Biochimica Sperimentale Serena Cucinotta 467345

Partialpurification,characterizationandkineticproperties ofLactateDehydrogenasefromrabbitliver
Lactate dehydrogenase (LDH; Llactate:NAD oxidoreductase, EC 1.1.1.27) is an essentialenzymeoftheanaerobicmetabolismofglucoseanditis ubiquitous among animals. LDH catalyzes the interconversion of pyruvate and lactate by the concomitant interconversionofNADHand NAD+.Whenoxygenisabsentorinshortsupply,this reactionregeneratesNAD+sotheglycolyticreactionscanoccur. PYRUVATE + NADH + H+
L-LACTATE

+ NAD+

LDHisahomoorheterotetrameroftwotypesofsubunits,HandM,whichareunder the control of two separate loci (Shaw and Barto, 1963). These loci are differently expressed depending on the tissue: the H subunit is predominant in myocardial tissues,whereasintheskeletalmuscletheMsubunitprevailsoverH;inrabbitliver theexpressionofthetwoformsispractically onbalanceandtheisoenzymeH2M2 is prevalent(Acerbaletal,1974). Thefiveisoenzymes(H4,H3M,H2M2,HM3,M4)showdifferentaffinitiesforthecommon substratesandadifferentallostericresponse. TheaimofthisprojectistoobtainapartialpurificationofLDHfromrabbitliverand tocharacterizesomeofitskineticproperties.

MATERIALSANDMETHODS
Sample:~10gofrabbitlivertissue. AssayBuffer:TRISHCl0.02MpH8.1. ExtractionBuffer:TRISHCl0.02MpH8.1with 1XPIC (ProteaseInhibitorCocktail, watersolublelyophilizedpowder,purchasedfromSIGMA). CibacronBlueAgaroseElutionSolutions: NAD+,2mMin16mMTRISHClBuffer,pH8withPIC NADH2mMin16mMTRISHClBuffer,pH8withPIC NaCl0,5MinMilliQH2O. Substratessolutions(inMilliQH2O): Sodiumpyruvate,30mM NADH,10mM SodiumLLactate,50mM NAD+10mM

Biochimica Sperimentale Serena Cucinotta 467345 BradfordProteinAssay Proteinlevelmeasurementswerecarriedoutaccordingto Bradford (1979), using 200 L of the 1X dye reagent (BioRad). A Bovine Serum Albumin(BSA,purchasedfromSigma)0.1mg/mlsolutionwasusedasastandard. Fig.1showsthecalibrationcurve,obtainedbyplottingtheabsorbanceat595nmwith fiveknownBSAamountsinduplicates,expresseding. LDHSpectophotometricAssay Basedontheknownmolarextinctioncoefficientfor NADHat340nm,itsoxidationtoNAD+ (whichdoesnotabsorbatthiswavelength) coincideswithadropinabsorbance.TheEU(EnzymeUnits)inthesamplescanbe easilycalculated. Thetemperaturewassetat37C. V ( A / min ) UE = ( dilution ) tot mL NADH V camp

(340=6,22103M1cm1)

[(

][ ]

StandardAssayMixtureVtot=1mL AssayBuffer:600L Pyruvate30mM:100L NADH10mM:20L LDHsample(Vcamp):10L H2OmilliQ:270L Thestandard mixture has been suitablymodified tofit kineticstudies, as detailed underResults.

Figure1.CalibrationcurvefortheproteinassayaccordingtoBradford(1979).

Biochimica Sperimentale Serena Cucinotta 467345

RESULTS
Homogenate.Tengramsofliverwerehomogenizedin3volumesof extractionbuffer (1:3w/v)withaPotterhomogenizerandthencentrifugedat10,000rpmfor1hat4 C. Thesupernatantwascollectedandmeasuredinaicecooledcylinderandlabeledas crudeextract(EG);100LofEGwereputasideforproteinandenzymeactivityassays (frozenat20C) Saltingout.TheEGwasfractionatedwithammoniumsulfate(AS)asfollows. Solid AS was added to the EG set on a stirring apparatus at 4 C to reach 35% saturation.Thesuspensionwasthencentrifugedat5,000rpmfor20min. Thepelletobtained(P35)wassuspended intheminimumvolumeof extractionbuffer. 50LofP35wereputasideforassays;thewholeP35fractionwasfrozenat20C. The supernatant obtained (S 35) was poured in a graduated cylinder (for volume measurement)andtheninacoolBeckeronthestirringapparatus,andaddedwithAS toreach70%saturation.Then,after20minsofcentrifugationat5,000rpm,thepellet wasresuspendedasabove(P70)andboththesupernatantandtheP 70weremeasuredin acylinderandthenfrozenat20C(50 Lofeachwere putinEppendorfftubes for assays). Gel exclusion chromatography A column was packed with Sephacryl200, a dextranpolyacrylamidegelthatseparatesproteinswithamolecularsizerangingfrom 5to250kDa;thegelwasequilibratedwith ExtractionBufferataflowrateofabout14 mL/h.Afterloadinga1mLaliquotofP70,fractionsof~2mLwerecollectedevery8 minovernightbyanautomatedcollector. Aftertheelution,80fractionswerecollected.Beginningfromthe20th,theabsorbance at280nm(A280)ofallfractionswasmeasured,inordertodeterminetheapproximate valueofproteincontent. Fractionswithhighopticaldensitywereassayedfor LDHactivity bythestandard assay. ThecurveshowninFig.2wasobtainedbyplottingtheA 280againstthenumberofthe fractions;inthesamegraph,usingasecondaryYaxis,theLDHactivityhadbeen plottedagainstthesameXaxis.Fractionswithhighactivity(fromn 49ton63)were mixedinapool(PoolS200). 2,5
2 1,5
Abs

0,9 0,6

0,75 0,45 0,3 0,15 0 20 30 40 50 60 70 80 90

#fractions A/min

1 0,5 0

Figure2ElutionprofileoftheS200chromatography.Absorbance=280nm; LDHactivity(A/min) 3

Biochimica Sperimentale Serena Cucinotta 467345 PseudoaffinityChromatography AsmallcolumnwaspackedwithafewmLof CibacronBlue 3GA Agarose, that is often used for purified dehydrogenases (M. Jahanshahietal,2006).Thebluegelwasequilibratedwith ExtractionBufferataflow rateof~16mL/h. TheS200poolwasloadedontheCibacronBluecolumnandfractionswerecollected immediatelyevery4minbyanautomatedcollector.Aftercompleteadsorptionofthe sample, elution was carried on with the Extraction Buffer until A280 of the eluted fractionsdropto<0.2AU.Then,thefollowingeluentswereadded(intheorder): 2mMNAD+(5mL) ExtractionBuffer(5mL) 2mMNADH(5mL) ExtractionBuffer(5mL)

TheA280ofallfractionswasmeasured,andthismonitoredtheproteincontentinthe firstphaseoftheelutionwiththe ExtractionBuffer.Thereafter,theabsorbancewas mainlyduetothecoenzymes. TheLDHactivitywasassayedinallfractionsafterNADHloading.Bothvalueswere plottedtogetherandFig.3showstheresultantplot. Highactivityfractionswerereunitedintwopools: Pool1V1=2,6mL Pool2V2=1,8mL

ThesepoolswerethenassayedforLDHactivityinordertodeterminatetheUE/mL. This step is important to evaluate the quality of purification. ( See the Purification TableTable2) Thepoolsweredividedinaliquotpartsof500 Leachandstoredat20C,forkinetic analysis. Theproteinconcentrationofallsampleswasmeasuredwith BradfordProteinAssay,as previouslystated.
3,5 0,7 3 0,6

2,5

0,5

0,4

1,5

0,3

0,2

0,5

0,1

0 0 10 20 30 40 50 60 70

Figure3ElutionprofileoftheCibacronBluepseudoaffinitychromatography.LeftY axis=A280 ();rightYaxis= A/min();Xaxis=fractionnumber. Note: ALL A280 VALUES EXCEEDING 3AU, DUE TO THE PRESENCE OF NICOTINAMIDE COENZYMES, WERE REPORTEDONTHEGRAPHAS=3AU. 4

Biochimica Sperimentale Serena Cucinotta 467345 The PurificationTable,shownin Table1,summarizestheresultsofthepurification procedure. Therelevantvaluesare: Specificactivity(AS= PurificationFactor(
UE ) mg

AS sample ) AS EG U sample ) U EG

Yield%ofpurification(

Volume (mL) Prot. (mg/mL) Tot. Prot (mg) Crude Extract 13 53,25 692,25 P35 5 34,02 170,1 P70 4 40,94 163,76 Pool S-200 23 1,37 31,51 Pool 1 2,6 0,212 0,551 Pool 2 1,8 0,112 0,202

UE/mL 149,5 38,26 123,7 2,68 33,78 12,88

UE tot AS 1943,5 2,81 191,3 1,13 494,8 3,02 61,64 1,96 87,83 159,34 23,18 115

Yield % Purif. Factor 100 1 9,84 0,4 25,46 1,07 3,17 0,69 4,52 57,71 1,19 40,93

Table1Summaryofdatafrompurificationprocedure SDSPAGE For electrophoresis, 10% polyacrylamide gels (Stacking gel and SeparatinggelLaemmlisystem)werepreparedbyastandardprotocol. Thefollowingsamplesmixedwithsamplebuffer(SB),togetherwithmolecularsize markers,werethenloadedandthegelwasrun. 1. 10,7LofPoolS200(+4,3LofH2OMilliQ)+3,75LofSampleBuffer(SB) 2. 20LofPool1+5LofSB 3. 20LofPool2+5LofSB SamplesandstandardswereloadedintheordershowninFig.4.

Figure4LoadingpatternforSDSPAGE. After running for 1 h at a constant current of 40 mA, the gel was stained with CoomassieBrilliantBlue(about45minwithgentlestirring)andthendestainedwith 50%methanol. 5

Biochimica Sperimentale Serena Cucinotta 467345 Thestandards(the molecularsizemarker,used toidentifytheapproximatesizeofa molecule run on a gel) were not clearly visible (Fig. 5). The LDH subunit can be identifiedasthemainbandthatappearsinthelanesloadedwithsamples2and3, althoughthisidentificationisonlytentative.

Figure5ImageoftheSDSPAGEgelobtainedbyascanner.

CharacterizationofsomekineticpropertiesoftheLDHpurifiedfromrabbitliver.

To characterize the enzyme preparation, its kinetics was studied experimentally to determineits KMandVmaxvaluesforallfoursubstratesintheforwardandbackward reaction.Forbothreactions,Pool1wasusedtomonitorLDHactivityasafunctionof [S], and the data were used to draw MichaelisMenten plots and LineweaverBurk plots. ForwardReaction Pyruvate The LDH kinetic analysis relative to this substrate was performed measuringtheinitialvelocityofthereaction,inmixtureswith10 LofLDH(1:20 dilutionofPool1)andincreasingconcentrationsofpyruvate(from0.025mMto0.75 mM),butatfixedconcentrationofthecoenzyme(0,2mM).

Figure 6 - Pyruvate Michaelis-Menten plot.

Figure 7 - Pyruvate Lineweaver-Burk plot.

Biochimica Sperimentale Serena Cucinotta 467345 NADHThiskineticanalysiswasperformedatafixedpyruvateconcentration(0.8 mM)andvaryingconcentrationsofNADH(from0.02mMto0.26mM).

Figure 8 - NADH Michaelis-Menten plot

Figure 9 - NADH Lineweaver-Burk plot

BackwardReaction Kineticstudiesonbackwardreactionrequireahigherconcentrationofenzyme(about 10timeshigher),becausethekcatofLDHforthisreactionismuchlowerthanthe kcatin theforwardreaction.Forthisreason,assayswererunwith100 LofPool1.This samplewasdiluted1:100topreventaninhibitionbytheNADHinsidetheenzyme preparation. LLactateKineticassaysonthissubstratewererunatfixedNAD +concentration(0.9 mM)andvaryinglactateconcentrations(from0.5mMto10mM).

Figure10LLactateMichaelisMentenPlot.Figure11LLactateLineweaverBurkplot.

Biochimica Sperimentale Serena Cucinotta 467345 NAD+ Assaysonthiscoenzymewererunatfixedlactateconcentration(10mM)and variousincreasingconcentrationofNAD+(from0.05mMto1mM).

Figure 12 - NAD+ Michaelis-Menten plot

Figure 13 - NAD+ Lineweaver-Burk plot

Table2summarizestheKMandVmaxvaluesobtainedfromeachsetofkinetic measurements.

KM (mM) Pyruvate NADH L-Lactate NAD+ 0,5925 0,229 0,0276 0,008 2,085 1,87 0,135 0,234

Vmax (mmol/min) 0,4073 0,09 0,073 0,008 0,0178 0,006 0,025 0,013

Table2SummaryoftheKMandVmaxvaluescalculatedfromthelinearregression bestfitsinFigures7,9,11,13.

Biochimica Sperimentale Serena Cucinotta 467345 DISCUSSION Thefinalgoaloftheexperiment,namelyapartialpurificationofLDH,wasreached, although at intermediate steps the specific activity did not increase as expected. However,duringthe procedure, an unaccountabledropinprotein contentoccurred, particularly after the exclusion chromatography step (as shown in Table 2). It is possiblethatthereisacorrelationbetweenthislossandthelowvaluesforspecific activity(therehasbeenalsoalossofenzymeunits).Thefinalstepseemsnevertheless successful, since it gave roughly an 80fold increase in specific activity over the previousstep. TheresultofSDSPAGEcanbehardlyevaluated,sincethemolecularsizemarkers werenotvisible,thushinderingtheevaluationofthemolecularsizeofthebandsin sample2and3.Interestingly,however,bothsamplesshowtwoprominentbands:itis possibletosuggestthatadifferentNADHdependentdehydrogenase,withadifferent subunitmolecularmass,wasisolatedtogetherwithLDH. ThekineticcharacterizationofLDHisalsotoconsidersuccessful.Someresultsare comparabletothosereportedintheliterature.TheBrendadatabase( http://www.brenda enzymes.info/) gives KM valuesof0.67mMforpyruvate(rabbitenzyme)and3.3mMfor lactate(pigenzyme). Moreover, substrate inhibition due to pyruvate was observed at concentration >0.6 mM,asdescribedbyBattellinoetal(1968). Thiskineticfeaturewasnotdeliberatelyinvestigated,butsomeofthedataobtained couldnotbeusedintheplotsofFigs.6and7sincetheydeviatedsystematicallyfrom Michaelis kinetics. All the data are instead plotted in Fig 14 as percent maximal activityagainsttheconcentrationofpyruvate,toshowthispeculiarkineticbehaviour.

Figure14 SubstrateinhibitionofrabbitliverLDHbyincreasingconcentrationsofpyruvate.

Biochimica Sperimentale Serena Cucinotta 467345 REFERENCES ShawC.R.,E.Barto(1963).Geneticevidenceforthesubunitstructureoflactate dehydrogenaseisozymes.PNAS196350(2)211214 Acerbal,C.,J.Castro,E.Melndez,andA.M.Municio(1974).Metabolicinfluenceson thelactatedehydrogenaseisoenzymepatternsinseveralrabbittissues. Int.J. Biochem.,5:2329. Battellino,L.J.,F.R.Jaime,andA.Blanco(1968).KineticPropertiesofRabbit TesticularLactateDehydrogenaseIsozyme.J.Biol.Chem.243:51855192. Bradford,M.M.(1979).Arapidandsensitivemethodforthequantitationof microgramquantitiesofproteinutilizingtheprincipleofproteindyebinding. Anal. Biochem.72:248254. Latner,A.L.,andA.W.Skillen(1964).Lactatedehydrogenaseisoenzymesinfoetal andneonataltissues.J.Embryol.Exp.Morph.12:501509. Pettit,S.M.,D.A.Nealon,andA.R.Henderson(1981).PurificationofLactate DehydrogenaseIsoenzyme5fromHumanLiver.Clin.Chem.27:8893. Scopes,R.K.(1977).PurificationofGlycolyticEnzymesbyusingAffinityElution Chromatography.Biochem.J.161,253263. Tymoczko,J.(s.d.).PurificationofLacticAcidDehydrogenase(LDH).Updatedby RebeccaBryant. http://www.acad.carleton.edu/curricular/BIOL/classes/bio380/lab_protocol.htm. Vesell,E.S.,J.Philip,andA.G.Bearn(1962).ComparativeStudiesoftheIsozymesof LacticDehydrogenaseinRabbitandManJ.Exp.Med.116:797. JahanshahiM.,T.C.Ling,A.GhoregshiandM.Khavarpour(2006)Analysisof performanceoftheanionexchangeandpseudoaffinitychromatographyfor intracellularenzymepurification.IranianJournalofChemicalEngineering,3:92107.

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