expression Such a system prevents the overexpression of a cloned protein that may be harmful to the host cells until expression is induced for an experiment Primer Design for directional cloning The primers used in this experiment are shown in Figure 74 The senseprimer has an Xbal restriction enzyme sequence attached at its 5 end while the antisense primer has a Sall restriction enzyme sequence attached at its 5 end These sites are chosen because they are available in the pASK75 MCS and do not exist in the BvdR gene The Forward primer sense primer The Xbal site is chosen to insert the start Methionine of the PCR product into the pASK75 MCS in proximity to the tetA promoter to transcribe the correct strand in the correct order for later translation to protein On the 3quotside of the Xbal site but on the 5 side of the ATG codon is the ribosome binding site RBS that allows the transcript to be translated It is included because the RBS available in the pASK75 MCS is lost along with the small segment between the XbaI and SalI sites after digestion with the XbaI and SalI restriction enzymes 74 The Reverse primer antisense primer The Sall site is chosen to insert the target sequence inframe with the strep tag sequence on the 3 side of the BvdR protein sequence The antisense primer sequence does not include the stop codon from the BvdR gene to permit translation through the strep tag The fusion of the BvdR protein with the streptag allows for detection of the translated protein The stop codon supplied at the end of the streptag sequence terminates translation To keep the BvdR sequence in fra1ne with the streptag sequence one extra nucleotide was added between the Sall site and the sequence complimentary to the BvdR sequence C in bold and grey highlighted pASK 75 vector map EcoR 210 Kpnl 222 M p B smumm mm Hindlll 291 promoter Streptag pASK75 3266bp Pvul 1379 Multiple Cloning Site 1 539 Egt CCATCGAATG GCCAGATGAT TAATTCCTAA TTTTTGTTGA CACTCTATCA TTGATAGAGT tetA promoter Xbal 61 TATTTTACCA CTCCCTATCA GTGATAGAGA AAAGTGAA ATG AAT AGT TCG ACA AAA ATC TAG tetA Met Asn Ser Ser Thr Lys Ile stop RES 124 ATAACGAGGGCAAAAA ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT OmpA Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly StuI ECORI Kpnl BamHI XhoI 181 TTC GCT ACC GTA GCG CAG GCC TGA GACCAGAATTCGAGCTCGGTACCCGGGGATCC CTCGAG Phe Ala Thr Val Ala Gln Ala stop Sa1I PstI HindIII 243 GTCGAC CTGCAG GC AGC GCT TGG CGT CAC CCG CAG TTC GGT GGT TAA TAAGCTT Egt 3 Streptag Ser Ala Trp Arg His Pro Gln Phe Gly Gly stop ampR Figure 73 Structures of the pASK75 plasmid and its Multiple Cloning Site 75 a Target DNA Sequence from Synechocystis sp strain PCC6803 only the sense strand is shown ATG start methionine codon TAG stop codon 539 GATTTTTCCCAAGGTACI g TCTG AAATTTTGCAGTTGCTACGCCGGTGCGGGTCGGAATTGTCGGTACTGGTTATGCGGCCCA ACGTCGGGCGGAAGTTTTCCGGGGCGATCGCCGTAGTCAATTGGTTAGTTTTTGGGGCAATAGTGAAGCCAATACAGCTAAATTTG CCGATACTTTTGGAGTTAGACCCCAGCAATCTTGGCAGGCATTAATTAATGATCCAGAGATAGATTTAGTGCTCATTGCCACCATT AACCAACTCCATGGGGCGATCGCCGAGGCGGCATTGCAAGCCGGTAAACATGTGGTGTTGGAATATCCTTTAGCGTTAACCTATGC CATGGGCAAAAAACTACAACAGTTAGCCCGGGAAAAAGGTAAATTACTGCATGTGGAACATATTGAACTATTGGGGGGAGTACACC AAGCCATTCGCCAGAACCTAGGCAAAATTGGTGAGGTTTTTTACGCCCGCTATAGCACCATCATGGGACAAAATCCCGCTCCCCAA CGTTGGACCTATCACCATCAGCAATTTGGCTTTCCTTTAGTGGCGGCCTTGTCCCGCATCAGTCGGTTTACGGATTTATTCGGTAC AGTACAGCAGGTGGATGCCCAATGTCGTTTTTGGGATCAGCCTAATCCGGAATATTTTCGGGCTTGTTTAGCCACCGCCTATCTCC AGTTTAATAATGGTCTTAAAGCGGAGGTTATCTATGGCAAAGGGGAAGTTTTTCACCAGAATGAACGGATTTTTACCCTCCATGGC GATCGAGGCACCTTAATTTTTGTCGGGGAAACAGGTAGGTTAATTCAGGGACAAACGGAAACTGAAATTACCGTTGGTAGTCGTCG AGGACTGTTCAGACAAGACACGGAAGCAGTGTTGGATTATCTAACCACTGGTAAGCCCCTTTATGTGGATTTAGAAGCTAGTTTAT ATGCTTTAGAAGTGGCGGATCTCTGTGCCCAAGCTTGTGGATATAAGGTTGAAAA1E CGGAAATATCGGCAG 339 b BVR1 S Sense primer forward primer 539CGTrCTAGATAAGGAGGGCAACATA T GT CT GAAAA TTTT GCA GTT G339 45 mer Tm 60 C Arrow indicates where Xbal will cut GAGG is the RBS site ShineDalgarno site ATG is the start Met c BVRIAS Antisense primer reverse primer 539GCGl TCGACCAITTT CAACCTTATA T CCACAAG339 32 mer Tm 60 C Arrow indicates where 39SilI will cut The C is added to keep the BVR sequence in frame with the streptag peptide Figure 74 a Target Locus Sir 784 b Sense primer BVRIS c Antisense primer BVRIAS Western Blotting and the Strepavidinalkaline phosphatase conjuagate detection system Western blotting involves the electrophoretic transfer of proteins om polyacrylamide gels onto a solid support such as a polyvinylidene di uoride PVDF The transferred proteins stick to the membrane see Figure 76 After electrophoretic transfer the membrane is rst washed with a buffer containing Tween20 that removes loosely bound proteins and proteins that may be binding nonspeci cally to the BVR protein Then avidin is added to bind and block biotin and biotinylated proteins which would also be recognized by the streptag detection system strepavidinalkaline phosphatase conjugate SA AP After blocking and washing the membrane is incubated with the SAAP that binds with high affinity and speci city to the streptag sequence Visualization is accomplished by subsequent incubation with a developing solution consisting of pnitrotetrazolium blue NBT and 5bromo4chloro3indolylphosphate BCIP BCIP is a substrate for alkaline phosphatase The dephosphorylation of BCIP and subsequent interaction with NBT creates a blue precipitate observable on the membrane Figure 75 8 CI 0 B C B Cl B Cl 0 0 CI B I 0 P 0quot AP pNw i 0 lzmlmncnsm X 0 2H YL 39 a39 t s OH L ltrquot HPOJ 39 395 quotquotquot1 I TT R H H NBT NBT lmmnznn H H BCIP 55 dshronm ll 039 nlutlulnm indigo white Figure 75 Chemical reaction of NBT and BCIP substrates with alkaline phosphatase AP BCIP is hydrolyzed by alkaline phosphatase to form an intermediate that undergoes dimerization to produce an indigo dye The NBT is reduced to the NBTfonnazan by the two reducing equivalents generated by the dimerization 76 substrates products U avidin strepavidinalkaline phosphatase other conjugate proteins PVDF membrane BvdR streptagged rsion protein biotin or biotinylated proteins Figure 76 Detection of the BvdR strep tagged fusion protein References 1 Sambrook J Fritsch EF Maniatis T Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press New York 1989 2 Watson JD Gilman M Witkowski J Zoller M Recombinant DNA Second Edition Scienti c American Books WH Freeman and Co New York 1992 260 pp 3 Fakhrai H and MD Maines 1992 Expression and characterization of a cDNA for rat kidney biliverdin reductase J Biol Chem 267 40234029 4 Kaneko T S Sato H Kotani A Tanaka E Asamizu et al 1996 Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp strain PCC6803 11 Sequence determination of the entire genome and assignment of potential proteincoding regions DNA Research 33 10936 5 Kutty RK and MD Maines 1981 Puri cation and characterization of biliverdin reductase from rat liver J Biol Chem 256 39563962 6 Maines MD BV Polevoda TJ Huang WK McCoubrey 1996 Human biliverdin IXalpha reductase is a zinc metalloprotein Characterization of puri ed and Escherichia Coli Expressed Enzymes Eur J Biochem 235 372381 7 Mullis KB and FA Faloona 1987 Speci c synthesis of DNA in vitro via a polymerasecatalyzed chain reaction Meth Enzymol 155 33550 8 Saiki RK DH Gelfand S Stoffel SJ Scharf R Higuchi et al 1988 Primer directed enzymatic ampli cation of DNA with a thermostable DNA polymerase Science 2394839 48791 9 Schmid R and AF McDonagh 1975 The enzymatic formation of bilirubin Annals of the New York Academy of Sciences 244 533552 10 Tabata S et al Cyanobase web site httpzfwwwkazusaorjpcyano 11 Schluchter WM Glazer AN 1997 Characterization of Cyanobacterial Biliverdin Reductase J Biol Chem 272 21 1356213569 12 httpwwwurn1crochesteredulabsMainesLabhistoryindexcfm 13 httpfwwwpiercenetcomfbrowsecfm dID01 041003 14 Morand LZ et al 1998 Alteration of the fatty acid substrate speci city of lysophosphatidate acyltransferase by sitedirected mutagenesis Biochem Biophys Res Commun 2441 7984 15 Pirone C et al Animal Pigment Bilirubin Discovered in Plants J Am Chem Soc Article ASAP D01 101021ja809065g Publication Date Web 10 February 2009 16 Prione C Bilirubin present in diverse angiosperms AoB PLANTS Vol 2010 Experimental Prelab period Part A Before the first lab period inoculate cultures of E coli cells in LBamp media to grow overnight 1 E coli cells with only the pASK75 plasmid BvdR gene and 2 E coli cells with the pASK75 plasmid BvdR gene First lab period Parts B C amp D Overnight E coli cultures are used for 1 plasmid preparations restriction enzyme digests and agarose gel analysis and 2 to inoculate 39esh LBamp media containing anhydrotetracycline to induce the protein expression of the BvdR streptag fusion protein after induction cultures are used for wholecell SDS protein extracts Second lab period Parts E amp F SDS PAGE and Western blot procedures are performed on the whole cell SDS protein extracts Third lab period Part G Development of the Western blot to detect the BvdR streptag fusion protein BIOHAZARD NOTES Since plasmids often contain antibiotic resistance genes it is necessary to prevent their spread Materials contaminated by plasmids and bacteria that contain plasmids are discarded into designated bags and autoclaved then discarded as trash Wear gloves throughout this experiment Use sterile techniques and discard liquid E coli cultures by rinsing test tubes or other glassware with bleach before putting them into the dishpans for machine washing PreLab Period Part A A INOCULATION OF OVERNIGHT CULTURES 1 On the day prior to the first full lab period for Experiment 7 label two sterile culture tubes one labeled for the plasmid containing the BvdR gene insert and the other for the plasmid without the BvdR gene insert 2 Transfer 2 ml LBamp media into each tube 3 Using sterile toothpicks 1 pick one E coli colony from the plate of cells containing plasmid plus the inserted gene and inoculate the tube and 2 pick one E coli colony from the plate of cells containing plasmid minus the inserted gene and inoculate the tube 4 Place the inoculated cultures in the 37 C incubator with shaking overnight First Lab Period Warts B C and D B INDUCTION OF PROTEIN EXPRESSION amp PREPARATION OF SDS WHOLE CELL PROTEIN EXTRACTS Induction of Protein Expression 1 Transfer 300 pl from of each Overnight culture into separately labeled culture tubes with 20 ml of fresh LBamp media containing 02 1lVl anhydrotetracycline SAVE the remaining overnight cultures for Part C below Incubate the two induced cultures for 3 hours in the 37 0C incubator with shaking During the 3 hour incubation perform Part C and begin Part D Preparation of hot SDS whole cell protein extracts 2 After the 3 hour incubation transfer 750 pl from each culture into separately labeled 15 ml tubes 3 Harvest the cells at maximum speed in a microcentrifuge for 20 sec 4 Remove and discard the supematants from each tube 5 Resuspend the cell pellets with 75 pl deionized water and immediately add 75 ul of 2X SDS sample buffer Mix by vortex and place the tubes in the 100 C dry heat block for 5 min 6 Remove cell debris from the SDS extracts in a microcentrifuge at maximum speed for 10 min Transfer the supematants to labeled microfuge tubes and store at 4 C for SDSPAGE and Westem blotting C PLASMID MINIPREPARATION The experiment is designed to use the pASK75 plasmid plus or minus the BvdR gene insert However continued propagation of E coli cultures may lead to the loss of the plasmid in a process called curing Curing is prevented by the selective pressure of keeping the antibiotic ampicillin in the media Also an uncommon yet sometimes observable problem is the alteration of an insert via natural processes in vivo such as recombination or repair Recombination can occur if the inserted DNA sequence shares signi cant similarity with a sequence within the host genomic DNA which may result in the loss or severe alteration of the insert For these reasons it is prudent to periodically check the plasmid for the presence and integrity of the insert by isolating the plasmids and analyzing them by restriction enzyme digests and39agarose gel analysis 1 The following solutions are available on the front table Solution I Solution H Solution III 25 mM TrisHCl pH 80 02 N NaOH 5 M potassium acetate pH 48 10 mM EDTA 1 SDS 5 mgml lysozyme 2 Transfer 1 ml of each overnight culture into separately labeled 15 ml microfuge tubes 3 Pulse microcentrifuge at maximum speed 15 sec to pellet the cells Discard supematant 4 Add 100 pl Solution I to each pellet vortex to fully resuspend the pellet 5 Add 200 pl Solution II to each suspension Mix well and gently by inverting the tubes several times 6 Add 100 pl Solution III to each suspension Mix well and gently by inverting the tubes several times 7 Microcentrifuge at maximum speed for 5 min 8 Transfer 300 pl of each supematant to separately labeled 15 ml micro ige tubes carefully avoiding the transfer of any white precipitate 9 Add 750 pl 95 ethanol to each supematant mix well and let sit on ice for at least 10 min 79 10 Microcentrifuge at maximum speed for 5 min to pellet the precipitated nucleic acids discard the supernatant Remove any remaining supematant by pulse microcentri igation to collect the liquid at the bottom of the tube and then pipetting out the solution 11 Resuspend the pellets in 100 pl 50 mM Tris pH 80 containing 20 pgrnl RNaseA RNaseA degrades the RNA that co precipitated with the plasmids Make sure pellets are 1lly resuspended vortex if needed 12 Add 10 pl 3 M sodium acetate pH 52 to each resuspended pellet and mix well 13 Add 300 pl 95 ethanol to each tube mix well and let sit on ice for at least 10 min 14 Microcentri lge at maximum speed for 5 min The pellet may be barely visible as a very small translucent strip of material on the bottom edge of the tube Discard the supernatant Remove any remaining supernatant by pulse microcentri lgation to collect the liquid at the bottom of the tube and then pipetting out the solution Leaving the caps open let air dry for 5 min 15 Resuspend the plasmid pellet in 40 pl TrisEDTA TE containing 10 pgml RNaseA D RESTRICTION ENZYME DIGESTION AND AGAROSE GEL ELECTROPHORESIS Restriction enzyme digestion 1 The BvdR gene om the cyanobacterium Synechocystis was cloned into the pASK75 vector at the Xbal and Sal I restriction enzyme sites see Figures 75 amp 76 To analyze the plasmid for the presence and integrity of this BvdR gene insert Xba I and Pst I restriction enzymes are used The Pst I site is on the 3 side of the Sal I site If the structure of the insert remains intact then successful restriction enzyme digests and agarose gel electorphoresis analysis should reveal two bands of the appropriate size one for the pASK75 plasmid and one for the BvdR gene insert 2 For better quality of results and conservation of material the TA will have prepared restriction enzyme cocktails for a Pst I digest an Xba I digest and an Xba I amp Pst I double digest Both the plasmids plus and minus BvdR gene insert are analyzed with each of these three restriction enzyme digests 3 Label six 05 ml microfuge tubes for the digestion of both plasmids under the three conditions Plasmid plus insert Plasmid minus insert lPst 2 Xba 3PstampXba 4 Pst 5Xba 6 PstampXba 4 Transfer 5 pl of the resuspended plasmid plus insert to the labeled tubes for each of the three restriction enzyme digests and transfer 5 pl of the resuspended plasmid minus insert to the labeled tubes for each of the three restriction enzyme digests 5 Transfer 15 pl of the appropriate restriction enzyme cocktail to the labeled tubes with the plasmid solution and mix well 6 Incubate the six tubes in a 37 C water bath for 10 minutes 710 Agarose gel electrophoresis 7 Dissolve 064 g of agarose in 80 ml of O5X TBE gel buffer 08 agarose by heating the mixture in a microwave oven for approximately 12 minutes Watch to ensure the solution does not boil over 8 Once the temperature is comfortable to touch add 4 pl of GelRedTM and swirl to mix then immediately pour the gel Insert the thicker side of the comb and let sit until the agarose has solidi ed 9 Once the agarose has solidi ed remove tray with the gel and reorient it 900 and add 400 ml 05X TBE electrophoresis buffer Then remove the comb 10 After the 10 minute incubation period retrieve the restriction enzyme reactions and add 4 pl 6X loading dye to each tube 11 Transfer 5 pl of both of the original ie uncut undigested plasmids into separately labeled microfuge tubes add 15 pl dH20 and then add 4 pl 6X loading dye 12 Obtain 20 pl of the 7 Hind III standards premixed with dye see Table 72 below 13 Load the agarose gel according to Table 71 below 14 Connect the electrodes with the lid of the apparatus and run the electrophoresis at 100 volts When the leading purpledark blue dye has migrated half the distance of the gel tum off the electrophoresis and photograph the gel Table 71 Lane Assignments for the 08 Agarose Gel Lane Volume pl Sample 1 2 10 uncut plasmid control 3 10 Pst I digest 4 10 Xba I digest plasmid minus insert 5 10 Pst IXba I double digest 6 10 uncut plasmid control 7 10 Pst I digest 8 10 Xba I digest plasmid plus insert 9 10 Pst IXba I double digest 10 20 ll Hind III standards supplied at front table 1 1 12 13 14 711 Table 72 L Hind III standards it phage uncut genomic DNA 47000 bp size and relative mass of DNA fragments generated from a Hind III digestion of 71 phage Length bp ng per band 250 ngotal DNA 23100 143 9400 58 6600 41 4400 27 2300 14 2000 12 560 34 Second Lab Period Parts E and F E SDS POLYACRYLAMIDE GEL ELECTROPHORESIS SDSPAGE 1 2 Each group will have one gel Setup the BioRad Electrophoresis Apparatus with the precast 12 Ready Gelm as shown by the instructor Fill the upper reservoir with reservoir buffer until the level is above the upper gel Check to be sure that there is no leakage of buffer from the upper reservoir to the lower Slowly and gently remove the comb If there is no leakage from the upper reservoir then ll the lower reservoir with reservoir buffer The lane assignments for loading the gel are shown in Table 73 Retrieve the samples prepared for the SDSPAGE reheat these samples in the heating block at 100 C for 2 min then cool to RT Refer to Table 72 for loading the samples the positive control and the molecular weight standards are ready to load take these sample only when ready to load the gel Connect the electrodes to the apparatus Start the electrophoresis at 200 V constant voltage Bubbles that begin to form indicate the apparatus is functioning properly The bromphenol blue dye present in the sample will become narrow and condense into a thin band as it migrates through the stacking gel Stop the electrophoresis when the dyefront reaches 05 cm from the bottom of the gel Turn off the power supply pour out the buffer and remove the plates Rinse the electrophoresis apparatus thoroughly Be very careful in handling the apparatus the electrode wires break easily Using the thin edge of the green spatula gently pry the plates apart Squirting a stream of water from the water bottle will help to separate the plates The gel should stick to one of the plates Cut the stacking gel away from the resolving gel using the green spatula and cut away the bottom of the gel at the dye ont properly discard the removed acrylamide Cut the gel in half using the green spatula Place onehalf of the gel in Coomassie stain in a labeled container leave until the next lab period Process the otherhalf of the gel for the Western blot as described below in Part F Table 73 Lane assignments for the SDSpolyacrylamide gel Lane Samples 1 10 pl Prestained SDS MW Standards refer to Table 74 2 10 pl pASK75 BvdR gene 3 10 pl pASK75 BvdR gene 4 10 pl Positive Control for Strepavidin Detection 5 empty 6 empty 7 10 pl Prestained SDS MW Standards refer to Table 74 8 10 pl pASK75 BvdR gene 9 10 pl pASK75 BvdR gene 10 10 pl Positive Control for Strepavidin Detection Table 74 Prestained Molecular Weight Markers F ennentas SM0671 Protein color MW Dal blue 1 70 blue 130 blue 95 RED 72 blue 55 blue 43 blue 34 blue 26 blue 17 GREEN 10 F ELECTROBLOTTING ONTO A PVDF MEMBRANE WESTERN BLOTTING 1 Wear gloves before handling the PVDF membrane a Label a corner of a dry PVDF membrane with pencil b Wet the PVDF membrane with methanol until translucent a few seconds c Rinse the membrane in deionized water d Rinse the membrane in transblot buffer 2 Assemble the BioRad transblot system in the following order ensuring that the components are fully wetted following each step and that air bubbles are gently chased out with the addition of each layer a Open the gel holder cassette like a book and place it in the dish b Place a wetted ber pad directly on top of the black side of the cassette c Place a wetted piece of Whatman paper atop the ber pad d Place the gel atop the rst Whatman paper e Place the wetted PVDF membrane atop the gel and gently smooth out air bubbles f Place a second piece of wetted Whatman lter paper atop the PVDF membrane g Place a second wetted ber pad atop the second Whatman iter paper h Holding all components rmly in place close the cassette like a book and clamp it closed i Insert the closed cassette into the transblot electrode chamber blackside to blackside 71 3 3 Place the transblot electrode chamber with the inserted cassette into the buffer reservoir with the black side of the cassette facing the black side cathode of the transblot electrode chamber 4 Quickly place an ice pack in the buffer reservoir and ll the buffer reservoir with cold transblot buffer place the assembled cassette into one of the two slots in the transblot electrode insert 5 Put the lid on the lled buffer reservoir and tum the power on for the electrophoretic transfer Set the power at 100 volts and rtm for 30 n1in 6 After 30 min tum off the power remove the PVDF membrane and rinse it in a dish of deionized water The PVDF membrane with transferred proteins can be stored dry between two sheets of Whatman 3MM paper in the refrigerator until the next lab period Third Lab Period Part G F continued DESTAINING THE SDSPA GE Recycle the staining solution by pouring the stain into the Used Stain container Rinse the gel with tap water to remove excess dye then soak it in 10 vfv acetic acid ie destain solution adding two sheets of Kimwipes to help remove the Coomassie Blue until stain is eliminated from gel place the container on the orbital shaker After destaining photograph the gel using the image processordestaining the acrylamide gel G WESTERN BLOT DEVELOPMENT 1 Rewet the PVDF membrane in MeOH for 5 sec 2 Rinse the membrane in deionized water 3 Equilibrate the membrane in TBS01 Tween in a large weigh boat for 5 min with shaking 4 Dump the solution then add 15 ml HS05 Tween containing 2 ugml avidin for 10 min with shaking 5 Add 4 ul strepavidin alkaline phosphatase conjugate directly to the solution and incubate at least 45 min with shaking use the orbital shaker set for a slow rotation 6 Wash the membrane with shaking as follows 30 ml per wash at room temperature discard each solution after wash a 5 min in TBSTween b 5 min in HSTween c 5 min in TBST ween 7 Add 30 mls TBS without Tween wash until the substrate in step 8 below is ready 8 When the rst group is ready a TA will prepare the 30 ml NBT and BCIP substrate solution 9 Incubate the blot in the substrate solution with shaking until satis ed with the color development 10 Transfer into a dish lled with distilled water for 5 minutes Remove the membrane and blot excess liquid using a clean paper towel and take a photo 714 Materials Equipment needed gloves eppendorf tubes shaking incubators micropipettors and tips sterile toothpicks power supply PVDF membranes chemical waste containers PAGEWestern blot apparatus Whatman 3MM paper biohazard waste containers Solutions and other materials required E coli containing plasmid pASK75 no BvdR gene 1 LBamp plate per section E coli containing plasmid pASK75 BvdR gene 1 LBamp plates per section LB 10 g bactotryptone 5 g bactoyeast extract 10 g NaCl per liter pH 75 sterile water LB with amp 3 tubes x 2mltube per group LB with amp and anhydrotetracyline 2 tubes x 2m1tube per group Plasmid Mini Prep Solutions I II and III 2Propanol 50 mM Tris RNase 30 M potassium acetate pH 52 95 Ethanol TE 05X TBE gel buffer 445 mM Tris 45 mM boric acid 0125 mM EDTA pH 80 Restriction enzymes and buffer Pst I and Xba I GelRedTM Biotium 2X SDSsample buffer with dye 025 bromphenol blue 025 xylene cyanol and 30 glycerol SDSPAGE buffer transblot buffer 25 mM Tris 192 mM glycine 20 methanol Turn in this page with the writeup Due on the last day of lab Name CALCULATIONS AND SOME POINTS FOR DISCUSSION OF YOUR RESULTS Agarose gel and Western blot analyses 1 Restriction enzyme digest and agarose gel analysis a Was an insert observed for the Pst IfXba I double digest b If yes then what approximate size in bp was observed c Is this approximately the expected size in bp What is the size of the Synechocystis sir 784 gene SDSPAGE and Western blot analysis a Was a protein band observed in the presence of the BvdR insert b If yes then what approximate molecular weight was observed c Is this approximately the expected size What number of amino acids makes up the Synechocystis BvdR protein plus what number of amino acids does the streptag fusion sequence contribute And the average molecular weight of an amino acid varies from 112 gmol to 120 gmol d What is the approximate molecular weight of the protein for the streptag positive control From Exp SE 3 Attach the Printed Alignment 4 How could an actual functions of the BvdR protein from Synechocystis be determined ie distinguish between Biliverdin Reductase and Homoserine Dehydrogenase in the lab 81 Experiment 8 Introduction to Internet Proteomics Tools Objectives These outofclass exercises are to introduce the use of use Internet accessible databases and tools used in many lifescience research endeavors including the BLAST search and sequence alignments Introduction Bioinformatics is a knowledgebased approach to research and biotechnology it is the cataloging storage and retrieval of information placed in databases on proteins genes and genomes regarding their structures biological mctions cellular localizations structural modi cations regulations of expression and mechanisms of action These databases might be research journals available online or a cataloging of information housed by the National Institutes of Health NIH and other institutions They are easily accessed through the Internet and are often crossreferenced Proteomics is the area of bioinformatics focused on proteins Biochemical research and biotechnological goals often involves deliberate changes to the primary structure of a protein by sitedirected mutagenesis to assess structurefunction relationships and these altered proteins must be puri ed and characterized Characterizations might include any of the following primary sequence determination Xray crystallography mass spectrometry NlVlR kinetic analysis relative expression levels in cells etc Traditionally the primary structures of puri ed proteins were obtained by amino acid sequence analysis Comparisons of amino acid sequences of different proteins provide insight into their function and evolutionary history Since the 1980 s the cloning of protein encoding genes has permitted the derivation of primary protein structures from sequencing DNA copies of their mRNAs ie cDNAs for many proteins not puri ed nor sequenced by traditional methods such as membrane proteins Automated amino acid and nucleic acid sequencing methods currently used have made rapid accumulation of primary structural information possible Indeed the advent of automated DNA sequencing and computerdriven information management permitted entire genomes to have been sequenced cataloged and made easily accessible using the Intemet Consequently puri cation is not wholly necessary to begin complex molecular biochemical and cellular analysis of protein function With just a agment of either an amino acid or a DNA sequence proteins can be identi ed from a search of genomic databases by sequence similarities with known sequences of related proteins There are many other cuttingedge techniques being developed as well as adapting new applications to established techniques to approach what were once intractable problems Bioinformatics Bioinformatics is founded on the annotation ie naming of molecular sequences and structures chie y for DNA RNA and proteins The design of experiments and predictions of results are often rst considered from the information accessed from these databases The success of bioinformatics owes much to the pioneering work of Frederick Sanger Sanger developed the initial chemistry for sequencing proteins 39om their Ntermini and published the primary structure of insulin in 1955 Later he turned his attention to sequencing DNA and in 1977 he published the sequence of the virus DX174 For both of these breakthroughs he received two Nobel Prizes both in Chemistry 1958 and 1980 The techniques used now are modi cations of those initially developed by Sanger Protein sequencing relies on the reagent developed by Pehr Edman and is called the Edman degradation method while DNA sequencing uses uorescent dyes for detection Both protein sequencing and DNA sequencing are automated and are usually outsourced to services provided by universities or by private rms 82 Automated Protein Sequencing Direct protein sequencing is accomplished by using the Edman degradation method The procedure labels the Nterminal amino acid with the reagent phenylisothiocyanate through the carbon noted by an arrow in Figure 81 The labeled Nterminal amino acid is cleaved off leaving a free Nterminus The released phenylthiohydantoin derivative of the amino acid PTHamino acid is easily detected Figure 92 The free Nterminus minus the rst amino acid is ready for another cycle of labeling and cleavage by the Edman reagent In a sequential marmer the Nterminal amino acids are removed one at a time The identities are determined by reverse phase column chromatography which separates the PTH amino acid derivatives by weak hydrophobic interactions with the colunm matrix against a standard set of all 20 PTHamino acid derivatives with known elution patterns 39r rs E og NH EH H CH2 c I cHcH3 Edman reagent CH3 Figure 81 The Edman reagent Figure 82 The phenylthiohydantoin phenylisothiocyanate derivative of the amino acid valine Sequencing a protein with the Edman procedure yields a 20 to 40 amino acid stretch of primary sequence data from as little as 10 to 100 picomoles of protein using microsequencing techniques Most proteins are of course much longer than 40 amino acids in length Once a primary sequence of signi cant length is determined the remaining sequence can be completed in one of two ways 1 If the genomic sequence of the organism 39om which the isolated and partially sequenced protein was obtained is known then the complete protein sequence is derived from the correlating gene sequence Literally hundreds of genome DNA sequences are nished representing viruses bacteria fungi plants and animals including organelles 2 For many organisms the complete genomic sequence remains unknown Thus the primary protein sequences from these organisms must be nished by the Edman procedure To nish the protein sequence the protein is cleaved into fragments exposing an Nterminus from an internal amino acid that is available for the Edman sequencing method Fragments are generated by chemical and enzymatic means that are then separated by reverse phase chromatography By sequencing overlapping sets of peptide fragments the entire primary amino acid sequence of a protein is obtained by aligning the sequence data Determining the primary structure of a protein is made easier using computer driven searches of biological databanks A partial sequence of 8 to 20 amino acids is often sufficient to identify related proteins of similar sequences Synthetic DNA primers can be designed based on sequence similarities between evolutionarily related proteins that are used to clone the gene for the protein of interest from a genomic library or cDNA library Hence sequencing an entire protein often is not necessary since obtaining partial peptide sequence data remains crucial for modern biochemical research Automated DyeTermination DNA Sequencing The determination of DNA sequences is accomplished by using the dideoxy chain termination technique Double stranded DNA is denatured and each complimentary strand serves as a template copied by DNA polymerase with a supply of the deoxynucleotide triphosphate substrates dATP dGTP dCTP dTTP or dNTP collectively The DNA polymerase requires a free 3 OH group to form the 83 phosphodiester bond with the dNTPs to extend the newly synthesized DNA strand so a synthetic DNA primer is provided The growing chains of DNA are terminated at different locations by the incorporation of dideoxynucleotide triphosphates ddNTP The concentrations of dNTP and ddNTP are balanced to allow for the statistical tennination at every base Figure 83 a ddNTPs terminate DNA synthesis b Using ddNTPs daughter strands of different length can be produced P P Template DNA 3 A C H B P P CGAAliTA39GGGAGTCTGGCAACT P P E 539 T 0 Base 539 3H2 O Base 339 339 OH Normal dNTP ddNTP extends DNA strand terminates synthesis Figure 83 Dideoxy termination method for DNA sequencing From Queens University of Charlotte Biology 103 DNA Technology When the ddNTPs are each labeled with a different uorescent molecule the identity of an incorporated ddNTP is easily detected and identi ed The terminated DNA strands of different lengths are separated by electrophoresis by size by a onebase difference and a uorescence detector records the elution pattern The pattern is printed along with the corresponding nucleotide identity Approximately up to 350 bases can be sequenced starting from the primer per run and up to 24 runs can be performed per day for a total of 8400 bases per day per automated system With several systems performing multiple runs per day it is now possible to sequence an entire bacterial genome in a day or two At the end of the sequencing run a new primer to extend the sequencing is based on the newly determined sequence Primers are also synthesized quickly by an automated system most labs that provide extensive DNA sequencing services will also have an automated primer synthesis system on site Contiguous sequences are properly aligned by overlapping sequence data Accessing bioinformatic pages for speci c protein sequences Information for structural parameters such as processed sequences and motifs are found within bioinformatic pages accessed through web portals hosted largely by NIH or various university sites eg several bioinformatic links are found at the following online site hosted by the University of Colorado mded by NSF httpwWwcoloradoeduchemistrybioinfoBioinformaticsLinkshtrn or sites accessed directly such as sites for proteomics tools httpcaexpasyorg Access is obtained using an accesion number which a tag or label used to identify a speci c protein or nucleic acid sequence If an accession number is not known a search can be started on some web sites by typing in the name of the protein being sought and often including a species too the response will include a list of proteins with the queried name along with their accession numbers The list can be looked over for the particular protein being sought The list of same or similar proteins that have been submitted and annotated ie named also may be of interest An example of accession numbers is shown in Table 81 below for cytochrome f from the photosynthetic cyanobacterium Synechocystis sp PCC6803 There are many other accession numbers for information such as 3D structure databases enzyme pathways databases phylogenomic databases 84 etc which are usually available as crossreferences on pages obtained from these primary accession numbers for protein and DNA sequences Table 81 Accession numbers for Synechocystis cytochrome f Acession Number Sequence Database P26287 Apoprotein UniProtKBSwissProt X58532 Genomic DNA EMBL BAO00022 Genomic DNA GenBank Alignment of protein sequences The alignment of two sequences is the mdamental operation of bioinformatics Aligning full length protein sequences or peptide sequences ie partial protein sequences was a crucial step in early structurefunction determinations of proteins Such alignments identi ed similarities and differences in amino acid sequences between mctionally related proteins For example peptide sequences of related proteins hemebinding cytochrome c from three photosynthetic species is shown in Figure 84 below Heme binding site R rubrum G A K L Q A L G G T C K A C H K E F K Alcaligenes sp II R A A F G D V G A S C K A C H D A Y C vinosum D T A F G D V G A A C K S C H K Y Figure 84 Alignment of peptide sequences containing the heme binding site for cytochrome c These proteins are functionally related thus the differences in sequences help the researcher to determine possible critical amino acids in the mction of the protein such as the amino acids required for hemebinding which is CXXCH X can be any amino acid the invariant K within the sequence is shown to be variant with additional sequences compared data not shown Some amino acids are identical between the two alignments and some are different Amino acids are designated as identical if the amino acid pairing is invariant Those amino acid pairings designated as similar are not identical but share similar properties For example a paring of glutamic acid with aspartic acid or a leucine paired with isoleucine or a methionine paried with isoleucine are all considered conservative changes and are designated as similar In Figure 85 below identical amino acid pairings are shown with an asterisks similarities are shown with a double dot greater conservation of structure and ftmction or a single dot lesser conservation of structure and function The abundance of protein sequence data from hundreds of species have allowed researchers to infer ftmctions and regulations of proteins not just from over all structure but also from structural motifs and structural domains Structural motifs or domains have identi able stretches of primary structures ie amino acid sequence found in proteins of similar function or similar regulation of their functions The heme binding site CXXCH is an example of a sequence motif see Figures 84 above and 85 below BLAST Search Program A powerful and widely used program for retrieving and analyzing protein or nucleic acid sequences is the BLAST search program Basic Local Alignment Search Tool The BLAST program is available via several web sites including the ExPASy site One result of the Genome Projects is the identi cation of Open Reading Frames ORF stretches of nucleic acid sequences that potentially code for a viable 85 protein The DNA sequence of an ORF or the translated protein sequence is submitted to a BLAST search as the query sequence and within a few seconds a results page appears listing the query sequence ie the submitted sequence being analyzed on top followed by other sequences from the most similar to less similar By comparing the similar known sequences a putative identity of the ORF can be assigned These similarities are based on how well they align with the query sequence For biochemists alignment of protein sequences is more informative than nucleic acid sequences There are four characters for aligning nucleic acids and the third character of most codons is degenerate resulting in mulitple sequences coding for the same amino acid and a stretch of nucleic acid sequence can be translated in six reading frames and decisions must be made as to the relevance of such results P2628 MRNPDTLGLWTKTMVALRRFTVLAIATVSVFLITDLGLPQAASAYPFWAQETAPLTPREA 60 CYFSYNY3 P46445 0 CYC6SYNY3 P26287 TGRIVCANCHLAQKAAEVEIPQAVLPDTVFEAVVKIPYDLDSQQVLGDGSKGGLNVGAVL 120 CYFSYNY3 P46445 MFKLFNQASRIFFG IALPCLI 21 CYC6SYNY3 P26287 MLPEGFKIAPPDRLSEGLKEKVGGTYFQPYREDMENVVIVGPLPGEQYQEIVFPVLSPDP 180 CYFSYNY3 P46445 FLGGIFSLGN TALAADL 38 CYC6SYNY3 P26287 AKDKSINYGKFAVHLGANRGRGQIYPTGLLSNNNAFKAPNAGTISEVNALEAGGYQLIL 239 CYFSYNY3 P46445 AHGKAIFAGNCAACH NGGLNAINPSKTLKMADLEANGKNSVAA 81 CYC6SYNY3 P26287 TTADGTETVDIPAGPELIVSAGQTVEAGEFLTNNPNVGGFGQKDTEVVLQNPTRIKFLVL 299 CYESYNY3 P46445 IVAQITNGNGAMPGFKGRISDSDMEDVAAYVLDQAEKGW 120 CYC6SYNY3 39 P2628 FLAGIMLSQILLVLKKKQIEKVQAAELNF 328 CYESYNY3 P46445 120 CYC6SYNY3 45 similarity 19 identity compared within the 120 amino acids of CYC6SYNY3 Figure 85 ClustalW Alignment of cytochrome f CYFSYNY3 and cytochrome c6 CYC6SYNY3 from Synechocystis sp PCC6803 Cytochrome f is a ctype cytochrome note the hemebinding sites CXXCH are not aligned Cytochrome f amino acids 144 are a signal sequence not present in the mature protein and amino acids 296313 are a transmembrane helix Cytochrome c6 is a soluble cytoplasmic protein The BLAST search program scores potential matches when aligning sequences by taking into account i Invariant amino acids ii Conservation acceptable substitutions while not changing the function charge size hydrophobicity iii Frequency re ects how often particular amino acids occur ie less frequently incorporated amino acids such as methionine and tryptophan generally are given more weight and iv Evolution scoring is designed to detect more closely related proteins or more distantly related proteins Notice in Figure 85 above the stretches of nonaligned sequences ie gaps There is much more probability and mathematical assessments behind these simple descriptions taking into account gaps penalties conservation a series of block comparisons comparing a block of amino acids and seeing how closely they align then comparing another block which overlaps considerably with the previous block and asking is it a better alignment A discussion of these treatments is beyond the scope of this introduction As endusers biochemists are interested in employing the BLAST function and other proteomic tools 86 Protein Processing and Cellular Localization Bioinformatic information pages regarding a protein often describes the sequences that are processed or transmembrane segments etc also described are molecular weights isoelectric points etc for an entire peptide sequence of a preprotein or aproprotein Due to the success of DNA sequencing of entire genomes many protein structures are inferred directly from DNA gene sequences or from cDNAs cDNAs are copies of mRNA sequences that include the coding sequences for the unprocessed newly translated proteins The processing events occur very quickly after translation so only the mature fonns of the proteins are isolated from cells Hence when using bioinformatic sites and programs it is important to check the annotations of a protein of interest to obtain appropriate information regarding the mature size and accompanying structural features of the mature protein The mature form of a protein may be a single protein or a subunit of a multisubunit holoenzyme or a structural subunit eg part of the cytoskeleton etc Protein translation begins with the start methionine codon AUG on mRN A which is nearly universal in all forms of life Thus every newly translated protein has a methionine at its Nterminus However many proteins are processed meaning one amino acid to long stretches of the Nterminal peptide sequence and or the Cterminal peptide sequences are removed Protein processing occurs extensively in eukaryotic organisms but also occurs in prokaryotes Processing converts the larger newly translated proteins referred as preproteins or aproproteins to the mature forms found in the cell There are several protein processing events in cells that allow for the regulation of trafficking of proteins cellular localization and the regulation of protein mction Looking again at the sequence comparison of the two cytochromes from the photosynthetic cyanobacterium Synechocystis in Figure 85 above there is a long Nterminal and Cterrninal sequences for cytochrome f not found in cytochrome c6 The Nterminal amino acids 144 of cytochrome f is a signal sequence directing the apocytochrome f to the photosynthetic membrane which are cleaved off whereas amino acids 296313 are retained which is a transmembrane helix anchoring the protein to the membrane Cytochrome c6 by contrast is a soluble cytoplasmic protein it is synthesized and released into the cytoplasm PART A CHARACTERISTICS or CALF INTESTINAL ALKALINE PHOSPHATASE Before work on a particular protein of interest might begin looking over the biochemical structures and properties of the protein of interest is usually undertaken The enzyme to be examined in Experiment 3 is Alkaline Phosphatase from calf bovine THE CALCULATED THEORETICAL MOLECULAR WEIGHT AND PI or BovINE INTESTINAL ALKALINE PHOSPHATASE DETERMINED BY ONLINE PRoTEoMICs TooLs 1 Open the web site expasyorg 2 The resulting page ExPASy Bioinformatics Resource Portal In the righthand box to the right of the box Query All Databases type in the accession number P1911 1 or the entry name PPBIBOVIN and select search 3 The results will appear on the same page Under Resource select UniProtKB 4 Under Entry select P191 11 87 5 Scroll up and down to become familiarized with the features and information available 6 Scroll up and down to become familiarized with the the features and information available 7 Scroll to the top of the page to the section Protein attributes Number of amino acids annotated 8 Now scroll to the section Sequence Armotations Features Note the Chain is the mature processed protein the accompanying graphics are helpful to see the gross anatomy of the protein structure 9 Scroll down to the section Sequences In the box to the right with the pulldown menu open the program Compute plMW and select Go 10 On the response page Compute pIMW Click on the link next to Chain 20506 this is the mature protein Print this page that includes the mature protein sequence theoretical pI and molecular weight PART B RESTRICTION ENZYME MAPPING OF THE GAF4 DoMAIN SEQUENCE FROM NOSTOC PUNCTIFORME N PR60124 USING A COMMERCIAL DNA SEQUENCE ANALYSIS TooL The primary purpose of Experiment 4 is to design PCR primers for the directional cloning of the Nostoc GAF4 domain DNA and then analyzing the results of the PCR reaction to verify that the correct segment of DNA is ampli ed The NEBcutter V20TM DNA analysis tool nds and displays the locations of hundreds of restriction enzyme recognition sequences The results are used to design the primers ie restriction sites to use and avoid for primer design and restriction enzyme sites that can be used to analyze the PCR reaction product 1 Open the web site httptoolsnebcomNEBcutter2 2 The resulting page NEBcutter V20 In the empty box paste the GAF 4 nucleotide sequence copied from Figured 410 in the manual Select All commercially available then select Search 3 Find the Hind III location on the resulting page using the mouse pointer identify the location of the cut site for Hind III Recognition sequence Cuts at 4 Print the resulting page that displays the linear sequence with the restriction site locations 88 PART C BLAST SEARCH AND CLUSTALW SEQUENCE ALIGNMENTS OF THE Nos39roc GAF4 DOMAIN Much of the current research efforts in biochemistry molecular and cellular biology are focused on dissecting the mechanistic aspects of protein protein interactions that regulate the functions of other proteins and that are involved in signal transduction pathways regulating cellular functions To understand the mechanistic details of such protein protein interactions requires dissecting ner details of protein structure The GAF Domain is found in proteins in all Kingdoms of life A protein domain is de ned as a structural unit that can fold independently of the larger protein Domains are often further identi ed by carrying speci c conserved sequence motifs while the larger domain primary sequence may not show the same level of sequence conservation when compared with other sequences of the same domain These exercises with the Nostoc GAF4 Domain illustrate some of these principles BLAST Searches I Search with the entire cyanobacteriochrome from Nostoc containing the GAF4 Domain 1 Open the web site expasyorg 2 The resulting page ExPASy Bioinformatics Resource Portal In the righthand box to the right of the box Query All Databases type in the accession number B2IUl4 and select search 3 Under Resource select UniProtKB 4 There is likely only I hit under the UniProtKB database hence the B2IUl4 protein is the only entry if not then select B2IU14 5 Scroll down to the subsection Sequences 6 On the right hand side under Tools select BLAST 7 On the resulting page select the boxes for the top four responses exclude the query make sure one checked box is B2IU14 the Nostoc cyanobacteriochrome protein that contains the GAF4 Domain 8 Select Align 9 Print the alignment there should be two pages the alignment and the second page has the names of the genes and the organisms Choose the print function om the browser II Search with the Nostoc GAF4 Domain sequence only 10 Return to the BLAST function select the BLAST tab at the top of the page Copy and Paste the GAF4 Domain sequence from Figure 47 reprinted below into the box the gaps and numbers do not need to be removed Then select RUN BLAST 11 The rst sequence again should be theNostoc protein B2IUl4 Click on B2IU14 located on the left side of the response page Scroll down to the Detailed BLAST Results subsection then below that are the Alignments There should be colored bars to the left of the gene accession numbers 89 12 Click on the stack of four colored bars to the left of the gene B2IUl4 the four colored bars indicate that the GAF 4 query sequence aligns with four stretches of sequences in the B2IU14 gene ie the Nostoc cyanobacetriochrome protein 13 Print the resulting pages These pages show a local alignment of the GAF 4 Domain to all four GAF domains within the larger Nostoc cyanobacteriochrome protein B2IUl4 EKAVTK ISNRIRQSSD 600 VEEIFKTTTQ EVRQLLRCDR VAVYRFNPNW TGEFVAESVA HTWVKLVGPD IKTVWEDTHL 660 QETQGGRYAQ GENFVVNDIY QVGHSPEHIE ILEQFEVKAY VIVPVFAGEQ LWGLLAAYQN 720 SGTRDWDESE VTLLARIGNQ LGLALQQTEY LQQVQGQSAK 760 Figure 47 The sequence for amino acids 585 760 of the Nostoc GAF 4 domain NpR60l2g4 The underlined C687 is the bilin attachment site UniProt accession number B21U14 PART D MOLECULAR WEIGHT ESTIMATION OF THE RECOMBINANT Nosroc GAF 4 DOMAIN The molecular weight of the Nostoc GAF 4 Domain polypeptide can be estimated by several available proteomics tools This is useful for having an expected size to compare with the actual results of the SDSPAGE analysis Experiment 6 of the puri ed Nostoc GAF 4 Domain Experiment 5 1 Open the website expasyorg 2 In the left hand column under Categories select proteomics 3 On the resulting page in the right hand colunrm under the Tools to nd ProtParam select ProtParam 4 Copy the amino acid sequence of the Nostoc GAF domain above and paste it into the box on the ProParam Tools page Remember this is the recombinant GAF 4 that contains added amino acids so add these onto the sequence 5 Select compute parameters 6 Print the resulting page PART E REEXAMINATION OF THE sLR1784 GENE LOCUS Open Reading Frames and Inferred Functions The sequencing of whole genomes presents unprecedented opportunity to study newly discovered genes including Open Reading Frames ORFS that potentially code for proteins It is often possible to infer biochemical functions for putative proteins encoded by ORFs through database comparisons resulting in 810 sequence similarity matches with proteins of known functions Such sequences are cloned for study In Experiment 7 the ORF from the genetic locus sir 784 of the cyanobacterium Synechocystis has been ampli ed by PCR and cloned into the plasmid pASK75 with E coli serving as the host BLAST Search to reassess the Synechocystis BvdR protein sequence and function Bioinoforrnatic databases are constantly updated and sometimes protein sequences with putative functions are reannotated ie renamed Thus while databases are powerful tools there is no substitute for actual controlled experimental analyses for testing hypotheses which the reason for performing Experiment 7 The ORF locus slr1784 from the cyanobacterium Synechocystis PCC6803 was identi ed in 1997 and given the putative function as a Biliverdin Reductase It is prudent to reexamine the gene sequence slrl784 against the databases and examine the results Re Examination of the ORF from PCC 6803 locus slr1784 1 Open the ExPASy web page expasyorg 2 On the ExPASy Resources Portal in the righthand box type in the slr1784 gene ORF accession number P72782 select search 3 The results will appear on the same page Under Resource select UniProtKB 4 Under Entry select P72782 5 Find the subcategory Sequences Under the Tools section select Blast then select go 6 In the Detailed BLAST subsection i What is the highest percent identity with the query sequence with the enzyme name Homoserine Dehydrogense From what organism ii What is the highest percent identity with the query sequence with the enzyme name Biliverdin Reductase From what organism 7 On the rst response page only select the boxes on the left for i the query sequence ii top 3 sequences named biliverdin reductase iii top 3 sequences named homoserine dehydrogenase Then click Align at the bottom of the page 8 Print this page for the Exp 7 writeup In Print Preview it might help to select landscape or highlight the alignment only and print selection or copy and paste the alignment on a separate MS Word document Mammalian BVR is a cytosolic enzyme with molecular mass of roughly 33 kDa The mammalian BVR is notable in that it utilizes either NADPH or NADH for the reduction of BV shown below A Thus BVR activity is monitored by the change of absorbance at 450 nm with time where absorbance of the BR product is maximum 8 BR 450nm 53000 M 1cmquot The reaction catalyzed by Homoserine 811 Dehydrogenase also uses either NADP or NAD B Neither homoserine nor aspartatesemialdehyde have a convenient visible absorbance hence the reaction can be followed at 340 nm 9 NADPH 340mn 6220 Mquotcmquot 0 NADF HHquot NADF H 0 iv H0 J0 B 0 NH 0 NH3 easpartatesemialdehyde h Omose n e Lhomoserine NADP 39quot Laspartate 4semialdehyde NADPH H References Edman P and Henschen A 1975 Protein Sequence Determination S B Needleman ed Vol 8 pp 232280 Esch F S 1984 Anal Biochem 136 pp 3947 Shively J E 1986 Methods of Protein Microcharacterization J E Shively ed pp 4186 Humana Press Williams and Stone 1995 Techniques in Protein Chemistry VI pp143152 Hellman et al 1995 Anal Biochem 224 pp 451455 Rosenfeld et al 1992 Anal Biochem 203 pp 173179 Best et al 1995 Techniques in Protein Chemistry VI pp 205213 Fernandez et al 1995 Techniques in Protein Chemistry VI pp 215222 Alberts et al 1994 The Molecular Biology of the Cell 3rd Ed pp 291334 Garland Publishing 10 Hartwell et al 2004 Genetics rom Genes to Genomes 2quot Ed pp 277361 McGraw Hill 1 1 Tateno Y and Barrero R 2005 htlpzlwwwddbjnigacjpddbjnewdcr2005cibact2 ehtm1 Section 2 Research Activities in CIBDDBJ 12 Genome Horne NCBI httpzlwwwncbinlmnihgovsitesentrezdbgenome 13 Altschul Stephen Fet al 1997 quotGapped BLAST and PSIBLAST a new generation of protein database search programsquot Nucleic Acids Res 2533893402 14 Vincze T Posfai J and Roberts RJ NEBcutter a program to cleave DNA with restriction enzymes Nucleic Acids Res 31 36883691 2003 15 httplmolbiol toolscaPCRhtm 16 Amino Acid Sequence of Cytochrome c from the Purple Photosynthetic Bacterium Rhodospirillum rubrum S1 1975 J Biol Chem 250 21 84168421 DOO39lOLhI3UJIJv 39
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