CHEMISTRY:MIRSKYANDPAULING6H.Poincare,Meth.Nouv.,1(1892),chap.III;G.D. - PDF document

CHEMISTRY:MIRSKYANDPAULING6H.Poincare,Meth.Nouv.,1(1892),chap.III;G.D.Birkhoff,loc.cit.'6H.Poincar6,op.cit.,2(1893),chap.XVII.ThefiguregivenbyPoincar6assumesthatpispositive.However,thecaseofanegativepmaybereducedtothecaseofapositivep,asseenfrom(6)bywritingv+1/27forv.M.J.0.Strutt,Math.Ann.,99,625-628(1928).8Cf.A.Wintner,Bull.Astr.,9,251-253(1936).9H.Poincare,op.cit.,3,346-351(1899).10H.Poincare,op.cit.,3,343-344(1899).11Cf.E.Holder,Sachs.Sitzber.,83,179-184(1931).12G.D.Darwin,ScientificPapers,4(1911),PartI.Cf.H.Poincar6,op.cit.,3,352-361(1899).ONTHESTRUCTUREOFNATIVE,DENATURED,ANDCOAGULATEDPROTEINSBYA.E.MIRSKY*ANDLINUsPAULINGGATESCHEMICALLABORATORY,CALIFORNIAINSTITUTEOFTECHNOLOGY,PASADENA,CALIFORNIACommunicatedJune1,1936Inthispaperastructuraltheoryofproteindenaturationandcoagulationispresented.Sincedenaturationisafundamentalpropertyofalargegroupofproteins,atheoryofdenaturationisessentiallyageneraltheoryofthestructureofnativeanddenaturedproteins.Initspresentformourtheoryisdefiniteanddetailedinsomerespectsandvagueinothers;refinementinregardtothelattercouldbeachievedonthebasisoftheresultsofexperi-mentswhichthetheorysuggests.Thetheory(somefeaturesofwhichhavebeenproposedbyotherinvestigators)providesasimplestructuralinterpretationnotonlyofthephenomenaconnectedwithdenaturationandcoagulationwhichareusuallydiscussed(specificity,solubility,etc.)butalsoofothers,suchastheavailabilityofgroups,theentropyofdenatura-tion,theeffectofultra-violetlight,theheatofactivationanditsdepen-denceonpH,coagulationthroughdehydration,etc.I.Theexperimentalbasisuponwhichthepresenttheoryrestswillbebrieflydescribed.1.Themostsignificantchangethatoccursindenaturationisthelossofcertainhighlyspecificpropertiesbythenativeprotein.Specificdifferencesbetweenmembersofaseriesofrelatednativeproteinsandspecificenzy-maticactivitiesofnativeproteinsdisappearondenaturation,asthefol-lowingobservationsdemonstrate:(a)Manynativeproteinscanbecrystallizedandthecrystalformischaracteristicofeachprotein.Nodenaturedproteinhasbeencrys-tallized.VOL.22,1936439 CHEMISTRY:MIRSKYANDPAULING(b)Mostnativeproteinsmanifestintheirimmunologicalpropertiesahighdegreeofspecificity,whichisdiminishedbydenaturation.'(c)Thenativehemoglobinsofcloselyrelatedanimalspeciescanbedistinguishedfromeachotherbydifferencesincrystalform,solubility,2gasaffinities,positionsofabsorptionbands,andotherproperties.3Ontheotherhand,inthedenaturedhemoglobinssomeoftheseproperties,theposi-tionsoftheabsorptionbands,forexample,canbesubjectedtoprecisemeasurement,anditisfoundthatdifferencesbetweenthevarioushemo-globinshavedisappeared.4(d)Anumberofenzymeshaverecentlybeenisolatedascrystallineproteins.Whentheseproteinsaredenaturedtheirenzymaticactivityvanishes.Inpepsinandtrypsin,whereanespeciallycarefulstudyhasbeenmadeofthisphenomenon,thereisaclosecorrelationbetweenlossofactivityandformationofdenaturedprotein.52.Strikingchangesinthephysicalpropertiesofaproteintakeplaceduringdenaturation.Atitsisoelectricpointadenaturedproteinisin-soluble,althoughthecorrespondingnativeproteinmaybequitesoluble.Itwasthelossofsolubilitythatfirstdrewattentiontothephenomenonofdenaturation,anddenaturationisnowusuallydefinedbythechangeinsolubility.Thedenaturedproteinafterprecipitationhastakenplaceiscalledacoagulatedprotein,theprocessofcoagulationbeingconsideredtoincludebothdenaturationandaggregationofdenaturedproteinintheformofacoagulum.IIfthedenaturedproteinisdissolved,byacid,alkali,orurea,thesolutionisfoundtobefarmoreviscousthanasolutionofnativeproteinofthesameconcentration.73.Changesintheavailabilityofsulfhydryl,disulfide,andphenolgroupsappearasaconsequenceofdenaturation.AlloftheSHandS-Sgroupsfoundinaproteinafterhydrolysiscanbedetectedinadenaturedproteinevenbeforehydrolysis,whileinthecorrespondingnativeproteinonlyafractionofthesegroupsisdetectable.InnativeeggalbuminnoSHorphenolgroupsaredetectable.Inothernativeproteins(hemoglobin,myosin,proteinsofthecrystallinelens,forexample)somegroupscanbedetected;newgroupsappearwhentheproteinismademorealkaline,althoughnotalkalineenoughtocausedenaturation,andthendisappearwhentheoriginalpHisrestored.Inallthedifferentwaysofcoagulatingaproteinaclosecorrelationbetweenappearanceofgroupsandlossofsolubilityhasuntilrecentlybeenobserved.8Now,however,ithasbeenfoundthatwhenmyosinisrenderedinsolublebydrying(orwhenwaterisremovedbyfreezing)thereisnochangeinavailabilityofitsSHgroups.9Furthermore,iftheinsolublemyosinistreatedwithatypicaldenaturingagent,suchasheatoracid,alloftheSHgroupsintheproteinbecomeavailable,althoughnochangeinsolubilityisobserved.Hithertoproteincoagulationduetodehydrationhasnotbeendistinguishedfromcoagulation440PROC.N.A.S. CHEMISTRY:MIRSKYANDPAULINGcausedbyotheragents,butthetestsforavailabilityshowthatincoagula-tionbydehydrationthechangeinproteinconstitutionisdistinctlydifferentfromthatcausedbyanyoftheknownproteincoagulatingagents.Inourtheoryofcoagulationbothtypesofchangewillbeconsidered.Probablybothtypesoccurbiologically.Ithasbeensuggestedthatwhenlightcon-vertsvisualpurple,aconjugatedprotein,intovisualyellowtheformerisdenaturedinmuchthesamewayasitisbyheat,alcohol,oracid.10Andithasbeenshownthatinthecourseoffertilizationandintherigorofmuscleacoagulationofproteinsimilartothatcausedbydehydrationtakesplace.11,124.Inalistofthelargenumberofdifferentagents,withapparentlylittleincommon,thatcausedenaturationareheat,acid,alkali,alcohol,urea,salicylate,surfaceaction,ultra-violetlight,highpressure.Thetemperaturecoefficientofheatdenaturationofmanyproteins(eggalbumin,ferrihemoglobin,trypsin,etc.)isabout600forariseintemperatureoftendegrees,andfromthistheenergyofactivationcanbecalculated.Oneithersideofapointbetweentheisoelectricpointandneutralitythetemperaturecoefficientofdenaturationbyheatisdiminished.'3Adrypreparationofeggalbuminisnotreadilydenaturedbyheat.65.Thedenaturationofcertainproteins,notablyhemoglobin,serumalbumin,andtrypsin,isreversible.'4Fromtheeffectoftemperatureontheequilibriumconstant,theheatofreactionandtheentropychangecanbecalculated.Inthedenaturationofhemoglobinbysalicylateandinthedenaturationoftrypsinbyheatoracid,theequilibriumbetweennativeanddenaturedproteinisnotaffectedbychangesinthetotalconcentrationofproteinpresent,'5fromwhichitcanbeinferredthatinthedenaturationofhemoglobinandtrypsinbytheseagentsnochangeinmolecularweighttakesplace.Sincetypicaldenaturationcanoccurwithoutchangeinthemolecularweight,itisimportanttodistinguishbetweendenaturationandthechangesinparticlesizeobservedbySvedberg.Althoughdenatura-tioncanoccurwithoutchangeinmolecularweight,undercertainconditions,asinthedenaturationofmyosinbyurea,denaturationmaybeaccom-paniedbydepolymerization.Weberfoundthemolecularweightofmyosintobeoftheorderofamillionandthatofmyosininureatobethirty-fivethousand."6Ontheotherhand,itisunlikelythatdepolymerizationisalwaysaccompaniedbydenaturation,forundersomeoftheconditionsofdepolymerizationdescribedbySvedbergitisimprobablethatdenaturation(lossofspecificity,lossofsolubility,orappearanceofpreviouslyinaccessiblegroups)takesplace.Inthecaseofhemocyanin,forexample,decompositionintoproductsl/aand'/16ofthesizeoftheoriginalmoleculereadilyoccurs,andthesesmallerparticlesseemtohavethepropertiesofnativehemo-cyanin.176.Theshapeofthenativeproteinmoleculeappearstohavelittlesig-VOL.22,1936441 CHEMISTRY:MIRSKYANDPAULINGnificanceforanunderstandingofdenaturation.Denaturationoccursinthesphericalmoleculesofeggalbuminandhemoglobin,intheelongatedpar-ticlesofsolublemyosinl8and(asindicatedbySHgroupsbecomingdetect-able)eveninmyosinthathasbeenformedintoinsolublefibresbydrying.97.Incertainconjugatedproteinsstabilityoftheproteinandpresenceoftheprostheticgrouparerelated.Inhemoglobin,theyellowoxidizingfermentofWarburg,andvisualpurple,itisnecessarytodenaturetheproteininordertodetachtheprostheticgroupwiththeuseofpresentmethods.'019Afterremovaloftheprostheticgroupitispossibletoreversethedenaturationofglobinandtheproteinoftheoxidizingferment,butthesenativeproteinsaremoreunstable(withrespecttodenaturation)thantheyarewhenconjugatedwiththeirprostheticgroups.Inhemoglo-bintheeaseofdenaturationdependsuponthestateoftheprostheticgroup.Carbonmonoxidehemoglobin,forexample,islessreadilydenaturedbyheat,acid,oralkalithanareoxyhemoglobinandferrihemoglobin.20Invisualpurplepresenceoftheprostheticgroupcausestheproteintobede-naturedbyvisiblelight.Denaturationinthiscaseappearstoberevers-ible.10II.Ourconceptionofanativeproteinmolecule(showingspecificproperties)isthefollowing.Themoleculeconsistsofonepolypeptidechainwhichcontinueswithoutinterruptionthroughoutthemolecule(or,incertaincases,oftwoormoresuchchains);thischainisfoldedintoauniquelydefinedconfiguration,inwhichitisheldbyhydrogenbonds2'betweenthepeptidenitrogenandoxygenatomsandalsobetweenthefreeaminoandcarboxylgroupsofthediaminoanddicarboxylaminoacidresidues.Weshallnotenterintoalongdiscussionofthepreciseconfigurationsofnativeproteins,aboutwhich,indeed,littlereliableinformationisavailable.Fromthex-rayinvestiga-tionsofAstbury22andhiscollaboratorsitseemsprobablethatinmostnativeproteinsthepolypeptidechain,withtheextendedoroneofthecontractedconfigurationsdis-cussedbyAstbury,foldsbackonitselfinsuchawayastoformalayerinwhichpeptidenitrogenandoxygenatomsofadjacentchainsareheldtogetherbyhydrogenbonds;severaloftheselayersarethensuperposedtoformthecompletemolecule,thebondsbetweenlayers(asidefromthecontinuationofthepolypeptidechainfromonelayertothenext)beinghydrogenbondsbetweenside-chainaminoandcarboxylgroups.Ingeneralnotallofthesidechaingroupswillbeusedinformingbondswithinthemole-cule;somewillbefreeonthesurfaceofthemolecule.Theimportanceofthehydrogenbondinproteinstructurecanhardlybeoverempha-sized.Nocompletereviewofthelargeamountofrecentworkonthisbondisavailable;weshallmentiononlythemoststrikingofitsproperties.2'Thehydrogenbondconsistsofahydrogenatomwhichbondstwoelectronegativeatomstogether(F,0,N),thehy-drogenatomlyingbetweenthetwobondedatoms.Thebondisessentiallyelectrostaticinnature.Thebondedatomsareheldmorecloselytogetherthannon-bondedatoms,theN-H-0distancebeingabout2.8A.Theenergyofastronghydrogenbondis5000to8000cal.permole,thelowervaluebeingapproximatelycorrectforanN-H-0bondas442PROC.N.A.S. CHEMISTRY:MIRSKYANDPAULINGinproteins.Side-chainbondsinproteinsweconsidertoinvolveusuallyanaminoandacarboxylgroup,thenitrogenatomformingahydrogenbondwitheachoftwooxygenatomsandholdingalsooneunsharedhydrogenatom.Inacidsolutionshydrogenbondsmaybeformedbetweentwocarboxylgroups,asinthedoublemoleculesofformicacid.23Thecharacteristicspecificpropertiesofnativeproteinsweattributetotheiruniquelydefinedconfigurations.Thedenaturedproteinmoleculeweconsidertobecharacterizedbytheab-senceofauniquelydefinedconfiguration.Astheresultofincreaseintem-peratureorofattackbyreagents(asdiscussedbelow)theside-chainhydrogenbondsarebroken,leavingthemoleculefreetoassumeanyoneofaverylargenumberofconfigurations.Itisevidentthatwithlossoftheuniquelydefinedconfigurationtherewouldbelossofthespecificpropertiesofthenativeprotein;itwouldnotbepossibletogrowcrystalsfrommole-culesofvaryingshapes,forexample,nortodistinguishbetweencloselyrelatedproteinswhenthemoleculesofeachproteinshowavariabilityinconfigurationlargecomparedwiththedifferencesinconfigurationofthedifferentproteins.Strongsupportofthisviewofthephenomenonofdenaturationispro-videdbytheknowndifferenceinentropyofnativeanddenaturedproteins,whichisabout100E.U.fortrypsin,theentropyofthedenaturedformbeingthegreater."Thisverylargeentropydifferencecannotbeascribedtoadifferenceinthetranslational,vibrational,orrotationalmotion,butmustbeduetoadifferenceinthenumberofaccessibleconfigurations.Itcorrespondstoabout1020accessibleconfigurationsforadenaturedproteinmolecule.Thelargeentropyofdenaturationthusshowsclearlythatthephenomenonofdenaturationconsistsinthechangeofthemole-culesofthenativeproteintoamuchlesscompletelyspecifiedstate.Themagnitudeoftheheatchange,entropychange,andactivationenergyofdenaturation(about30,000cal./mole,100E.U.,and150,000cal./mole,respectively,fortrypsin)canbeinterpretedintermsofourhydrogen-bondpictureoftheproteinmolecule.Weconsiderthenativeproteinmoleculetobeheldinitsdefiniteconfigurationbyside-chainhydrogenbonds,aboutfiftyinnumberforthisprotein(correspondingtoabouttwenty-fiveaminoandtwenty-fivecarboxylsidechains),eachwithabondenergyofabout5000cal./mole,asinsimplersystems.Theactivationenergyof150,000cal./moleshowsthatinorderforthemoleculetoloseitsnativeconfigura-tionaboutthirtyofthebondsmustbebroken.Someoftheside-chaingroupsthenagainformhydrogenbonds;theheatofdenaturationshowsthatontheaveragethereareaboutsixfewersuchbondsinthedenaturedmoleculethaninthenativemolecule.(Theactivationenergyfortransitionofadenaturedproteinmoleculefromoneconfigurationtoanotheriswithoutdoubtsmallerthantheactivationenergyofdenaturation,asitVOL.22,1936443 CHEMISTRY:MIRSKYANDPAULINGinvolvesbreakingasmallernumberofhydrogenbonds,sothatatatempera-tureatwhichtherateofdenaturationisappreciablethedenaturedmole-culewouldrunrapidlythroughitsvariousconfigurations.)Themagnitudeoftheentropyofdenaturationalsofitsintoourpicture;itcorrespondstothenumberofconfigurationsobtainedbyforminghydrogenbondsatrandombetweenabouttwentyaminosidechainsandtwentycarboxylsidechains.Thereagentswhichcausedenaturationareallsubstanceswhichaffecthydrogen-bondformation.Alcohol,urea,andsalicylatearewell-knownhydrogen-bond-formingsubstances;theyformhydrogenbondswiththeproteinsidechains,whicharethuspreventedfromcombiningwitheachotherandholdingtheproteininitsnativeconfiguration.Acidsactbysupplyingprotonsindividuallytotheelectronegativeatomswhichwouldotherwiseshareprotons,andbasesbyremovingfromthemoleculetheprotonsneededforhydrogen-bondformation.Thisconceptionprovidesanexplanationof.thefactsthattheisoelectricpointofaproteinshiftstowardtheneutralpointondenaturation24andthatthepHatwhichtheactivationenergyfordenaturationhasitsmaximumvalueisingeneralnotattheisoelectricpointofthenativeprotein,butbetweenthispointandtheneutralpoint.25Inthenativeproteinmoleculeoftheusualtypesomeaminoandcarboxylside-chaingroupsarepairedtogetherbyforminghydrogenbonds.Theacid-baseproperties-ofthemoleculeareinthemaindeterminedbythegroupswhichareleftfree.Ondenaturationsomeofthepairedgroupsarefreed,aminoandcarboxylinequalnumbers,andinconsequencetheisoelectricpointofthedenaturedproteinisshiftedtowardneutrality.Wehavepicturedtheprocessofdenaturationasinvolvingtheruptureofalargenumberofhydrogenbonds,toformalabileacti-vatedmolecule.Thislabilemoleculeisstabilizedbyactionofbaseonitsfreecarboxylgroupsorofacidonitsfreeaminogroups,theactivationenergythushavingamaximumatapHvaluebetweentheneutralpointandtheisoelectricpointofthenativeprotein.Theactionofultra-violetlightmustbedifferent.Itisnotpossibletoformulateareasonablemechanismwherebyaquantumoflightcanbreaktwentyorthirtyhydrogenbonds.Insteadthelightmustattackthemole-culeinadifferentplace,probablybreakingthemainpolypeptidechainafterabsorptioninatyrosineorotherphenolicresidue,assuggestedbyMitchell.26Thatthisoccursisindicatedbytheobservation27thatafterilluminationwithultra-violetlightinthecolddenaturationoccursonlyonwarming,thoughthenatalowertemperaturethanwithoutillumina-tion;itisclearthatilluminationinthecoldcausesabreakinthemole-cule,which,however,isrestrainedtoconfigurationsneartoitsnativecon-figurationbytheside-chainhydrogenbonds.(Thatsomelooseningofthemoleculeoccursisshownbytheobservationofanincreaseintheavailable4g44PROC.N.A.S. CHEMISTRY:MIRSKYANDPAULINGgroupsineggalbuminafterilluminationinthecold.)Onwarming,thesebondsarebroken;becauseofthebreakinthemolecule,however,itcanbedenatured"inparts,"andhenceatalowertemperaturethanbeforeillumination.Wepredictthatitwillbefoundthatdenaturationbyil-luminationwithultra-violetlightisingeneralnotreversible.Inaconjugatedproteintheprostheticgroupplaysapartinholdingthemoleculeinthenativeconfiguration(hemoglobin,yellowoxidizingferment,visualpurple).Itispossibleinsuchaproteinforreversibledenaturationtotakeplaceafterabsorptionoflightbytheprostheticgroup,nopermanentdamagebeingdonetothemolecule.Inaproteincoagulumside-chainhydrogenbondsholdadjacentmole-culestogether.Nativeproteinsdonotcoagulatebecausemostofthesidechainsareinprotectedpositionsinsideofthemolecule;denaturedproteins(attheisolectricpoint)docoagulatebecausetheyhavealargernumberoffreesidechainsandbecauseinthecourseoftime,asthemoleculeassumesvariousconfigurations,allofthesidechainsbecomefree.Theincreaseofviscosityofproteinsolutionsondenaturationweattributetothechangefromthecompactconfigurationofthenativeproteinmoleculestomoreextendedconfigurations.Anativeproteinmoleculeofsmallmolecularweightmayhavefreesidechainssoarrangedastopermitittocombinewithsimilarmoleculestoformapolymerwithpropertiesdifferinglittlefromthoseofthesmallmolecules.TheobservationsofSvedbergandhiscollaboratorsindicatethatthisisthecaseforhemocyanin,casein,andcertainotherproteins.Ourtheoryofdenaturationleadstodefinitepredictionsregardingtheavailabilityofgroupstoattackbyreagents.Inthelargecompactmole-culeofanativeprotein,oftheorderofmagnitudeof50Aindiameterandhavingthesamestructureaseveryothermoleculeoftheprotein,allgroupswouldbeprotectedfromattackbyreagentsexceptthoseonthesur-faceornearthesurface.Afterdenaturationoftheprotein,however,themolecule(insolution)wouldinthecourseoftimeassumevariousconfigura-tions,andeverygroupinthemoleculewouldbecomeavailabletoattack.Thesestatementsareincompleteagreementwiththeexperimentalresultsregardingsulfhydryl,disulfide,andphenolgroupsmentionedabove.Theobservationthatthenumberofavailablegroupsisincreasedwhenthesolutionismadealkaline,thoughnotalkalineenoughtocausedenaturation,showsthatundertheseconditionssomeofthehydrogenbondsintheproteinmoleculearebroken,causingittoassumeaconfigurationsomewhatmoreopenthanitsoriginalconfiguration.Althoughadenaturedproteinmoleculeinacoagulumisnotfreetoas-sumeallconfigurations,beingrestrainedbybondstoitsneighbors,ingeneralitsconfigurationwillbesoopenastomakeallgroupsaccessibletoattack.However,asmentionedabove,thereisnochangeintheavail-VOL.22,1936445 CHEMISTRY:MIRSKYANDPAULINGabilityofgroupsinmyosinwhenitisrenderedinsolublebydryingorwhenwaterisremovedbyfreezing.Weinterpretthisasshowingthatcoagula-tionofthistypeisnotaccompaniedbydenaturation;thatis,themoleculesdonotlosetheiruniquelydefinedconfigurations.Ondehydrationofthenativeproteinthesurfacesidechainsofadjacentmoleculesformbondssufficientlystrongtoproduceacoagulumofnativeproteinmolecules.Itwouldbeexpectedthatthemechanicalbolsteringeffectofadjacentmole-culesinthiscoagulumwouldaidthemoleculestoretaintheirnativeconfigurations;thisisobservedtobethecaseforeggalbuminandsomeotherproteins,whichinthisstatearedenaturedbyheatonlyatatempera-tureconsiderablyhigherthanbeforedehydration.6Somefeaturesofthetheoryofproteinstructurediscussedabovehavebeensuggestedbefore.Manyinvestigatorshavecorrelatedthespecificpropertiesofnativeproteinswithdefinitelyspecifiedmolecularconfigura-tionsandthelossofspecificpropertiesondenaturationwithchangeincon-figuration.Astburyandhiscollaboratorsinparticular,indiscussingtheirx-rayinvestigations,whichhaveprovidedsomuchvaluableinformationonproteinstructure,havestated22thatdehydrationandtemperaturede-naturationleadtodestructionoftheoriginalspecialconfigurationofthenativeprotein,givingadebrisconsistingsimplyofpeptidechains.Ourpictureagreeswiththeirsexceptinregardtotheeffectofdehydration,whichwebelievetoconsistprimarilyinthecoagulationofmoleculeswithessentiallyunchangedstructure.Thehydrogenbondswhichwepostulatetoexistbetweenside-chaincarboxylandaminogroups(eachnitrogenatomattachedbyhydrogentotwooxygenatoms,withthedistancesN-H-Oequalto2.8A)arearefinementoftheside-chainsalt-likelinkagesdiscussedbyAstbury.Ourpictureofthespreadingofproteinfilmsonwater,involvingtheunfoldingofcompactmoleculestolayersoneamino-acid-residuethick,isnotessentiallydifferentfromthatofGorterandNeurath,whohavesuggestedunfoldinginconnectionwithfilmformation,thoughnotwithdenaturationingeneral.28Inthispaperwehavediscussedinsomedetailaverydrasticchangeinconfigurationofproteinmolecules,thatconnectedwithdenaturation.Wehavepointedoutthatthelargeentropyofdenaturationprovidesstrongsupportforoursuggestedstructuresofnativeanddenaturedproteinmole-cules,andthatmanyotherphenomenacanalsobeinterpretedinasimplewayfromthispointofview.Therealsohasbeenputforthrecentlysomeevidencethatsmallchangesinconfigurationofproteinsoccur,whichplayanimportantpartinproteinbehavior.ThusNorthropandhiscollabora-torshavepreparedanenzymaticallyinactiveproteinwhichonslighthydrolysisbytrypsinistransformedintonativetrypsin,21andanotherwhichissimilarlytransformedbypepsinintonativepepsin,"andGorter"hasshownthatmyosinspreadsonwateronlyafterslighthydrolysis.The446PROC.N.A.S. CHEMISTRY:MIRSKYANDPAULINGstructuralinterpretationofthesephenomenacanbemadeonlyafterfurtherexperimentalinformationisavailable.*OnleaveofabsencefromtheHospitaloftheRockefellerInstitute.1Zinsser,H.,andOstenberg,Z.,Proc.NewYorkPath.Soc.,14,78(1914).2Landsteiner,K.,andHeidelberger,M.,Jour.Gen.Physiol.,6,131(1933).3Barcroft,J.,TheRespiratoryFunctionoftheBlood,PartII,CambridgeUniv.Press,1928.4Anson,M.L.,andMirsky,A.E.,Jour.Physiol.,60,50(1925).5Northrop,J.H.,Jour.Gen.Physiol.,13,756(1930);16,33,323(1932).6Chick,H.,andMartin,C.J.,Jour.Physiol.,40,404(1910);43,1(1911-12);45,61,261(1912-13).7Anson,M.L.,andMirsky,A.E.,Jour.Gen.Physiol.,15,341(1932).8Heffter,A.,Mediz.nat.Arch.,1,81(1907);Arnold,V.,Zeit.Physiol.Chem.,70,300(1911);Mirsky,A.E.,andAnson,M.L.,Jour.Gen.Physiol.,18,307(1934-35);19,427,439(1936).9Mirsky,A.E.,unpublishedexperiments.10Wald,G.,Jour.Gen.Physiol.,19,351(1935);Mirsky,A.E.,Proc.Nat.Acad.Sci.,22,147(1936).1Mirsky,A.E.,Jour.Gen.Physiol.,19(1936),inpress.12Mirsky,A.E.,unpublishedexperiments.13Lewis,P.S.,Biochem.Jour.,20,978,984(1927);Loughlin,W.J.,Biochem.Jour.27,1779(1933).14Anson,M.L.,andMirsky,A.E.,Jour.Gen.Physiol.,9,169(1925);Jour.Phys.Chem.,35,185(1931).15Anson,M.L.,andMirsky,A.E.,Jour.Gen.Physiol.,17,393,399(1934).16Weber,H.H.,Ergeb.d.Physiol.,36,109(1934).17Svedberg,T.,Science,79,327(1934).18vonMuralt,A.,andEdsall,J.T.,Jour.Biol.Chem.,89,315(1930).19Theorell,H.,Biochem.Zeitschr.,278,263(1935).20Hartridge,H.,Jour.Physiol.,44,34(1912).21Latimer,W.M.,andRodebush,W.H.,Jour.Am.Chem.Soc.,42,1419(1920);Pauling,L.,Proc.Nat.Acad.Sci.,14,359(1928);Sidgwick,N.V.,TheElectronicTheoryofValency,OxfordUniversityPress,1929;Bernal,J.D.,andMegaw,H.D.,Proc.Roy.Soc.,AISI,384(1935).22Astbury,W.T.,andStreet,A.,Phil.Trans.Roy.Soc.,A230,75(1931);AstburyandWoods,H.J.,Ibid.,A232,333(1933);AstburyandSisson,W.A.,Proc.Roy.Soc.,Al50,533(1935);AstburyandLomax,R.,Jour.Chem.Soc.,1935,846;Astbury,Dick-inson,S.,andBailey,K.,Biochem.Jour.29,2351(1935);seealsoMeyer,K.H.,andMark,H.,DerAufbauderHochpolymerenOrganischenNaturstoffe,AkademischeVerlags-gesellschaft,M.B.H.,Leipzig,1930.23Pauling,L.,andBrockway,L.O.,Proc.Nat.Acad.Sci.,20,336(1934).24Heidelberger,M.,andPederson,K.O.,Jour.Gen.Physiol.,19,95(1935);Pederson,K.O.,Nature,128,150(1931).?ALoughlin,W.J.,Biochem.Jour.,27,1779(1933).26Mitchell,J.S.,Nature,137,509(1936).27Bovie,W.T.,Science,37,373(1913).28Gorter,E.,andGrendel,F.,Proc.Acad.Sci.Amsterdam,29,371(1926);Neurath,H.,J.Phys.Chem.,40,361(1936).29Kunitz,M.,andNorthrop,J.H.,Science,80,505(1934).3'Herriott,R.M.,andNorthrop,J.H.,Ibid.,83,469(1936).31Gorter,E.,Nature,137,502(1936).VOL.22,1936447