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Biochemical Methods I

by: Michael Reilly

Biochemical Methods I CHEM 153L

Michael Reilly
GPA 3.71

S. Kim

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S. Kim
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This 10 page Class Notes was uploaded by Michael Reilly on Friday September 4, 2015. The Class Notes belongs to CHEM 153L at University of California - Los Angeles taught by S. Kim in Fall. Since its upload, it has received 123 views. For similar materials see /class/177977/chem-153l-university-of-california-los-angeles in Chemistry at University of California - Los Angeles.


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Date Created: 09/04/15
7252008 BIOCHEMICAL M ETH 0 DS SUMMER 2008 PROFESSOR STEVEN J K M TA MAUR CE SECHON 1C GROUP MOOOOO Graehl Heather Section 1C Group M00000 Introduc on Lactate dehydrogenase LDH catalyzes the conversion of pyruvate to lactate with coenzyme NADH to NAD In muscle tissue LDH is crucial to maintaining levels of NADH and NAD especially under anaerobic conditions Glycolysis is the only source of ATP in the absence of oxygen though it consumes large amounts of NADtto fuel this process Lactate dehydrogenase serves to replenish this NADt for glycolysis so that it can continue ATP production LDH exists as a tetramer of several isoforms that catalyze the same reaction but vary slightly in kinetic properties as to better function in the tissue they reside Experiencing Biochemistry We aim to purify LDH from cow shank muscle determine its subunit composition and study kinetics of two types of inhibitors Purification is achieved by homogenization ammonium sulfate fractionalization and affinity chromatography though an additional step of ion exchange chromatography could provide useful for separation of LDHA and LDHB isoforms SDSPAGE and GEC verified LDH as a tetramer and served as diagnostic of purity The kinetics of purified LDH were then studied to determine its turnover number and the modes of inhibition by oxamate and oxalate Materials and Methods Experimental procedures are outlined in Experiencing Biochemistry lab manual and Course Reader Companion with a few notable modifications During homogenization to obtain the crude extract cheese cloth was used instead of wool glass as a filtering device When running the affinity column approximately 15ml of AMPagarose stationary phase was used instead of the outlined 10ml Also load and rinse fraction assays were not performed at all for enzyme assays and not in duplicate for protein assays due to limited time and their insignificance in a properly run affinity column enzyme assays assays enzyme assays assays enzyme enzyme Page 2 Graehl Heather Section 1C Group M00000 ResultsDiscussion a Crude Extract Cow shank muscle was butchered to eliminate excess fat and homogenized in a blender with phosphate buffer in order to lyse cells releasing LDH into a slurry of tissue components Centrifugation separated membranes nuclei and other large cellular components to a pellet leaving a supernatant of crude product Controlling temperature was a major consideration after homogenization since not only did this step release LDH from the cell but it also released proteases that can now interact to degrade the LDH Keeping samples on ice precooling the phosphate buffer and avoiding excess kinetic energy through conservative blending were methods to minimize activity of these proteases Excessive blending also increases LDH s exposure to air that can cause oxidation ofthe enzyme or it can cause surface denaturation in which LDH folds differently due to the atmosphere interacting with LDH instead of its native aqueous environment To improve yield alternative methods of cell lysis to homogenization could be investigated to avoid surface denaturation using inert gas could avoid atmospheric oxidation and protease inhibitors could combat unwanted protease activity The presence of LDH was con rmed via enzyme assays through monitoring a rate of decrease of 340nm absorbance corresponding to the conversion of NADH A340nm to NAD The assay provided initial reaction rates and through Beer s Law the total enzyme was calculated to 7000100units Table l b 40 ammonium sulfate Highly soluble ammonium sulfate salt is added to the crude extract as to outcompete unwanted impurities for water solvation As increasing amounts ofammonium sulfate are added the weakly soluble proteins membranes and fats are outcompeted for hydration and precipitate from solution Centrifugation yields a pellet of these impurities and a 40 supernatant containing LDH The addition ofammonium sulfate must be done slowly with stirring as to avoid microenvironments ofhigh concentration that would prematurely precipitate LDH into the 40 pellet and decrease yield Page 3 Graehl Heather Section 1C Group M00000 To monitor progress throughout puri cation steps both enzyme and protein assays are utilized to con rm the sample is becoming more concentrated with LDH and signi cant loss has not occurred Protein assays are a way to determine protein concentration but these proteins are not speci c to LDH The protein concentration is obtained through use ofCoomassie Blue dye which at low pH binds to hydrophobic pockets ofproteins to form a dyeprotein complex that absorbs at 595nm Sample serial dilutions are assayed until a reliable range at 595nm with Coomassie blue is met and then interpolated on a standard curve ofknown BSA concentrations with Coomassie blue When combining both enzyme and protein assay results an assessment can be made ifthe enzyme to protein ratio or speci c activity has increased with each puri cation step The 40 ammonium sulfate step had a slight decrease in protein amount from 22020mgml to 17020mgml and relatively no change in enzyme activity from 7000100unitsml to 7100200unitsml Table 1 This is consistent with the theory ofsalting out since 0 to 40 saturated solution will keep most to all of LDH in solution while precipitating fats membranes and less soluble proteins Though this step doesn t increase speci c activity signi cantly from 323unitsmg to 435unitsmg table 1 it is crucial since af nity column chromatography would be crippled by the presence of bulky fats Furthermore speci c activity is only a calculation based on protein removal which doesn t give any quantitative consideration to removal to nonprotein contaminants like the fats and membranes removed in this step Yield was 1034 table 1 which is suspiciously high but can be excused within delta values Protein and enzyme assays were repeated from new reaction mixtures in duplicate to con rm this high yield was not in error c 60 Pellet The 60 ammonium sulfate step has the same theory as the previous step in which ammonium sulfate competes for hydration which precipitates out less soluble components however in this step LDH is targeted for precipitation rather than impurities The resulting 60 pellet theoretically contains most of the original LDH which is resuspended in about tenfold less volume to create a more pure and concentrated sample The puri cation factor of 1302 is not particularly impressive considering such a Page 4 Graehl Heather Section 1C Group M00000 large loss ofenzyme and yield of 676 table 1 but this step is still required since it separates out LDH from salts and nucleic acids lfthe salts were not removed they would interfere with subsequent steps such SDSPAGE causing signi cant gel smiling and ifnucleic acids were not removed they could absorb at 280nm during GEC causing additional or overlapping peaks This step was expected to have no more than 33 loss and with a yield of677 table 1 the experimental enzyme loss was considerable This can be accounted for by unequal splitting of two pellets in which the assayed pellet caused an underestimate of enzyme activity This could be tested by performing an enzyme assay on the second 60 pellet sample to see if it has a significantly higher enzyme activity than the rst pellet Another source ofloss could be attributed to degradation ofenzyme since itwas stored for several days between assaying the 40 supernatant and 60 pellet The loss ofenzyme cannot be blamed on not obtaining high enough saturation to precipitate all the LDH since enzyme assays on the 60 supernatant showed insigni cant amounts of enzyme present Ideally all assays would be performed in the same lab period to avoid possibility of enzyme degradation during prolonged storage as well as splitting of pellets could be performed in a more precise manner and checked via enzyme assays d Affinity Column The af nity column serves as a very effective purification step that manipulates LDH s af nity toward specific ligands for puri cation The 60 cut is loaded onto a column containing AMP agarose beads The AMPagarose beads temporally bind and release LDH as well as other AMPbinding proteins slowing their descent down the column while other proteins pass through the mobile phase with high velocity Phosphate buffer is loaded to ensure all nonAMP binding proteins have completely passed through A NADHpyruvate adduct is used to elute LDH very effectively because it contains both substrates that LDH binds which brings LDH into the mobile phase for elution while other AMPbinding proteins remain bound to the AMPagarose beads Fractions were collected and initially tested for enzyme presence qualitatively by tetrazolium spot plate color change An elution pro le was constructed using protein and enzyme assays offractions gure 2 Page 5 Graehl Heather Section 1C Group M00000 Protein assays were performed on all fractions while enzyme assays were only performed for elute fractions however overload was observed in which tetrazolium spot plate detected some enzyme activity in fractions load 3 and rinse 4 Since enzyme assays were not performed on these overload fractions the spot plate results can be qualitatively compared to similar degree of color change to elute fractions 13 and 14 which did have enzyme assays Though this method is has high error the amount of overload enzyme is so low that it is less than the delta for peak fraction 11 and thus this qualitative method is acceptable to determine enzyme amount ofoverload fractions Fraction 11 was selected as the peak fraction with highest speci c activity of 49040unitsmg table II Yield after affinity column was 607 which indicates very little loss since previous step was at 676 yield table 1 This was by far the most effective purification step with a puri cation factor of8509 though previous puri cation steps were still necessary as they provided a way to remove components that would clog the column Though overload did not contribute much to enzyme loss it could be prevented by further diluting the 60 cut prior to loading and using a larger column with more AMPagarose beads e SDSPAGE Now that the enzyme has gone through several purification steps SDSPAGE is employed to evaluate progress ofpuri cation as well as determine subunit molecular weight ofLDH SDSPAGE is an electrophoresis technique that separates proteins based on size by use ofa porous acrylamide gel and electric eld An aliquot ofpurified LDH is denatured so that LDH exists as its individual subunits instead of a tetramer Since different proteins have varying charges negatively charged SDS is employed due to its ability to bind to proteins at a constant one SDS per two amino acids This negates the initial charge a protein might have held since all proteins will have a similar charge to mass ratio with SDS bound Since velocity is dependent on this charge to mass ratio which is made relatively constantwith SDS the proteins will separate by size There is an exponential relationship between distance traveled through the gel and molecularweight By running a set of known subunit Mr proteins a linear trend can be observed when plotting long versus Page 6 Graehl Heather Section 1C Group M00000 distance figure 4 The column puri ed LDH is interpolated to this graph in the linear range to obtain subunit molecular weight Two different isoforms might be present as 36598Da LDHA or 36724 Da LDHB SwissProtTrEMBL database Both isoenzymes are very similar size but since LDHB heart isoform is more negatively charged than the LDHA muscle isoform it would travel slightly further Lane 5 has a very faint single band while lane six is twice as concentrated and a second overlapping lower band can be observed gure 3 This could be due to the existence of LDHB in much lower concentration than LDHA so that the LDHB lower band only shows at higher concentration lnterpolating the LDHB band would not be very useful since the isoform separation due to charge difference and not size so it is not reliable to distinguish their molecular weights this way Gel stacking was already employed so to further enhance resolution a larger percent acrylamide resolving gel longer running time and standards closer in molecular weight to LDH could be used Other methods such as ion exchange chromatography HPLC or PAGE without SDS could be used better distinguish LDHA and LDHB forms The observed interpolated molecular weight of37931 Da table III is slightly higher than the 36kb isoenzyme published values One explanation is that the published values are unprocessed precursors while puri ed LDH will have glycosylation and other post transcriptional modi cations though this won t likely significantly increase the molecular weight Another explanation is that the Long versus Distance figure 4 relationship is truly exponential so the linear relationship is prone to error especially as standards become further from the molecular weight of the interpolated LDH The drawn best t line including BSA results in 37931 Da while neglecting this largest 66kb protein adjusts the interpolation of LDH subunit molecular weight to 36083 Da table III which is closer to published values In addition to determining subunit molecular weight SDSPAGE was also run with previous puri cation steps to determine if LDH is pure since speci c activity only provides a gage of relative increases in purity The column puri ed LDH appears pure as it shows a single band that can be matched in all previous puri cation samples gure 3 Crude extract is not possible to read though luckily this is the least important Page 7 Graehl Heather Section 1C Group M00000 lane Oddly the 60 pellet has several additional high molecular weight weak bands that the previous 40 supernatant step did not show though this can be explained since the 60 pellet is suspended in a tenfold smaller volume making these components more concentrated and weakly visible in 60 and not in the 40 lane figure 3 f GEC Similarly to SDSPAGE a set ofstandards are used to create a log Mr plot to interpolate a molecular weight ofLDH though GEC aims to determine a native molecularweight instead GEC uses a different mode ofsize separation in which porous beads target to separate intermediate molecular weight A linear range exists where these intermediate size standard proteins elute so LDH was interpolated to obtain a native molecular weight of 159294 Da Table IV Ferritin was removed in constructing a linear range since it has a molecular weight of 770000 Da which is far outside the resolution capability of5 250kD and arti cially increases the molecularweight determination to 200kD which is nowhere close to published values of 140kD Holbrook et al 1975 Dividing GEC native molecularweight by SDSPAGE subunit molecular weight gives a value of44 which corresponds to number ofsubunits in LDH Dividing published 140kD Holbrook et al 1975 and 36kb SwissProtTrEMBL database gives a value of39 which establishes LDH as a tetramer An additional modi cation to the GEC best t line figure 5 could improve comparability to published values through neglecting hemoglobin standard which would give a native Mr of 143244 Da and number of subunits to 40 Hemoglobin cannot be rejected based on being out of resolution though it does not fit well on the best t line Little was known about the condition standard proteins and they were not handled directly though it is possible the hemoglobin was degraded to smaller Mr causing time to elute to increase g Kinetics With a modi cation to enzyme assays performed in purification steps the kinetics of LDH and two inhibitors were investigated Purified LDH is assayed with varying pyruvate concentrations with and without inhibitors Inhibitors oxamate and oxalate are assayed at high and low concentrations to determine their identity and mode ofinhibition Page 8 Graehl Heather Section 1C Group M00000 Inhibitor A s double reciprocal plot identi es it as competitive inhibitor oxamate due to 1Vmax remaining relatively equal in the presence and absence of inhibitor gure 7 Oxamate binds to LDH preventing formation of ES complex which lowers the apparent Km At theoretical infinite substrate concentrations the inhibitor would be outcompeted to produce the same Vmax value Since Km is de ned as substrate concentrations at 12 Vmax the Km change is only apparent represented by d Kr ofoxamate calculated using 02mM and 004mM inhibitor concentrations and alpha to average a Ki disassociation constant of 149x10392 table V Inhibitor B s double reciprocal plot shows only uncompetitive nature even though this inhibitor must be oxalate which is mixed inhibition gure 8 Uncompetitive inhibition binds to enzymesubstrate complex making this complex more stable decreasing Vmax and thus an apparent Km decrease The slope VmaxKm remains constant at 0003 with both low and high oxalate concentrations indicating strong uncompetitive inhibition In order to observe the true mixed inhibition by oxalate lower than 004mM oxalate could be used to demonstrate its more competitive nature The Kr ofoxalate is solved to be 0154 table V Oxalate does not bind to ES complex as strong as oxamate binds to E based on Kr and Ki values though these cannot be compared directly since they act on different sites LDH binding affinity of ES to ES appears greater in oxalate than oxamate by observation of oxalate s lower Km values which is logical since oxalate stabilizes the ES complex reducing the k4 On the other hand oxalate has lower observed Vmax than oxamate which can be explained because available Etotal is lowered as oxalate binds and creates a stable ESl complex In oxamate increasing substrate can outcompete the inhibitor so that Vmax is unaffected though with oxalate increasing substrate does not hinder the formation ofan ESl complex thus Vmax is lower in oxalate Table V K2 or Kcai of LDH was determined to be 29400 catalytic events per enzyme site per second Table V h Purity Assessment The affinity column elution pro le gure 2 has one de ned elution peak but this doesn t necessarily mean it is pure since it could be possible for another component to bind to the NAD Page 9 Graehl Heather Section 1C Group M00000 pyruvate adduct GEC shows one peak corresponding to LDH elution gure 5 though a similar native weight protein in low concentration would not be detectable SDSPAGE is most useful when assessing purity because it gives suggestion that both LDHA and LDHB forms are present Lane 5 is puri ed LDH from elute fraction 11 and shows a faint single band Lane 6 is twice as concentrated and a second lower band appears due to low concentration LDHB figure 3 It is known that LDHB has a lower Km or higher binding af nity so LDHB would also bind to the elution adduct stronger than LDHA Thus elution fraction 10 likely has more LDHB while laterfractions like 12 and 13 likely have more LDHA This hypothesis can be con rmed by performing a PAGE without SDS on all elute fractions to compare strength ofbands To obtain complete purity of LDHA without any LDHB ion exchange chromatography could be performed as a nal puri cation step to separate the two based on their charge differences Page 10


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