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EXAM REVIEW - AMAZING This is one of the best reviews I've ever made... it not only contains the background info for each of the 6 labs, but it also includes explanations of certain steps in the procedure, as well as what is SUPPOSED to happen for results

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Miguel Rodriguez

BIOCHEMISTRY LAB EXAM REVIEWLAB 1 ENZYMATIC CHARACTERIZATION OF HEART TRANSAMINASES BACKGROUNDTransaminases catalyze the transfer of an amino group from one amino acid to a keto acid The reaction occurs without yielding free NH3 the amino group is retained through the transfer to the vitamin B6derivative pyridoxal phosphate PLP a prosthetic group present within the transaminase The reaction involves the formation of a Schiff base intermediate between the amino acid and the PLP GarrettRH and Grisham CM 2004 GlutamateOxaloacetate Transaminase GOT and GlutamatePyruvate Transaminase GPT are responsible forthe transfer of an amino group from glutamate to oxaloacetate and pyruvate respectivelyThe transamination reaction is reversible Go0 and can be initiated from either direction by supplying theappropriate substrates The equilibrium constant of this reversible reaction can be determined by measuring the concentration of eachcomponent at equilibrium Transaminases play an important role in amino acid metabolism1 deamination most amino acids transfer their amino group to ketoglutarate to form glutamate 2 The glutamate is converted by the action of GOT into aspartate a precursor of the urea cycle 3 The second amino group in the urea comes from the deamination of glutamate through the glutamatedehydrogenase reaction urea excretion is the pathway used for elimination of nitrogen by most terrestrial vertebrates In the muscle GPT is responsible for the conversion of pyruvate into alanine which is delivered to the liver whereit is used for glucose synthesis The amino group of alanine ends up in the urea cycle Ratner S 1977Voet D and Voet JG 2004 Other transaminases are involved in the transfer of the amino group from glutamate to different keto acidprecursors of some amino acids synthesis Transaminases are intracellular enzymes and are present in high concentrations in muscle and liverUnder conditions of extensive tissue damage necrosis and cell breakdown such as hepatitis andmyocardial infarction these enzymes leak into the blood stream Thus high serum levels of GPT and GOTare indicators of those diseases COUPLED ASSAY OF TRANSAMINASESAssays of transaminase activity are usually performed in the direction which generates pyruvate GPT oroxaloacetate GOTbecause it is easy to measure pyruvate using the coupled oxidation of NADH to NADIn an incubation buffer with an excess of NADH and lactate dehydrogenase LDH malate dehydrogenase MDHin the case of GOT assay the pyruvate produced by the GPT transferral of NH2 from alanine to ketoglutarate is reduced to form lactate This reduction can be measured by the decrease in 340 nmabsorbance of NADH upon oxidation to NADAt high levels of NADH and LDH the conversion of pyruvate into lactate is not rate limiting and the rate ofNADH oxidation represents the rate of pyruvate production in the GPT catalyzed reaction Since the LDH reaction is irreversible pyruvate is removed and the GPT reaction becomes unidirectional As long as NADH is in excess the reaction proceeds till complete depletion of either substrate KG or alanine Usually the substrates of the first reaction KG and alanine are at saturating concentrations and thusthe initial velocity of the reaction corresponds to the Vmax of the transaminase Similarly for the GOTMDH system this mating of two or more enzyme reactions to make use of an easilymeasured component to measure more difficultly measured compound is known as a COUPLED ASSAYIn some instances the mitochondrial enzyme glutamate dehydrogenase GDH can interfere with this coupledassay by using NADH for the reduction of ketoglutarate to glutamate PROTOCOL AND RESULTS EXPERIMENT 1 ISOLATION OF GOT FROM HEART HEART PREPARATIONpotassiummaleate EDTA buffer Polytronfor homogenization physical breakup of tissueHEAT DENATURATIONGOT is resistant to heat inactivation especially in the presence of alphaketoglutarate therefore heat shockdenatures other enzymes in the protein sample including GPT odone by incubation at 55C with the addition of alphaketoglutarate cooling and centrifugation used to obtain supernatant with protein sampleDEVELOPMENT OF SALT FRACTIONATIONammonium sulphate usedfirst precipitation contaminating proteins precipitate while GOT remains in solutionsecond precipitation with more salt the desired enzyme is precipitated where other proteins remain in solution the optimal salt concentrations for two successive precipitations are empirically determined through enzymeactivity and salt concentrationsavoid foaming the solutions because this introduces oxygen into solution which denatures the proteins PROTEIN DETERMINATIONDone through Bradford Coomassie Bluesee Lab 5KINETIC ASSAY OF TRANSAMINASE ACTIVITYkinetic assay determines how much functional enzyme in this case GOT is in the samplesdone through coupled assayLaspartate MDH and phosphate buffer with NADH and transaminaseGPT also assessed to measure functional GPT done through coupled assayLalanine LDH and phosphate buffer with transaminasenote that the homogenate is the sample that was saved before heat and salt treatmentSamples created GPT SAMPLE Pure Homogenate Heat ASSAY enzyme treatment DILUTION 110 110 ACTIVITY Highest Hightrace GOT SAMPLE Pure Homogenate Heat HeatHeatHeatHeatHeatASSAY enzyme treatment 50 60 70 80 85 salt salt salt salt salt DILUTION 110 110 12 14 14Table IGOT enzymatic assay analysis using different ammonium sulfate salts saturations resulting in different activities of GOT Tube Salt Saturation Activity Activity umol of NADHmin 1 NA 3600100002 50 109 304 3 60 139387 4 70 6671860 5 80 18405110 6 85 16704650SUMMARY OF RESULTSGPT ASSAYPURE ENZYMEcontrol high activity HOMOGENATEactivity shows that active GPT is in sample HEAT TREATMENTvery trace amounts shows that heat treatment deactivates GPTGOT ASSAY PURE ENZYME control high activity HOMOGENATEactivity HEAT TREATMENTactivity 100 activity HEAT TREATMENTSALTwith increased salt concentration comes increased activity but slightdecrease after 85 optimal saturation is 80however highest specific activity was through 50 saturation 1680Figure 1Percents of protein activity and specific activity as measured by the 1470GlutamateOxaloacetate Transaminase 1260 GOT coupled assayGOT activity and 150Spec protein percentage were calculated as a0840activitpercentage where tube 1 was 100protein 0630Specific activity was obtained by dividingy or0420activity byprotein Theprotein values activity were obtained from the Bradford Protein 0210Assay00 5060708085 saturation Sample calculation for activity Ae x l x c
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