Assayofsuperoxidedismutaseactivitybycombiningelectrophoresisanddensitometry

Abstract.Amodifiedtechniquewasdevelopedtoassaysuperoxidedismutase(SOD)activitybycombiningpolyacrylamidegelelectrophoresisanddensitometry.Afterelectrophoresisonnativepolyacrylamidegels,thenegativebandingcorrespondingtotheSODactivitywasvisualizedbysoakingthegelsinnitrobluetetrazoliumthenriboflavin,andfinallyexposingtolight.EffectsofthebandingofSODactivityinducedbydifferentsoakingdurationsandlightintensitieswereevaluatedinthissystem.Theoptimalsoakingdurationwasdeterminedtobe15minforeachofthetwosoakingsteps,whiletheoptimalexposurewas30mEm-2s-1for15min.Thegelswerethenimmediatelyscannedwithalaserdensitometer,andthereADIngsofthesamplescorrespondingtotheirtotalSODactivitywereobtainedbyprocessingtheimage.AstandardcurvewaspreparedwithaserialdilutionofpartiallypurifiedSOD,whoseactivitywaspreviouslydeterminedbyusingaspectrophotometricmethod.ThetotalSODactivityofanunknownsamplecouldbeobtainedbyinterpolatingitsreadingtothestandardcurve.TheactivityofasingleSODisozymeofasamplecouldalsobeobtainedwiththesameprocedure.Thetechniquewastentimesmoreefficientthanthespectrophotometricmethod.Theinterferencecomingfromnon-SODsubstancesinthecrudeextractcouldberemovedbyelectrophoresis.ThestandarddeviationsoftheSODactivityofthecrudeextractsfromriceseedlings,papaya,andtobaccoleavesmeasuredwiththetechniquewerelessthan9%,7%,and8%(foreachn=6,on6gels),respectively.

Keywords:Densitometry;SODisozyme;Superoxidedismutase.

Abbreviations:DETAPAC,diethylenetriaminepentaaceticacid;ED,electrophoretic-densitometry;NBT,nitrobluetetrazolium;SOD,superoxidedismutase;TEMED,tetramethylenediamine.

Introduction

Superoxideradical(•O2_)isgeneratedasaby-productinaerobicorganismsfromanumberofphysiologicalreactionssuchastheelectronflowinthechloroplastsandmitochondriaandfromsomeredoxreactionsincells.Itcanreactwithhydrogenperoxide(H2O2)toproducehydroxylradical(•OH_),oneofthemostreactivemoleculesinthelivingcells.Hydroxylradicalcancausetheperoxidationofmembranelipids,breakageofDNAstrands,andinactivationofenzymesincells(forreviews,seeBowleretal.,1992;Mehdy,1994).Toamelioratethedamagecausedbyhydroxylradicalformedfromsuperoxideradicalandhydrogenperoxide,organismshaveevolvedmechanismstocontroltheconcentrationofthetworeactants.Superoxidedismutase(SOD,EC1.15.1.1)isagroupofisozymesfunctioningassuperoxideradicalscavengerinthelivingorganisms.ThereactionofSODisasfollows:2H++2•O2_®H2O2+O2

theproducedhydrogenperoxideisthendetoxifiedbycatalaseorperoxidase.

TheexpressionofSODgenesisregulatedbothspatiallyanddevelopmentallyatleastinmaize(ZhuandScandalios,1993)andrice(unpublisheddata).SODactivityisalsoinducedbydiversestresses(Bowleretal.,1992),presumablybecauseoftheincreaseintheconcentrationofsuperoxideradicalincellsunderthoseconditions.Obviously,SODisanimportantenzymefamilyinlivingcellsformaintainingnormalphysiologicalconditionsandcopingwithstress.However,thestudyofSODgeneexpressionregulationattheendproductlevelhasbeenhandicappedbythelackofaconvenientmethodforquantifyingtheactivityassayoftheisozymes.MostoftheworkconcerningSODgeneexpressionregulationhasbeenbasedonRNAgelblotanalysis.LittleisknownabouttheactivitychangeoftheSODisozymesofplantsinthedevelopmentalcourseandinresponsetostresses.SODactivityiscommonlyassayedspectrophotometrically,e.g.,themethodfirstdefinedbyMcCordandFridovich(1969)andmodifiedbyOberleyandSpitz(1985).Butitisbothlabor-intensiveandtime-consuming.WehavedevelopedaconvenienttechniqueforassayingtheSODactivitybycombiningelectrophoresisanddensitometryandhavecalledittheEDscheme.ThistechniqueisbasedonBeauchampandFridovich"smethod(1971),butisquantitativeratherthanqualitative.TheEDschemeismorethantentimesmoreefficientthanthespectrophotometricmethod.TheSODactivityofunknownsamplescanbederivedfromcomparingwithaSODstandardwhoseactivitywasmeasuredbythespectrophotometricmethod.Inadditiontoitsefficiency,thetechniquecanassaytheactivityofasingleSODisoformonthepolyacrylamidegelandexcludetheinterferencecomingfromnon-SODmoleculesinthetissuecrudeextract,whichisnotpossIBLeusingthespectrophotometricmethod.

MaterialsandMethods

Nitrobluetetrazolium(NBT),diethylenetriaminepentaaceticacid(DETAPAC),xanthineandxanthineoxidasewerepurchasedfromSigma.Riboflavinandtetramethylenediamine(TEMED)werepurchasedfromServaChemicalCompany.

ForthepreparationofsamplesfortheassayofSODactivity,3gofriceseedlings,tobaccoandpapayayoungleaveswereharvestedandfrozenwithliquidnitrogen,groundintoafinepowder,andthenmixedwith3mlofextractionbuffer(0.15MTris,pH7.5).Thesampleswerecentrifugedat4°C,14k×gfor10min.Centrifugationwasrepeated2_3timestoclearallthedebris.Thesupernatantwasthentransferredintomicrotubes,storedat-20°C,andcentrifugedagainbeforeuse.SODstandardwasapartiallypurifiedmungbeanSODobtainedfromtheKingCarFoodIndustrialCooperation.

Electrophoresiswascarriedoutat4°CaccordingtoamodifiedprocedureofGabriel(1971)with1.5mmof10%polyacrylamidemini-slabgelinstandardtris-glycinebuffer(pH8.3).Sampleswereloadedintoeachwellandthenelectrophoresedat80Vthroughthestackinggelfor15minand120Vthroughtheseparatinggelfor60min.Afterelectrophoresis,amodifiedphotochemicalmethodofBeauchampandFridovich(1971)wasusedtolocateSODactivitiesongels.Thegelwasfirstsoakedin25mlof1.23mMNBTfor15min,brieflywashed,thensoakedinthedarkin30mlof100mMpotassiumphosphatebuffer(pH7.0)containing28mMTEMEDand2.8×10-2mMriboflavinforanother15min.Thegelwasbrieflywashedagain,andthenIlluminatedonalightboxwithalightintensityof30mEm-2s-1(measuredbyLI-CORLI1000)for15mintoinitiatethephotochemicalreaction.Alltheprocedureswerecarriedoutatroomtemperature,andthetwosoakingstepswereshakenat75rpm.

Thegelwasscannedwithalaserdensitometer(MolecularDynamics)immediatelyafterthephotochemicalreaction.TomeasurethetotalSODactivity,eachsamplelanewasindividuallyframedwithasuitablesizerectanglewhenprocessingtheimageonthecomputer,ensuringthateverySODbandofalanefellintotherectangle.Inadditiontothesamplerectangles,anumberofblankrectangleswereframedonthesamegelforcalibratingthebackgroundofthesamplerectangles.ThereadingcorrespondingtotheSODactivityinonesamplerectanglecouldbecalculatedaccordingtotheformula:ReadingofSODactivity=(averagepixelreadingoftheblankrectangles)×(thepixelnumberofthesamplerectangle)_(thetotalreadingofthesamplerectangle).TheoutcomerepresentsadifferentdegreeofstainingresultingfromthetotalSODactivityofeachsample.AsingleSODisoformcanbeprocessedinasimilarwaybyframingthelocationofthedesiredisoformasthesamplerectangle.

SpectrophotometricassayofSODactivitywascarriedoutbyadaptingtheprocedureofOberleyandSpitz(1985).Fora20-cuvetteassay,thefollowingreagentswereaddedtoatesttube:13.8mlof50mMpotassiumphosphatebuffer(pH7.8)containing1.33mMDETAPAC;0.5mlof2.45mMNBT;1.7mlof1.8mMxanthine.Thetotalvolumewas16ml,enoughtodispense0.8mlintoeachofthe20cuvettes.

CuvetteholdersinthesamplechamberofthespectrophotometerwereThermo-controlledat25°C.Fortheblanktest,100mlof50mMpotassiumphosphatebuffer(pH7.8)wasaddedintoacuvettewith0.8mlofworkingsolution.Asuitableconcentrationofxanthineoxidasewasdiluted,and100mlofitwasaddedintothecuvettetoinitiatethereaction.Alinearcurvewithaslopeof0.025absorbanceperminintimescanwasobtainedbyadjustingtheconcentrationofxanthineoxidase.Thetimescanlasted5min.ThephosphatebufferwasthenreplacedbyaserialdilutionofaSODsampletoobtaindifferentdecreasedslopes,andeachreactionwasperformedatleasttwice.AtleastthreedilutionsofaSODsamplewerescannedinthedefinedreaction,oneforthemaximumcompetition(i.e.,thelowestslopethatcouldbeobtainedbyaddingthesampleintothereaction),theothertwoweredilutedtoapproachhalfofthemaximumcompetition.Theslope-sampleamountdatawerethenconvertedtoaplotofslopeagainstsampleamount.Maximumcompetition(lowestslope)andalinearcurve,includingthepointfortheblanktest,werethetwoelementsintheplot.Aregressionfunctionwasobtainedforthelinearcurve.Thesampleamountcorrespondingtohalfofthelowestslopewasobtainedthroughinterpolatingorextrapolatingtotheregressioncurve.OneunitofSODactivitywasdefinedastheamountofSODwhichproducedonehalfofthemaximumcompetitionagainstNBTinthespecifiedsystem.

Results

Thetwo-stepsoakingofthegelwasfollowedbyillumination.Thesoakingperiod,illuminationduration,andlightintensitywerecriticalfactorsindevelopingthenegative-stainedbandsofSODisozymes.Inordertosavetimewhilehavingwelldevelopedgels,thetimecourseexperimentwascarriedoutforthetwosoakingstepsandtheillumination.Theresultsindicatedthat15minforeachsoakingandilluminationfor15minwithalightintensityof30mEm-2s-1forphotochemicalreactionweresuitableforthecolordevelopment.

TheEDschemewasdemonstratedwithfourplantsamples.AsshowninFigure1,thereadingswereproportionaltotheamountofenzymepreparations,andthecurveofthepartiallypurifiedmungbeanSODwasusedasastandard.ThecurveofmungbeanSODislinearbelowthereadingof1,300.Similarresultswereobtainedfortheotherthreesamples,however,withawiderlinearrange.

Interferences,includingpossibleendogenousSODinhibitorsandagentscausingthereductionofNBTintheplantcrudeextract,couldberemovedbypolyacrylamidegelelectrophoresis.Oneoftheinterferencescomingfromnon-SODsmallmoleculeswasdetectedasthesmearsatthebottomsofpapayaandtobaccolanes(Figure2).These

Table1.ComparisonofSODactivitiesofthreeplantsamplesassayedviatheelectrophoresis-densitometricmethod(EDscheme)andbythespectrophotometricmethod.

SODactivity(unit/ml)

CrudeextractByEDschemeBythespectrophotometricmethod

Riceseedlings2.081.19

Papayaleaves2.482.03

Tobaccoleaves1.132.02

Foreachofthethreesamples,n=6on6gelsassayedviatheEDscheme.Thereadingsobtainedbydensitometrywere1001.5±87.44,1066.5±68.02,and820.5±59.39(mean±SD)forrice,papayaandtobacco,respectively.Inthespectrophotometricmethodgroup,theassaywasperformedtwiceforeachofthesamples.

interferencescouldresultininaccuratereadingsofSODactivityinthespectrophotometricmethod.ComparisonsofplantSODactivitiesmeasuredwiththeEDschemeandthespectrophotometricmethodareshowninTable1.

Discussion

SODactivityhasbeendeterminedspectrophotometricallyforovertwentyyears—eversinceitsfunctionwasfirstunderstood(McCordandFridovich,1969).Becausesuperoxideradicalisunstable,itisdifficulttodeterminetheactivityofSODbydependingonthedynamicconcentrationofthesubstrate.Dependingontheproductlevelisalsonotfeasiblebecausehydrogenperoxideisalsonotstableinthepresenceofcertainmetalions.Catalaseandperoxidaseexistinginthetissuecrudeextractcanalsointerferewiththeresult.Inthisspectrophotometricmethod,xanthineoxidaseandxanthinewereusedtogenerateasuperoxideradicalflux,whilecytochromecfunctionedasacompetitoragainstSODonthesuperoxideradicalandasacolorindicator.CytochromecwaslaterreplacedbyNBTbecausetheresultcouldbeinterferedwithbysomeenzymessuchascytochromeoxidaseandcytochromeperoxidaseexistinginthetissuecrudeextract(BeauchampandFridovich,1971).Themethodissensitive,butwithsomedisadvantages.First,itistime-consuming.Ineighthoursofwork,aboutfoursamples"SODactivitycanbedetermined(fivedatapointsfortheplotofslopeagainstsampleamountwitheachdatapointperformedtwice).Second,theactivityofthedilutedxanthineoxidase,whichinitiatesthereaction,decreasesduringtheworktime,affectingtheprecisionoftheslopedatafromthetimescan.Theslopeoftheblanktestintheassay,whichmeasuresthesuperoxideradicalfluxgeneratedbythexanthineoxidase,iscriticaltoobtainingtheactivityunitofaSODsample.Thelowertheslope,thelesstheunit.ThesameeffectwasassumedtoexistinthereactionswhenSODsampleswerescanned.Thefinaldisadvantageofthetraditionalspectrophotometricmethodisthatitcannotexcludepossiblemoleculesinthesampleextract,whichcaninterferewiththedeterminationofthegenuineactivityofSOD,contributingtoanincorrectestimationoftheSODactivity.

Figure1.CurvesofdensitometricreadingscorrespondingtoSODactivityagainstproteinquantitiesofthefourplantsamples.ThefoursampleswerepartiallypurifiedmungbeanSOD,andcrudeextractsofriceseedlings,papaya,andtobaccoleaves.AfterelectrophoresisandSODactivitystaining,thegelswerescannedwithalaserdensitometer.Thereadingswereobtainedbyprocessingtheimages.InthecurveofmungbeanSOD,foreachdatapointn=4,8,12,12,8,and4fromlowtohighreading,respectively.Allthedatapointsinthecurvesofrice,papaya,andtobaccowereperformedfourtimesinfourpolyacrylamidegels.

Figure2.ElectrophoresisandnegativestainingofSOD.Lane1_5areserialdilutionsofpartiallypurifiedmungbeanSOD.Lane4and5areduplicated.Lane6_8arecrudeextractsofriceseedlings,papayaleaves,andtobaccoleaves,respectively.Atthebottomofpapayaandtobaccolanestherearedifferentlightyellowsmears,presumablycausedbyinterferencemoleculesinthecrudeextracts.

BotanicalBulletinofAcademiaSinica,Vol.37,1996

ThemethodusingNBTasasuperoxideradicalcompetitorandacolorindicatorwasalsoexploredtoqualitativelylocateSODonpolyacrylamidegelsatthetimewhencytochromecwasreplacedbyNBT.IntheNBTnegativestainingsystem,afterthegelshavebeensoakedwithNBTthenriboflavin,exposingthemtolightcausestheriboflavintogenerateasuperoxideradicalfluxinthepresenceofoxygenandTEMED.NBTandSODinthegelscompeteforthesuperoxideradicalatthesametime.AtthelocationswhereSODexists,thegelremainstransparent,incontrasttothoseareaswithoutSODwherethegelbecomespurple-blueduetoreducedNBT.Althoughthestainingsystemwassetupmorethantwentyyearsago,ithasnotbeenmodifiedandoptimizedtoquantifySODactivity.OurexperienceandsomereportsshowedthepotentialtomodifythequalitativeSODstainingsystemtoquantifytheactivityofSOD.TheisozymesoftheSODfamilycanbeseparatedbyelectrophoresis,theirbandingintensitiesareproportionaltotheloadedamountofSODsamplesinfixedconditions,andthedevelopedgelscanbeeasilyscannedwithalaserdensitometer.ThesethreefactorsprovidedthebasisforassayingtheactivityofthetotalorasingleSODisozymeonagelwiththeEDscheme.Furthermore,inthemini-slab-gelsystem,torunfourgelswhichhavetenwellseachatthesametimeiseasy.Thisprovidedtheefficiencyforthetechnique.LoadingasuitablequantityofsampleineachwellisacrucialfirststepintheEDscheme.Ifthesampleisoverloaded,someSODisozymewithhigheractivitywillbeunder-estimatedduetothesaturationeffect.Thus,theloadedsampleamountshouldfallintothelinearrangeofboththestandardcurveandthesamplecurve.BecausethecurvesofcrudeextractsshowedawiderlinearrangethanthatofpurifiedSODduetothepresenceofmoreisozymes,readingsofsamplesfallinginthelinearrangeofthestandardcurvepreparedwithapurifiedSODareconsideredtobesafe.However,thevariationofthereadingswasalsoquitehighwhentheloadedamountwaslow.Inthiswork,thesuitablerangefortheinterpolationwasbetween300and1,300.

TheintensityofSODbandswasaffectedbythe2-stepsoakingandilluminationprocedure.Thelongerthesoakdurations,thedarkerandblurriertheSODbands.SomeSODminorbandsdisappearedduetoaprolongedduration(40minutes)ofsoakforeachofthetwosteps.Forreasonsthatarestillunclear,differentSODisozymeswereobservedtohaveadifferentresistancetothesoakingeffect(datanotshown).

Intheilluminationstep,thelightintensityofthelightboxisveryimportant.AhighlightintensitycausesagreatoutputofsuperoxideradicalfluxfromtheriboflavinandTEMED,resultinginarapiddarkeningofthegel.Therefore,allbandsmightdisappearunderhighlightintensities.Lowlightintensitiesarelesseffectiveincolordevelopmentandrequiremoretime;moreover,theyaresuseptibletointerferencefromanenvironmentallightsource.Accordingly,30mEm-2s-1wasdeterminedtobesuitablefortheillumination.

Inthedensitometry,100micronperpixelsizeand12bitsofdigitalresolutioninthescanningsystemwereused.Thesamplelaneswereframedassmallaspossible.Thebackgroundofthegelswerenotevenespeciallyatthemarginalareaofthegel,potentiallycontributingtoavariationinthereadings.Ideally,blankframesshouldbelocatedontheupperandlowerpartsofthelanestogiveabetterrepresentationofthebackground.Itisnotknownwhetherexposingthegeltothelaserbeamduringthescanningcauseschangetothestaining.Nevertheless,fewgelsneededtobescannedtwice.

Theoretically,measurementbyinterpolationtoastandardcurvewouldnotobtainthetruevaluesofthesamples.Butitisconvenienttoshowtherelationship.TheSODactivityofriceseedlingcrudeextractassayedviatheEDschemewashigherthanthatbyusingthespectrophotometricmethod,presumablybecausethesuperoxideradicalfluxinthetwomethodsisdifferent.Crudeextractofpapayaleavesshowedasimilarsituationbutwithcloservalues.Crudeextractoftobaccoleaves,incontrast,showedadifferentpicture.Electrophoreticpatternsshowedthattherewerelightyellowsmears,presumablyinterferencemolecules,atthebottomofthetobaccolaneandtoalesserextentinthepapayalane,whilesuchaphenomenonwasunobservedinthericelane.Activityassaywiththespectrophotometricmethodshowedthatthecrudeextractoftobaccoleaveswasabletoincreasetheabsorbanceinthereactionsysteminenzymaticandnonenzymaticwayswithouttheadditionofxanthineoxidase.Thissuggestedthatsomemoleculesinthecrudeextractcouldinitiatethesuperoxideradicalgenerationsystem.Extractfrompapayaleavesshowedasimilarsituation,buttoalesserextentwhilenosuchreactionwasobservedinriceseedlingextract(datanotshown).Thetwocasesindicatedthatthecrudeextractoftobaccoleavescontainedthelargestamountofinterferingsubstancesinthethreesamples,resultinginthelargestover-estimationoftheSODactivitybythespectrophotometricmethod,followedbythecrudeextractofpapayaleaves.RiceseedlingscontainedfewinterferingsubstancesintheSODactivityassay,resultinginminimalover-estimation.Inthespectrophotometricmethod,theseinterferenceswereaddedtothegenuineSODactivity.

TheexpressionofSODgenesareinvolvedinmanylifeaspectsincludingdevelopmentalcourseandinresponsetoenvironmentalstress.MostworkonunderstandingtheexpressionregulationofSODgenesbasedonRNAgelblotanalysishassufferedfromthelackofaconvenientmethodtoassaySODactivity.WiththeEDscheme,notonlythetotalSODactivitybutalsotheactivityofasingleisozymecanbedeterminedefficiently.ThistechniquecanprovideresearcherswithaneyetoseehowaSODgeneisexpressedattheenzymeactivitylevel.

Acknowledgments.ThisworkwassupportedinpartbytheNationalScienceCounciloftheRepublicofChina,underGrantNo.NSC83-0211-B-002-293.TheauthorsaregratefultoDr.Chia-YinTsaiforhelpfuldiscussionandcriticalreviewofthismanuscript.

ChenandPan—Assayofsuperoxidedismutaseactivity

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