676BIOCHEMISTRYGIVOLETALPROCNASPhinneyTheresearchwassupportedby - PDF document

676BIOCHEMISTRY:GIVOLETAL.PROC.N.A.S.Phinney.TheresearchwassupportedbygrantsfromtheNationalScienceFoundationandtheNationalInstitutesofHealth.1Bishop,J.,J.Leahy,andR.Schweet,thesePROCEEDINGS,46,1030(1960).2Dintzis,H.,thesePROCEEDINGS,47,247(1961).3Warner,J.R.,P.M.Knopf,andA.Rich,thesePROCEEDINGS,49,122(1963).3aRich,A.,J.R.Warner,andH.M.Goodman,inSynthesisandStructureofMacromolecules,ColdSpringHarborSymposiaonQuantitativeBiology,vol.28(1963),p.269.4Gierer,A.,J.Mol.Biol.,6,148(1963).5Gilbert,W.,J.Mol.Biol.,6,374(1963).6Wahba,A.H.,C.Basilio,J.F.Speyer,P.Lengyel,R.S.Miller,andS.Ochoa,thesePROCEED-INGS,48,1683(1962).7Borsook,H.,C.L.Deasy,A.J.Haagen-Smit,G.Keighley,andP.H.Lowy,J.Biol.Chem-196,669(1952).8Allen,E.H.,andR.S.Schweet,J.Biol.Chem.,237,760(1962).9Rossi-Fanelli,A.,E.Antonini,andA.Caputo,Biochim.Biophys.Acta,30,608(1958).10Razzell,W.E.,andH.G.Khorana,J.Biol.Chem.,234,2114(1959).11Hilmoe,R.J.,J.Biol.Chem.,235,2117(1960).12Naughton,M.A.,andH.Dintzis,thesePROCEEDINGS,48,1822(1962).13Diamond,J.M.,andG.Braunitzer,Nature,194,1287(1962).14Smith,J.D.,J.Mol.Biol.,8,772(1964).15Doty,P.M.,R.E.Thach,andT.Sundararajan,personalcommunication.DISULFIDEINTERCHANGEANDTHETHREE-DIMENSIONALSTRUCTUREOFPROTEINSBYDAVIDGIVOL,*FRANCESCODELORENZO,tROBERTF.GOLDBERGER,ANDCHRISTIANB.ANFINSENLABORATORYOFCHEMICALBIOLOGY,NATIONALINSTITUTEOFARTHRITISANDMETABOLICDISEASES,NATIONALINSTITUTESOFHEALTHCommunicatedJanuary28,1965ThecrystallographicstudiesofKendrewandPerutzandtheircolleagueshavedemonstratedthatthemyoglobinsandhemoglobinsofseveralspeciespossessuniquetertiarystructures.1'2Suchstructuralhomogeneityprobablycharacterizesmostorallproteins,asindicatedbythelargebodyofphysicalandchemicalinforma-tionnowavailableonmanypurifiedpreparations.Ithasbeensuggestedthatthethree-dimensionalconformationsofproteinsarecompletelydefinedbytheinforma-tionpresentinthelinearsequencesofaminoacidsthatmakeupthecorrespondingpolypeptidechains.3Thissuggestionhasbeensupportedbyanumberofstudiesonthereversibledenaturationofproteins(summarizedinref.4).Ithasbeenshownthattherenaturedmolecules,boththosedevoidofcovalentcrosslinkages5-7andthosepossessingdisulfidebondswhichhavebeenreductivelycleavedpriortorenaturation,8-2exhibitphysicalandbiologicalpropertiesindistinguishablefromthoseofthenativemolecules.Thesestudiesindicatethatthetertiarystruc-tureandspecificdisulfidebondsofanativeproteinmoleculerepresentthemoststableconformationofitspolypeptidechainunderphysiologicconditions.Iftheinformationrequiredforthepairingofhalf-cystineresiduesinapolypep-tidechainisinherentintheaminoacidsequence,onemayaskwhetherinterrup- VOL.53,1965BIOCHEMISTRY:GIVOLETAL.677tionofthechainbycleavageofoneormorepeptidebondsmightseriouslymodifythisinformation.Thiscouldbethecase,forexample,foraproteinthatissyn-thesizedasonepolypeptidechain(e.g.,chymotrypsinogen)andfunctionalasamultichainedprotein(e.g.,chymotrypsin).Wehaverecentlyshownthatamicrosomalenzymewhi

chacceleratesthere-activationofthereducedformofbovinepancreaticribonuclease(RNase)andofeggwhitelysozymel"isinvolvedinthecatalysisofsulfhydryl-disulfideinterchange.14Thus,theinactiveproductobtainedbyoxidationofreducedRNaseinurea,con-tainingrandomsetsofhalf-cystinepairs,israpidlyconvertedtoactiveRNasebytheenzyme.Ifthe"correct"disulfidebondsofaproteinareacorollaryofnativetertiarystructure,anenzymethatwouldcatalyzetheirrearrangementcouldbeusedtotestthestabilityofthesequence-directedpairingofhalf-cystineresidues.Wepresent,inthiscommunication,theresultsofstudiesontheeffectofthedi-sulfideinterchangeenzymeonseveralRNasederivatives,chymotrypsin,andinsulin.Theresultsareconsistentwiththeideathatthespecificdisulfidebondsofthesepro-teinswereformedaccordingtotheinformationpresentinsingle-chainedpre-cursorswhichweresubsequentlyconverted,bypeptidebondcleavage,tothemeta-stablemultichainedproteins.MaterialsandMethods.-Bovinepancreaticribonuclease(SigmaChemicalCo.)wasreducedasdescribedpreviously'5andwasallowedtooxidizeeitherspontaneouslyin8MureaatpH8.2for100hr,'6orbyincubationfor10minwith10-3Mdehydroascorbicacid(DHA)(NutritionalBiochemicalCorp.)in0.05Mbicarbonatebuffer,pH7.4.14AfterremovaloftheureaortheDHAunderacidicconditions,'4bothpreparationswerefoundtocontainnofreesulfhydrylgroupsandtobeenzymicallyinactive.The"C-protein"derivativeofRNasewaspreparedbycleavageofmethionylbondswithcyanogenbromideandsubsequentremovaloftheNH2-terminaltridecapeptidebygelfiltrationthroughacolumnofSephadexG-25(Pharmacia).17Theextentsandratesofreactivationofre-ducedRNase,andoftheoxidizedRNasepreparationsdescribedabove,weredeterminedundervariousconditionsbyassayofRNaseactivityatpH5.0.18ChymotrypsinogenAanda-chymotrypsin(3Xcrystallized)werepurchasedfromtheWor-thingtonBiochemicalCorp.Chymotrypsinogenwasactivatedbyincubationwithtrypsin(seeResults).Theesteraticactivityofa-chymotrypsinwasdeterminedbythehydrolysisofbenzoyl-tyrosyl-ethylester(DetermatubeBTEE,WorthingtonBiochemicalCorp.).'9Thein-creaseinabsorbancyat256muwasfollowedduringthefirst4minofthereactioL#'Trypsin(2Xcrystallized,WorthingtonBiochemicalCorp.)wastreatedwithdiisopropyl-fluorophosphatetodestroyresidualchymotrypticactivity.20Beefinsulin(lowzinc;lot#4096836997)andtheoxidizedBchainandAchainofinsulinwereagiftfromEliLillyLaboratories.BeefinsulinlabeledwithIF3(AbbottLaboratories)wasusedinasolutionof10mgug/mlin0.025Mphosphatebuffer,pH8,containing50mgofbovineserumalbumin(ArmourCo.)perml.Pepsin(2Xcrystallized)wasobtainedfromWorthingtonBiochemicalCorp.Thedisulfideinterchangeenzymewaspreparedfrombeeflivermicrosomesasdescribedpre-viously,exceptthatSephadexG-200filtrationwasintroducedintotheprocedurefollowingtheCM-Sephadexstep.'4Theenzymewasassayedroutinelybyitseffectontherateofreactiva-tionofreducedRNase.3SephadexG-25andG-200,DEAE-Sephadex,andCM-SephadexwerepurchasedfromPhar-macia.Urea(BakerChem.Co.)wasrecrystallizedfrom95%ethanolbeforeuse.f3-Mercapto-

ethanol(EastmanCo.)wasusedwithoutfurtherpurification.ImmunoassayofI131-labeledporkinsulinwasperformedessentiallyasdescribedbyBersonetal.,21exceptthatascendingchromatographyin0.025Mphosphatebuffer,pH8,wasusedin-steadofelectrophoresis.Thesamples(0.5mlcontaining0.5mugofI'31-labeledinsulin)wereincubatedwith0.05mlofguinea-pigantibovineinsulinantiserum(diluted1:40with0.025Mphosphatebuffer,pH8.0,containing50mgbovineserumalbuminperml)for48hrat40and0.2 678BIOCHEMISTRY:GIVOLETAL.PROC.N.A.S.mlwaschromatographedonWhatman3MMpaperfor31/2hrat4°.Radioactivitywasmeas-uredwitha4-Piautomaticwindowlesspaperchromatogramscanner(AtomicAccessories,Inc.).Changesinthepairingofhalf-cystineresiduesininsulinwereexaminedqualitativelyonpeptidemapsafterpepticdigestion.Theproteinwasdissolvedin5%formicacidataconcentrationof10mg/mlandpepsin(0.1mgin10IA5%formicacid)wasadded.After6hrofincubationat370asecondaliquotofpepsinwasadded.Thereactionwasstopped,afteratotalincubationtimeof16hr,byfreezingandlyophilization.Thelyophilizedmaterialwasdissolvedinwateratacon-centrationof50mg/ml,and0.05mlwereappliedtoWhatman3MMpaper.Descendingchroma-tographywasperformedintheorganicphaseofbutanol:aceticacid:water(4:1:5),andelectro-phoresiswascarriedoutat2500vinpyridineacetatebuffer,pH3.6,for2hr.Thepeptidemapswerepreparedinduplicate.Onewasstainedwithninhydrinandtheotherwithcyanide-nitro-prussideforthedetectionofpeptidescontainingdisulfidebonds.22OxidationwithperformicacidwascarriedoutaccordingtothemethodofHirs.21ABeckman/Spincomodel120aminoacidanalyzerwasutilizedforaminoacidanalyses.Sam-plesforanalysiswerehydrolyzedinconstantboilingHClinevacuated,sealedtubesfor22hrat1100.ProteinconcentrationsweredeterminedbythemethodofLowryetal.24Freesulfhydrylgroupsweredeterminedwith5,5'-dithiobis(2-nitrobenzoicacid)(AldrichChemicalCo.).25Results.-Ribonucleasederivatives:Theenzymicallyinactiveproductsob-tainedbytheoxidationin8MureaorwithDHAhavebeenshowntoberapidlyactivatedbythedisulfideinterchangeenzymeinthepresenceof,B-mercaptoethanol(optimalconcentration,10-3M)(Fig.1,curves1and2),ascontrastedwiththelongperiods(16-24hr)requiredwithouttheenzyme.'6Therequirementfor,B-mercaptoethanolinthereactivationmixturecouldbeabolishedbypreviouspartialreductionofthesemolecules(Fig.1,curve3).14Sincewehaveshownthattheenzymedoesnotacceleratetheoxidationofsulfhydrylgroups,'4andsinceitacti-vates"incorrectly"cross-linkedRNaseai,-ellasfullyreducedRNase,itmaybecon-cludedthattheprocesscatalyzedisasulfhydryl-disulfideinterchange.Theeffectoftheenzymeonthe"C-protein"derivativeofRNasedescribedbyGrossaidWitkop'7isillustratedinFigure2.TheC-proteiniscomposedofthreepolypeptidechainsheldtogetherbyoneintrachainandthreeinterchaindisulfidebonds,allpresentintheoriginalRNasemolecule.Theaggregationandprecipita-tionproducedbytheenzymeinthepresenceof10-3M3-mercaptoethanolwassorapidthatthefl-mercaptoethanolconcentrationhadtobediminishedto10-4M.Atthel2tterleveltheconcentrat

ionoffl-mercaptoethanolwaslessthan5percentthatofhalf-cystineresiduesintheC-proteinemployed(4mgprotein/ml).Thus,theprecipitationcannotbeduetosimplereduction.Noprecipitationoccurredovera24-hrperiodinthepresenceofeitherenzymealoneorf-mercaptoethanolalone(10-3or10-4M)undertheseconditions(0.1MTris,pH7.2).(Precipitationdidoccurafter1hratpH7.8inthepresenceof10-3M,B-mercaptoethanolalone.)Theenzyme-catalyzedaggregationandprecipitationofC-proteinispresumablycausedbydisulfideinterchange,whichleadstorandompairingofhalf-cystineresiduesformingacross-linkednetworkofchains.InpreviousexperimentsonthereactivationofreducedRNaseitwasdifficulttodemonstrateenzymeactivityatlowratiosofenzymetoreducedRNase"3sincecon-centrationsofthesubstrate(reducedRNase)greaterthan0.02mg/mlledtoex-tensiveintermoleculardisulfidebonding.IntheexperimentswithC-proteinandwithinsulin,describedbelow,catalysisbythedisulfideinterchangeenzymeiseasilydemonstratedatweightratiosofenzymetosubstrateoflessthan1:100.Chymotrypsin:Chymotrypsinrapidlyinactivatesthedisulfideinterchangeen- VOL.53,1965BIOCHEMISTRY:GIVOLETAL.679123100~~~~~~~~60of-~~~~~~~~~~~~~~~~~~~~~~~~~6FIG1-eatiatonofoxdiedR~seervaivs.Cuve1:DH-oidze,.80e1-i4020.43~~~~~~~~~~~~~~~~~~~~~~~~~~~420~~~~~~~~~~~~~~~~~~~20510IS520241020020MINUTESMINUTESMINUTES123FIG.1.-ReactivationofoxidizedRNasederivatives.Curve1:DHA-oxidizedRNase,10-3M,5-mercaptoethanol.Curve2:Urea-oxidizedRNase,10-3Mj3-mercaptoethanol.Curve3:Urea-oxidizedRNasecontaining2-4sulfhydrylgroupspermole.'4Curve4:DHA-oxidizedorurea-oxidizedRNasewitheither10-3M,3-mercaptoethanolorthedisulfideinterchangeenzyme.Allincubationswerecarriedoutat370in0.1MTris,pH7.5,andcontained20fgofRNasederiva-tivepermland(exceptasindicatedforcurve4)20,gofthedisulfideinterchangeenzymeperml.FIG.2.-Treatmentof"C-protein"withthedisulfideinterchangeenzyme.Theincubationmixturecontained4mgof"C-protein"and80/Agofthedisulfideinterchangeenzymein1mlof0.1MTris,pH7.2,10-4MiB-mercaptoethanol.TurbiditywasfollowedwithaCarymodel15spectrophotometer.FIG.3.-Inactivationofchymotrypsinbythedisulfideinterchangeenzyme.Theuppercurve(e-*)wasobtainedwithanincubationmixturecontaining10jgofchymotrypsin,dilutedfromaureasolution(seetext),in1mlof0.1MTris,pH7.5,withorwithout0-mercaptoethanolatacon-centrationof10-3M.Thelowercurve(O.-)wasobtainedwithanincubationmixturepreparedasabove(withg-mercaptoethanol)plus100sAgofthedisulfideinterchangeenzyme.Aliquotsof0.1mlwereremovedfromtheincubationmixturesforassayofchymotrypsinactivity.100�.,VX60°040--[10420£402020.40.60.81.0-MGENZYMEPREPARATIONMINUTES45FIG.4.-Inactivationofchymotrypsinbythedisulfideinterchangeenzymeattwostagesinitspurification.ConditionsofincubationwereasdescribedinthelegendforFig.3(with100Mgofdisulfideinterchangeenzymeand10-3M,-mercaptoethanol).Thepreparationsofdisulfidein-terchangeenzymewereobtainedafterfiltrationonSephadexG-200(.-.)andafterchromatog-raphyonDEAE-Sephadex(A-,).FIG.5.-Treatme

ntofinsulinwiththedisulfideinterchangeenzyme.Theincubationmixturecontained4mgofinsulinpermland80Mgofthedisulfideinterchangeenzymepermlin0.1MTris,pH7.2,10-3Mfl-mercaptoethanolatroomtemperature.Theturbidityofthissolutionwasfol-lowedwithaCarymodel15spectrophotometer,using,inthereferencecuvette,thesameincubationmixturebutwithoutenzyme(uppercurve,left-handscale).Thecontentofsulfhydrylgroupsin1-mlaliquotsoftheincubationmixtureafterremovalof6-mercaptoethanolisshowninthelowercurve(right-handscale). 680BIOCHEMISTRY:GIVOLETAL.PROC.N.A.S.zyme,asdemonstratedbylossintheabilityofthelatterenzymetocatalyzethereactivationofreducedRNase.KoshlandandMozerskyhaveshown27thatchymotrypsinishighlyresistanttospontaneousdisulfideinterchange,withonlyslowinterchangeoccurringin7.5Murea.Theactionofthedisulfideinterchangeenzymeonchymotrypsinwasthereforestudiedafterthelatterenzymehadbeenexposedto8Murea.FollowingMartinandFrazier,28chymotrypsin(1mg/ml)wasincubatedin8Murea,containing0.2MCaCl2and0.1Macetatebuffer,pH4.0,for20min.Undertheseconditionstheinactivationisreversibleand,inourhands,dilutionwith100volof0.1MTrisbuffer,pH7.5,withorwithout10-3Mfi-mercaptoethanol,rapidlyrestored85percentoftheoriginalchymotrypsinactivity.Whenthedilutingsolutioncontainedthedisulfideinterchangeenzymeand10-3M,3-mercaptoethanol,rapidinactivationofchymotrypsinwasobserved.AsisshowninFigure3,theinactivationwasdependentonthepresenceoff-mercapto-ethanolandratherlargequantitiesofthemicrosomalenzyme.Thefactthatin-activationofchymotrypsinoccurredmainlyduringthefirstminutesuggeststhattheurea-treatedproteaseremainsinadenaturedandinactiveformforonlyaveryshortperiodafterdilution.TheinactivationbytheenzymeattwostagesinitspurificationisshowninFigure4.AsimilardifferenceinspecificactivitieswasfoundwhenthetwopreparationsweretestedfortheirabilitytocatalyzethereactivationofreducedRNase.Iftheinactivationofchymotrypsinisduetodisulfideinterchangeandtheproduc-tionofrandominterchainandintrachainsetsofdisulfidebonds,anecessarycontrolexperimentistotesttheeffectoftheenzymeonchymotrypsinogen,whichpresum-ablycontains,initssinglepolypeptidechain,theinformationfortheformationofthe"correct"disulfidebonds.ChymotrypsinogenAwasincubatedin8Murea,asinthecaseofchymotrypsin,diluted100-foldintoTrisbuffercontaining10-3M,B-mercaptoethanolanddisulfideinterchangeenzyme(finalconcentrations:1mgenzymeand10,gchymotrypsinogen/ml),andincubatedfor20minatroomtemperature.Trypsin(0.5,gg/ml)wasthenadded,andtheappearanceofchy-motrypsinactivitywasfollowedbyperiodicassays.Thetheoreticallevelofchy-motrypsinactivitywasachievedafter7hrat240,demonstratingthatchymotryp-sinogenisnotsusceptibletoinactivationbythedisulfideinterchangeenzyme.Insulin:Theaggregatingeffectofthedisulfideinterchangeenzymeleadstoprecipitationofinsulin(enzyme:insulin,w/w,1:50;10-3M,B-mercaptoethanol,0.1MTrisbuffer,pH7.2),asillustratedinFigure5.Thephysicalpropertiesoftheprecipitatechangedwithtime,becomingp

rogressivelylesssolublein8Arureaor1percentsodiumdodecylsulfate.Samplesof1ml,containing4mgofinsulin,takenatthetimesindicatedinFigure5,wereprecipitatedbytheadditionof10volofacidacetone(1NHCl:acetone,1:39),washedtwicewithacidacetone,anddissolvedin0.1MTrisbuffer,pH7.8,containing1percentsodiumdodecyl-sulfateandDTNB(10-3M).AsshowninFigure5,freesulfhydrylgroupsap-pearedslowlyduringtheincubation,reachingafinallevelofapproximately0.3freeSHgroup/moleofinsulin.Theprecipitateformedattheendof1hrofincubationwaswashedthreetimeswithwater,oxidizedwithperformicacid,andsubjectedtohydrolysisbytrypsin(1:100w/w)in0.1MNH4HCO3at370for8hr.Suchtreat-mentwouldbeexpectedtohavenoeffectontheoxidizedAchainofinsulinbuttoyieldfreealanineandtwopeptidefragmentsfromtheoxidizedBchain.Theresults VOL.53,1965BIOCHEMISTRY:GIVOLETAL.681TABLE1AMINoACIDANALYSES*OFPRECIPITATESFORMEDBYACTIONOFDISULFIDEINTERCHANGEENZYMEONINSULIN50-Min2-HrInsulinAchainBchainAminoacidprecipitateprecipitateobserved(theory)(theory)Lyst(1)(1)(1)01His1.731.951.9902Arg1.041.191.0701CYSO33.655.57Asp1.912.133.2521Thr1.101.100.9501Ser1.771.692.6721Glu4.825.647.6143Pro1.291.241.1201Gly3.754.234.2413Ala2.682.893.12121/2Cys5.4842Val3.764.444.5223Met00000Ilu0.260.340.5010Leu5.006.116.0624Tyr1.902.853.9022Phe3.183.492.9103*Uncorrectedfordestructionduringhydrolysis.tTherelativeamountsoftheaminoacidswerecalculatedonthebasisoflysineas1.00.ofhigh-voltageelectrophoresisofthedigestsoftheoxidizedprecipitateshowedthecomponentsexpectedfrombothchains.Asecondportionoftheoxidizedprecip-itatewashydrolyzedforaminoacidanalyses.Theresults,showninTable1,indicatethattheprecipitateformedinthepresenceoftheenzymecontainsbothAandBchainsofinsulin.However,someenrichmentforthoseaminoacidspresentonlyintheBchainisevident,particularlyintheearly(50-min)precipitate.Thisfindingsuggeststhatduringtheprocessofinterchangenewintrachaindisulfidebondsareformed,leadingtoseparationoftheAandBchains.Thisconclusionisconsistentwiththegreatereaseofreductionofinterchaindisulfidebondsininsulin2andthelowersolubilityoftheBchainatpH7.2.30Aqualitativeestimationoftherateandextentofdisulfideinterchangecatalyzedbytheenzymewasobtainedbythepreparationofpeptidemapsofpepticdigestsofinsulinandofprecipitatesandmaterialremaininginsolutionafter50minandafter2hrofincubation(6mginsulinand0.06mgenzyme/ml,0.1MTris,pH7.2).Thesolublefractionswereprecipitatedandwashedwithacidacetoneasweretheprecipitates.Afterremovaloftheacetone-HClinvacuo,10-mgsamplesofeachfractionandofuntreatedinsulinweredissolvedorsuspendedin5percentformicacidanddigestedwithpepsin.Thedigestsweredilutedtenfoldwithwater,lyophilized,and2.5-mgaliquotswereappliedtoWhatman3MMpaperforpep-tidemapping.Duplicatemapsofeachfractionwerestainedasdescribedabove.Thepeptidemapswereidenticalexceptforthosecomponentsstainingpositivelyfordisulfidebonds.Thedisulfide-positivecomponentscharacteristicofinsulinwerestillfaintlyvisibleinthesolubleandinsolublefract

ionsafter50minofincuba-tionalthoughanumberofnewpeptidescontainingdisulfidebonds,havingdifferentmobilities,werealreadypresentinlargeamounts.After2hrofincubation,digestsofboththesolubleandinsolublefractionsshowedaconsiderablefractionofthedisulfide-positivematerialremainingattheorigin,whiletherestwaspresentinnewlocationsonthemap.Evidenceforchangeinthethree-dimensionalstructureofinsulinaftertreatment 682BIOCHEMISTRY:GIVOLETAL.PROC.N.A.S.:withthedisulfideinterchangeenzymewasobtainedbymeasurementsofanti-/l^fly;;genicity.AsolutionofI'll-labeledin-----\*_---/~@~-sulin(1mug/ml)containingbovineserumalbumin(50mg/ml)wasincu-batedwithandwithoutenzyme(50.......s--;z-g/ml),andinthepresenceandab-A~'senceof10-Mfl-mercaptoethanol.Incubationswerecarriedoutin0.1Mp_---J-\Trisbuffer,pH7.2,at370for30min.Attheendofthisperiod,0.05mlofthedilutedantiserumwasaddedtohalfoftheincubationmixture.After48hrat40,thematerialthathadbeenFIG.6.-Immunoassayofinsulintreatedwithtreatedwithantiserumandthecontrolthedisulfideinterchangeenzyme.CurveA:in-fractionwerechromatographed,andsulinalone.CurveB:insulinandj3-mercapto-thedistributionofradioactivitywasethanol.CurveC:insulin,,3-mercaptoethanol,anddisulfideinterchangeenzyme.CurveD:in-scannedoneachchromatogram(Fig.sulinanddisulfideinterchangeenzyme.CurvesA',B',C',andD'arethesameasthecorrespond-6).ingcurvesexceptthatantiserumwasaddedafterUndertheconditionsofchromatog-30minofincubationat37°.Thequantitiesofthevariouscomponentsaregiveninthetext.raphythesmallamountofinsulinusedremainsattheorigin,while,inthepresenceofantibodies,theantibody-boundinsulinmoveswiththefront(Fig.6,A,A').Theadditionofenzymealonehasnoeffect(Fig.6,D,D')andonlyasmalleffectresultswithf3-mercaptoethanolalone(Fig.6,B,B').Inthepresenceofbothenzymeand,3-mercaptoethanolthechromatographicpatternofinsulinismarkedlychanged(Fig.6,C).However,thispatternisnotaffectedbythepresenceofantibodies(Fig.6,C')andalmostnoantibody-boundinsulinisob-served.Theseobservations,togetherwiththosedescribedabove,indicatethatextensivedisulfideinterchangehastakenplace,withdisruptionoftheantigenicallyspecificstructureoftheinsulinmolecule.Discussion.-Theinformationnowavailableindicatesthattheenzymeusedinthesestudiescatalyzesaprocessofsulfhydryl-disulfideinterchange.Forexample,anRNasederivative,preparedbyoxidationinureawhichcauses"incorrect"pairingoftheeightsulfhydrylgroupsofthereducedpolypeptidechain,israpidlyconvertedtothenativeproteinbytheenzyme.Theprocessrequiresthepresenceofsmallamountsofathiolreagentsuchasj3-mercaptoethanolor,alternatively,theintroductionintotherandomlydisulfide-bondedderivativeofafewsulfhydrylgroupspriortoadditionoftheenzyme.Itisknownthatthedisulfideinterchangereactioniscatalyzedbysmallamountsofthiolcompounds.hiReactivationofthefullyoxidizedinactiveRNasederiva-tiveoccursinthepresenceof,3-mercaptoethanolalone,althoughatamuchslowerratethaninthepresenceofenzyme.Thedisulfideinterchangeisaccompaniedbyar

apidrearrangementoftertiarystructure,thesoledrivingforceforwhichappearstobethehighlyfavorablefreeenergyofconformationofthenativestructureascomparedwiththoseofotherthree-dimensionalarrangements.Ifboththetertiarystructureandthepairingofhalf-cystineresiduesinaprotein VOL.53,1965BIOCHEMISTRY:GIVOLETAL.683areapredeterminedconsequenceoftheaminoacidsequence,cleavageofoneormorepeptidebondsmightbeexpectedtoupsetthedelicatelybalancedsetofinter-actionsrequiredtoachievethenativestructure.Aswehaveobservedinthesestudies,nativeRNaseandchymotrypsinogenarenotalteredbythedisulfideinter-changeenzyme.Ontheotherhand,thethree-chainedstructuresofthechemicallyproduced"C-protein"derivativeofRNase,andtheproductofenzymicactivationofchymotrypsinogen,chymotrypsin,arerapidlymodifiedbytheenzyme.Theseresultssuggestthatinformationpresentintheoriginalsingle-chainedprecursorsismissingafterfragmentationofthechains.Thusthedisulfideinterchangeenzymeappearstoconstituteauseful"thermodynamicprobe"fortestingtheintrinsicstabilityofdisulfide-bondedpolypeptidesingeneral.Ourresultswithinsulinsupporttheviewthatthehormoneisoriginallysyn-thesizedasasingle-chainedproteinandlaterconvertedtothetwo-chainedformbyazymogen-likeconversion.SuchamechanismwouldbeconsistentwiththerecentobservationsofMarkusontheconformationalchangesinducedininsulinbynondenaturingelectrolyticreduction,32andwithreportsoflowyieldsofinsulinfollowingoxidationofmixturesofreducedAandBchains.33-36WewishtothankDr.FrankTietzeforthesamplesofI"3'-labeledinsulinandantiseraandforhishelpfuladviceconcerningtheimmunoassayofinsulin.WearealsogratefultoMrs.DianaTrundleforhertechnicalassistance.*Visitingscientist,onleavefromWeizmannInstituteofScience,Rehovoth,Israel.tInternationalfellow,USPHS;onleavefromUniversityofNaples,Italy.1Kendrew,J.C.,Science,139,1259(1963).2Perutz,M.F.,Science,140,863(1963).3Anfinsen,C.B.,inEnzymeModelsandEnzymeStructure,BrookhavenSymposiainBiology,No.15(1962),p.194.4Epstein,C.J.,R.F.Goldberger,andC.B.Anfinsen,inSynthesisandStructureofMacromole-cules,ColdSpringHarborSymposiaonQuantitativeBiology,vol.28(1963),p.439.5Fraenkel-Conrat,H.,andB.Singer,Biochim.Biophys.Acta,33,359(1959).6Anderer,F.A.,Z.Naturforsch.,14b,642(1959).7Harrison,S.C.,andE.R.Blout,J.Biol.Chem.,inpress.8White,F.H.,Jr.,J.Biol.Chem.,236,1353(1961).9Imai,K.,T.Takagi,andT.Isemura,J.Biochem.,53,1(1963).10Goldberger,R.F.,andC.J.Epstein,J.Biol.Chem.,238,2988(1963)."Isemura,R.,T.Takagi,V.Maeda,andK.Yutani,Biochem.J.,53,155(1963).12Frattali,V.,R.F.Steiner,D.B.S.Millar,andH.Edelhoch,Science,199,1186(1963).13Goldberger,R.F.,C.J.Epstein,andC.B.Anfinsen,J.Biol.Chem.,239,1406(1964);Vene-tianer,P.,andF.B.Straub,Biochim.Biophys.Acta,67,166(1963).14Givol,D.,R.F.Goldberger,andC.B.Anfinsen,J.Biol.Chem.,239,3114(1964).15Anfinsen,C.B.,andE.Haber,J.Biol.Chem.,236,1361(1961).16Haber,E.,andC.B.Anfinsen,J.Biol.Chem.,237,1839(1962).17Gross,E.,andB.Witkop,J.Biol.Chem.,237,1856(1962).18Anfinse

n,C.B.,R.R.Redfield,W.L.Choate,J.Page,andW.R.Carroll,J.Biol.Chem.,207,201(1954).19Hummel,B.C.W.,Can.J.Biochem.Physiol.,37,1393(1959).20Potts,J.T.,Jr.,D.M.Young,C.B.Anfinsen,andA.Sandoval,J.Biol.Chem.,239,3781(1964).21Berson,S.A.,R.S.Yalow,A.Bauman,M.A.Rotschild,andK.Newerly,J.Clin.Invest.,35,170(1956).22Toennies,G.,andJ.J.Kolb,Anal.Chem.,23,823(1951).23Hirs,C.H.W.,J.Biol.Chem.,219,611(1956). 684PATHOLOGY:GILDENETAL.PROC.N.A.S.24Lowry,0.H.,N.J.Rosebrough,A.L.Farr,andR.J.Randall,J.Biol.Chem.,193,265(1951).26Ellman,G.L.,Arch.Biochem.Biophys.,82,70(1959).26Epstein,C.J.,R.F.Goldberger,M.D.Young,andC.B.Anfinsen,Arch.Biochem.Biophys.,Suppl.1,223(1963).27Koshland,D.E.,andS.M.Mozersky,FederationProc.,23,609(1964).28Martin,C.J.,andA.R.Frazier,J.Biol.Chem.,238,3268(1963).29Markus,G.,inProtidesoftheBiologicalFluidsII,ed.M.Peters(1963).30Tomizawa,H.H.,FederationProc.,20,190(1961).31Ryle,A.P.,andF.Sanger,Biochem.J.,60,535(1954).32Markus,G.,J.Biol.Chem.,239,4163(1964).33Dixon,G.A.,andA.C.Wardlaw,Nature,188,721(1960).34Meienhofer,J.,E.Schnabel,H.Brenner,0.Brinkhoff,R.Zabel,W.Snoka,H.Keostermeyer,D.Brendenberg,T.Akuda,andH.Zahn,Z.Naturforsch.,18b,1120(1963).35Niu,C.,Y.Kung,W.Huang,L.Ke,C.Chen,Y.Chen,Y.Du,R.Jiang,C.Tsou,S.Hu,S.Chu,andK.Wang,Sci.Sinica,13,1343(1964).36Tietze,F.,personal.communication.THENATUREANDLOCALIZATIONOFTHESV4O-INDUCEDCOMPLEMENT-FIXINGANTIGEN*BYR.V.GILDEN,R.I.CARP,F.TAGUCHI,ANDV.DEFENDItTHEWISTARINSTITUTE,PHILADELPHIA,PENNSYLVANIACommunicatedbyK.F.Meyer,January4,1965Neoplastictransformationofmammaliancellsbyanumberofoncogenicvirusesisaccompaniedbytheappearanceofnew"cellular"antigens.Thesearede-tectablebytheirabilitytoinduceresistancetotumorimplantationinsusceptibleanimals'orbytheirabilitytofixcomplementincombinationwithserumfromtumor-bearinganimals.2-5Therelationshipbetweenthetransplantationantigenandthecomplement-fixing(CF)antigenremainsunknown;however,considerableevidenceinthecaseofSV40andpolyomavirusesindicatesthattheyaredistinctfromovertantigensoftheintactvirus.3'IIndeed,thenewantigenspersist,ap-parentlyindefinitely,asthesoleevidenceofanoriginalinfectionafterviruscannolongerbedetected.TheSV40-inducedCFantigen(SV40ICFA)isvirus-specificandnotspecies-specific;i.e.,serumfromhamstersbearingSV40tumorsreactsspecificallywithcellsofdifferentspeciestransformedbySV40virus.3'4(Inordertoallowforthepossibilitythattheantigensdemonstratedbythecomplement-fixingandtrans-plantationtestsaredistinct,theformerisidentifiedasSV40ICFAandthelatterasSV40ITA.)Itwouldappear,therefore,thatsynthesisofthisantigenrequiresthepersistenceofatleastsomepartoftheSV40genome.OnemightthuspresumethatSV40ICFArepresentsan"internal"virusantigen,aviralprecursor,oranewnonviralproteincodedforbytheviralgenome.Althoughourresultstodatefavorthelastofthesepossibilities,thereisasyetnoevidencebearingonthebio-logicalfunctionofthisantigen.MaterialsandMethods.-Cells:TheSV40-transformedhumancellswerethelinesde