Review Sheet for BIOC 463A at UA
Review Sheet for BIOC 463A at UA
Popular in Course
Popular in Department
This 18 page Class Notes was uploaded by an elite notetaker on Friday February 6, 2015. The Class Notes belongs to a course at University of Arizona taught by a professor in Fall. Since its upload, it has received 15 views.
Reviews for Review Sheet for BIOC 463A at UA
Report this Material
What is Karma?
Karma is the currency of StudySoup.
Date Created: 02/06/15
BIOC 463A Expt 2PNP Tit n Expt 2 Spectroscopy and Determination of pKa of PNP Goals 1 Learn how to use spectroscopy as a powerful tool in biochemical studies 2 Perform a pH titration of paranitrophenol PNP using changes in the visible spectrum to determine the pKa 3 Determine if the polarity of the solvent alters pKa by adding alcohols of increasing number of carbons Spectroscopy Over View Spectrophotometry the process that determines the amount of light at a given wavelength absorbed by chromophores o Chromophore any material that absorbs light PNPOH and PNPO39 Protein amino acids Trp Tyr cystines Phe and nucleotide bases Cofactors and prosthetic groups Hemes flavins FeS Cuzquot NADINADH BIOC 463A Expt 2PNP Tit n Uses of Spectroscopy in Biochemistry Product identification 0 Changes in structuralphysical properties 0 Determining concentration 0 Measuring rate of product formation steady state kinetics 0 Enzyme activity assays o Ligand binding assays Absorption Process Absorption of light electromagnetic radiation occurs because the energy of some physical transition in the chromophore the energy of the light Most common forms of light in biochemical studies 0 UVvisible 200 300 nm electronic transistions rr 9 11 and some a 9 0 Infrared 1o3 105 nm vibration rotation about bending of covalent bonds Radio NMR and EPR 106 and 101 nm spin orientation of nuclei and unpaired electrons BIOC 463A Expt 2PNP Tit n Review of Light and Energy E hv where h 663 x 103934 J s and v frequency in Hz s391 Hz of light the number of waves passing a fixed ptsec Wavelength A the peak to peak distance in cm 10392 m or nm 10399 m Ehvhg c299x108ms391 nA n refractive index 1 NOTE E is inversely proportional to A As A increases the Energy of that light decreases BIOC 463A Expt 2PNP Tit n For a mol of photons 1 Einstein at 410 nm E 663 x 103934 Js299 x 101 cms602 x 1023mol 410 x 10395cm E 29 x104 Jmol 29 kJmol This represents a lot of free energy if it can be captured For a species that absorbs light at 410 nm such as PNPO39 as we shall see the energy of that light represents the difference the energy levels in the Trelectronic configuration in that molecule ie the energy of light absorbed the energy difference between orbitals Absorption of UVvisible light energy results in electronic transitions in 1T 9 1T and o 9 o MO s BIOC 463A Expt 2PNP Tit n Liqht Intensitv Transmittance and Absorbance Transmittance llo and varies from 1 9 0 If nothing is in the beam then I lo but if a cuvette containing a solution is in the beam then generally llo lt 1 Cuvette may absorb light Cuvette scatters light Buffer solution scatters light This DOES NOT take into consideration absorption of light by the solution This is why you ALWAYS run a baseline spectrum BIOC 463A Expt 2PNP Tit n Path Lenqth and Concentration Effects on llo If a solution is in cuvette that absorbs light of specific A then there is a path length and concentration dependency for l Lambert Relationship Consider the cell to be divided into layers with the same concentration of solution in each layer Then there is an llo value lt1 for each layer Then llo DECREASES EXPONENTIALLY with each layer 1st 9 2nd 9 3rd 9 4th 9 9 nth mo 9 llo29llo39lllo499 mon BIOC 463A Expt 2PNP Tit n Beer s Relationship llo DECREASES EXPONENTIALLY with INCREASING CONCENTRATION in similar manner Combined BeerLam bert Relationship T M0 1oquotcL logT cL A absorbance a is an absorptivity absorption coefficient dependent upon the species 0 c is the concentration 0 L is the path length How Does T and A Relate llo T A lho 1 0 09 0046 05 03 Believable 01 1 001 2 0001 3 Doubtful Very low A values limited by Signalnoise ratio Very high A values limited by sensitivity of detector You pay a lot of money to optimize these two BIOC 463A Expt 2PNP Tit n About a o Commonly called an Extinction or Absorptivity Coefficient o Molar M391cm391 milliMolar mM391cm391 microMolar uM391cm391 o Is specific for chromophore A and conditions pH oxidation state etc o It is the absorbance that a 1 M solution would have in a 1 cm path length cell Determination of Extinction Coefficient Determination of 8 mM 1 cm391 at 260 nm 15 for ATP wg 10 U C U Y Q I 6 u g 05 Slope 154 mlvr1 Note Fit must pass through origin 00 39 I 000 005 010 ATP mM Measure absorbance at specific wavelength as a function of concentration Determine a from slope BIOC 463A Expt 2PNP Tit n Values of a for Bioloqical Chromophores Chromophore A nm a M391 cm391 RSSR 280 600 Tyr 280 1000 Trp 280 10000 FeSox 400500 60008000 Flavinsox 440 14000 Hemes 400450 80000120000 BIOC 4A Expt 2PNP Tit n Examples of Visible Spectra Mitochondrial c ochrome c 100 l l l l yi41enm 1 5 158 mMquot cmquot 75 cythez39 a l E A m r 12 050 7 mama m n lt 025 000 39 400 450 500 550 500 06 cyt uFe wequot a 04 u 1 1 g Azm22mwr an 3 02 a lt 1 j I 02 400 450 500 550 Wavelength nm 600 Top panel absolute spectra of reduced and oxidized cytochrome c Bottom panel Reduced minus Oxidized difference spectrum of cytochrome c Note that the intensity is expressed as AAbsorbance 10 BIOC 463A Expt 2PNP Tit n Obtaininq Spectra of Samples Every spectrum ALWAYS corresponds to a difference between the A of the sample and A of a reference or baseline solution Afinal Asample 39 Areference Done either electronically or in computer memory Precom puter spectrophotometers Com puterdriven spectrophotometers 11 BIOC 463A Expt 2PNP Tit n On Cary 50 scanning generates a numerical array of numbers AA 0 Baseline numerical array for reference solution Takes into account absorption scattering properties of cuvette and solution 0 As you scan sample a temporary array of signal coming from detector is generated 0 Spectrum seen on screen Sample Temporary array Baseline array 12 BIOC 463A Expt 2PNP Tit n Absolute vs Difference Spectrosconv AbSOIUte ASample 39 ABuffer Absolute Spectra Absorbance 300 350 400 450 500 Wavelength nm The absolute spectra of PNPOH and PNPO39 are shown lP corresponds to the isosbestic point IF the wavelength at which both spectra have equal absorbance values see below At each wavelength A for PNPOH AA APNP0HPNPOHL For PNPO39 AA APNPOPNPOL If PNPOH PNPO what do we know about EPNPQH and Epr0 at 13 BIOC 463A Expt 2PNP Tit n Difference Sample in final state Sample in initial state PN PO minus PN POH Difference Spectrum 04 02 r Delta Absorbance 3h 0 350 400 450 5 0 Wavelength nm The difference spectrum from the pH titration of PNP was obtained by subtracting the spectrum of PNPOH initial state from that of PNPO39 final state Note that at some wavelengths AA lt 0 At each 9 AAA AAPNP0 39 AAPNP0H APNP0PNPOL APNPOHPNPOHL lf PNPO PNPOH then AAA APNP0 39 APNPOHPNPOL AExXIIPNPOXL 14 BIOC 463A Expt 2PNP Tit n Advantaqes and Uses of Two Types of Spectra Absolute 0 Determine Amax and Amiquot values 0 Determine concentration 0 Can easily find isosbestic points Difference 0 Allow you to monitor small changes in concentration of sample easily 0 Determine if you have simple chemical reaction 0 Almost all kinetic measurements are made using Difference Spectroscopy Fixed wavelength monitoring Stopped flow and laser flash measurements 15 BIOC 463A Expt 2PNP Tit n Following Spectral Changes in Reactions In this week s pH titration experiment we are looking at the conversion of PNPOH lt gt PNPO39 Absolute spectra Percent Titration quot40 100 035 90 03 75 025 50 020 25 Absorbance 015 10 010 0 005 000 39 300 350 400 450 500 Wavelength nm For each spectrum AA AAPNPOH AAPNPO APNP0HPNPOHL APNPOPNPOL How would you modify this eqn to take into account PNPJtotah instead of individual concentrations At the IP absorbance is constant throughout the titration at every other 7 it changes 16 BIOC 463A Expt 2PNP Tit n Difference Spectra PNPO39 minus PNPOH Percent Titration 0394 39 100 90 03 75 02 50 OJ AAbsorbance O 1 o2 I I I I 300 350 400 450 500 Wavelenth nm At every A you are looking at AA AAA EAPNPO39 EAPNPOHPNP039L ASAPNP039L When sApNPO39 lt EAPNPOH then AAA lt 0 When sApNPO39 EAPNPOH then AAA 0 When sApNPO39 gt EAPNPOH then AAA gt 0 17 BIOC 463A Expt 2PNP Tit n So how does one make use of all these spectroscopic concepts An example using single wavelength difference spectroscopy to follow product formation in an electron transfer reaction Figure 52 Reduction of Sul te Oxidase M The signal at t 0 takes 12 W Reoxidation into account the sample K before the laser flash quot 39 therefore is difference 03 r llillwill 02 Initial Reduction Ml Asignal55 Volts a m 00 r 02 41002 0000 0002 0004 0006 0000 0010 Time ms 0 In this experiment sulfite oxidase was rapidly reduced by in a laser flash photolysis experiment resulting in rapid increase in absorbance at 555 nm 0 There is a second slower oxidation reaction by another cofactor resulting in a decrease in A555 0 Two useful pieces of information gained from trace 1 The rate constants for the reduction and oxidation of the heme AND the sequence of these reactions 2 The equilibrium distribution of the electron between the two cofactors thus Keq 18