CATALYZED HYDROLYSIS OF AMIDE AND PEPTIDE BONDS IN PROTEINS 1

Transcription

1 U. S. DEPARTMENT OF COMMERCE NATONAL BUREAU OF STANDARDS RESEARCH PAPER RP153 Pat f Junal f Reseach f the N:atinal Bueau f Standads, Vlume 29, N:vembe 1942 CATALYZED HYDROLYSS OF AMDE AND PEPTDE BONDS N PROTENS 1 By Jacint Steinhadt and Chales H. Fugitt 2 ABSTRACT The ates f hydlysis by dilute acids f bth a disslved ptein (egg albumin) and an insluble ptein (wl) ae shwn t depend nt nly n the tempeatue and acidity but als n the acid used. When hydlyzed at 65 C by cetain stng mnbasic acids f high mlecula weight, the amide and the peptide bnds ae bken ve 1 times as fast as when they ae hydlyzed with hydchlic acid. Even amng the cmmn mineal acids, lage diffeences appea. These diffeences in hydlytic effectiveness paallel diffeences in the affinities f the anins f the acids f ptein. A futhe easn f attibuting t his effect t the anins is the attainment, with anins f high affinity, f a maximum ate f amide hydlysis at elatively lw cncentatins, stichimetically equivalent t the sum f the amin plus the amide gups. A simila limiting anin cncentatin maximum ate f hydlysis f t he much me numeus peptide gups is nt bseved. On the basis f details f the dependence f the ate f hydlysis n cncentatin f effective anins and hydgen ins, a mechanism which invlves cmbinatin f the gups hydlyzed with hydgen ins, is ppsed. At lw cncentatins f effective anins, amide hydlysis is catalyzed me stngly than peptide hydlysis. By keeping the cncentatin slightly belw stichimetic equivalence t the sum f the amin plus amide gups the amide gups may be apidly hydlyzed withut extensive hydlysis f the peptide bnds in the ptein. Pactical applicatins ae suggested. CONTENTS Page 1. ntductin 316. Expeimental pcedue Expeimental cnditins Analytical methds 316. Results and discussin Relative effectiveness f diffeent acids Dependence f eactin ate n cncentatin f catalytically effective anins Dependence f eactin ate n cncentatin f hydgen ins Dependence f eactin ate n cncentatin f effective anins and n tempeatue at highe cncentatins f acid Results btained with a sluble ptein 323 V. Mechanism f the catalysis 324 V. Ptential fields f applicatin 326 V. Refeences 327 A bief accunt f this wk was pesented at the Bstn meeting f the Ameican Sciety f Bilgical Chemists in Apil Reseach Assciates at the Natinal Bueau f Standads, epesenting the Textile Fundatin. 315

2 l 316 Junal f Reseach f the Natinal Bueau f Standads 1. NTRODUCTON The ate f hydlysis f pteins in dilute slutins f stng acid s has lng been knwn t depend n tempeatue and n cncentatin f acid. R ecently it has been fund that the ates f hydlysis f bth an insluble ptein (wl) and a sluble ptein (egg albumin) als depend n the chice f hydlyzing acid [7).3 Since lage diffeences in hydlytic effectiveness appea amng acids which ae ttally dissciated, it is evident that the ate f hydlysis is influenced stngly by anins. This catalytic influence is futhe demnstated by the expeiments descibed in the pesent pape. The esults yield infmatin cncening the mechanism f hydlysis, and suggest that pteins can be hydlyzed unde cnditins cnsideably milde than thse custmaily emplyed.. EXPERMENTAL PROCEDURE 1. EXPERMENTAL CONDTONS Tw pteins, puified wl and cystallized egg albumin, wee hydlyzed at tw tempeatues (65 C and 75 C,±O. ). Details f the puificatin f bth pteins and, with few exceptins,4 f the eagents used, have been descibed elsewhee [8, 9]. The kinetic pcedues wee as fllws: F each measuement with wl a ptin was immesed in a quantity f slutin, peviusly bught t the tempeatue f the themstat. The pptin f 98 ml f slutin pe g f dy wl was always emplyed. With egg albumin, the cncentatin f ptein, afte mixing a stck slutin with the eagents, was.71 pecent. Glass-stppeed flasks wee used j they wee sealed as sn as the wl was thughly wetted, t pevent evapatin. Afte the desied time had elapsed, the flasks wee cled apidly, pened, and aliquts f 5 1 ml wee emved and analyzed as descibed in the next sectin. Althugh the egg albumin is almst instantly denatued at the cncentatin f acid (.53 M) used in the expeiments with this ptein, it emains in slutin at all cncentatins f sdium ddecylsulfnate abve.1 M. Hweve, as the pducts f hydlysis accumulate, they pecipitate, unless the cncentatin f ddecylsulfnate is abve.16 M. 2. ANALYTCAL METHODS A distinctin has been made expeimentally between the hydlysis f amide bnds (RCO-NH2 ) and f peptide bnds (RCO-NHR'). The fist may be fllwed with bth pteins by measuing the evlutin f ammnia, wheeas the secnd may be studied by a diect methd (the fml titatin) nly with the sluble ptein. Hweve, the methds which indicate the extent f peptide hydlysis ae available, and wee used with bth pteins, as descibed late. The ammnia evlved was adsbed n pemutit befe distillatin t emve it fm much f the the disslved mateial pesent, thus deceasing the dange f secnday pductin f ammnia duing the, Figues in backets indicate the liteatue efeen ces at the end f this pape. 'The sulfate half-estes (ROSO,H) wee supplied thugh the cut.esy f E. 1. du P nt de Nemus & C., nc. Mixtues f these half-estes have been used t denatue pteins [1]. (

3 L Oatalyzed Hydlysis f Pteins 317 distillatin with alkali. n the expeiments with egg albumin, deteminatins f ammnia wee als made n ptein-fee filtates, which wee btained by pecipitating the unchanged ptein with tichlacetic acid as explained belw. The esults btained by the tw methds ageed clsely. The hydlysis f peptide bnds in egg albumin was measued by detemining the incease in the fml titatin value, as in Nthp's mdificatin [4) f Sensen's methd. Since the incease cespnds t the nnmbe f cabxyl gups libeated by the' hydlysis f bth amide and peptide gups, the amunt f hydlysis f the latte is given by the diffeence between the incease in the fml tite and the amunt f ammnia evlved. As its peptide bnds ae hydlyzed, pat f the wl disslves. The amunt disslved, expessed in tems f its nitgen cntent as detemined by Kjeldahl analysis, has theefe been used as a cnvenient measue f the ttal extent f hydlysis f the wl. Subtactin f the ammnia evlved gives a quantity designated as "nn ammnia nitgen," which depends nly n the hydlysis f peptide bnds. Since patially hydlyzed egg albumin is nt pecipitated by tichlacetic acid, Kjeldahl analyses f the slutins f this ptein, afte mixing with tichlacetic acid, have been used as an additinal indicatin f the extent f peptide hydlysis in this ptein. The tem "disslved nitgen" is als applied t this expeimental quantity. The pesence f sdium ddecylsulfnate and cetain the salts intefes with the pecipitatin f ptein by tichlacetic acid in the cncentatins f this eagent custmaily used. Hweve, tests shwed that in the pesence f.3 M sdium ddecylsulfnate, the amunt f ptein pecipitated was pactically independent f the cncentatin f tichlacetic acid at cncentatins abve.47 M. This cncentatin has been used thughut. Wheneve the cncentatin f sdium ddecylsulfnate was belw.3 M, enugh f the salt was added t the slutin just befe sampling it t bing the cncentatin up t this value befe additin f the pecipitant.. RESULTS AND DSCUSSON 1. RELATVE EFFECTVENESS OF DFFERENT ACDS The elative effectiveness f vaius anins has been chiefly detemined by measuing the ate at which ammnia is libeated by hydlysis f amide linkages. This is the ate f a definite chemical eactin invlving the beaking f nly ne bnd. Th esults f many such measuements made with wl ae shwn in figue 1. t is bvius that diffeent acids vay enmusly in thei effectiveness in hydlyzing the amide bnds f this ptein. Thus the ates given by hydchlic acid and cetylsulfnic acid,5 diffe by a fact f 114. All degees f effectiveness between these extemes ae epesented by the the acids studied. Thei elative effectiveness as hydlyzing agents tends t paallel the affinity f thei anins f pteins, as peviusly epted [9J. A numbe f the acids have been studied which ae nt included Because f its lw slubility, cetylsulfnic acid was tested at a lwe cncentatin (.2 M), but the ttal cncentatin f acid was.5 M, as in the the expeiments

4 318 Junal f Reseach f the Natinal Bueau f Standads.8.5 M ACD.5 M He1 PLUS.5 M NO SALT HYDROCHLORC NAPtlTHALEl'iESULFOHC (!J SulfURC O?HENYlSULFONATE e PCRC OCTYLSULFATE,.'.- p-ophenylbenzeti SUlFOHC ff OOOECVLSUlFOHATE WOOL ;t NA?HTHOL OSlJLf"ONATE 65 C Eli ORANGE n 1'5 DOOEC YLSULFATE 4J P DOOECYSULFONC CETYLSULFONATE (.2 "' DOOECYLSULFijRC --- <t.6 ffi "- ::; -' ".4 -' <t Z <t.2 () TME - HOURS FGURE.-Relative effectiveness f vaius stng acids in hydlyzing the pimay amide bnds f wl. All the acids ae cmpaed at the same cncentatin f hydgen in (.5 M) and at the same tempeatue (65 ). Pints cssed by a shilling mak epesent measuements btained with equimla mixtues with hydchlic acid f the sdium salts f the anins indicated. in figue 1. Amng them is the hmlgus seies f sulfate halfestes (ROSOaH), f which cmpaative data ae given in table 1. Hee a maximum hydlytic effect appeas t be eached with the 14-cabn acid. This effect is a cnsequence f the lwe hydgenin cncentatin in slutins f the highe hmlgues, a esult f an inceasing tendency t fm mlecula aggegates as thei mlecula weight inceases [1]. The esults btained with the sulfate halfestes ae futhe cmplicated by the fact that as thei mlecula weights incease they ae inceasingly apidly hydlyzed at 65 C t give sulfuic acid and the alchls. 6 Thus with these acids the ate f the hydlysis is affected by a simultaneus incease in acidity and decease in the cncentatin f effective anins as hydlysis pceeds. This cmplicatin des nt affect the data btained with the sulfnic acids (RSOaH), which ae shwn in figue 1. The half peids f this self hydlysis, in the case f the highest membes f the seies, ae less than 24 hus.

5 Oatalyzed Hydlysis f Pteins 319 T ABLE.-Relative effectiveness f Va1'ius sulfate half-estes in acceleating the acid hydlysis f wl at 65 C All the slutins cntained.5 M Hel plus.5 M sdium salt f the half-estes Sulfate half-este n-octyl- n-decyl- n-ddecyl- n-tetadecyl- n-hexadecyl- n-octadecyl- Amide hydlysis. Time equied t libeate.3 millimle ammnia. pe gam HOUTS Peptide hydlysis. Time equied t libeat 1. millimle f nn ammnia nitgen pe gam Hus , 2. DEPEN DENCE OF REACT ON RATE ON CONCENTRATON OF CATALYT CALLY EFFECTVE ANONS The influence f the me effective anins may be appaised, apat fm the simultaneus influence f hydgen ins, by vaying the cncentatin f the sdium salt f a stngly effective anin, at a fixed cncentatin f ne f the least effective acids, i. c., hydchlic acid. This pcedue is justified because the effect f the pesence f sdium and chlide ins is elatively small (f. data f ddecylsulfnate in figue 1) ,..,, , ,.-, WOOL - 65 C M HC PLUS DODECYLSULFONATE :; " : : w Q, (/) W..J :; :: w..j z ",6.4.5 M ".3 M (J).2 M e.15 M.1 M 4. z :::; c z j) < ;: ;= 2. C ;: (f) " " lj,j.'''''''-+-t 1. i! ;:: 25 5 TME - HOURS FGURE 2.-Hydlysis f wl in.5 M hydchlic acid at 65 C, as affected by the cncentatin f ddecylsulfnate in. The hydlysis f amide bnds is epesented in the left-hand fame, and the evlntin f disslved nitgen, which is a functin f the hydlysis f peptide as well as amide bnds, is epesented in the fame n the ight. n the latte, expeiments with each cncentatin f anins ae epesented by a pai f cuves, the aea between which is shaded. The uppe cuve f each pai epesents the ttal disslved nitgeu, and the lwe cuve epesents the nnammnia nitgen, which efes t the hydlysis f peptide bnds nly,

6 32 Junal f R eseach f the Natinal B ueau f Standads The esults ne suchexpeiment with sdium ddecylsulfnate at 65 C ae shwn in figue 2. The measuements f amide hydlysis (left-hand fame) ae paticulaly significant. The ate f hydlysis inceases shaply as the cncentatin f ddecylsulfnate is inceased until a limit is eached at a cncentatin between.15 M and.2 M. Beynd.2 M n incease in ate ccus.7 An the limit exists at vey lw cncentatins «.8 M, nt shwn in the figue). This limit is pactically identical with the cuve shwn in the figue f hydchlic acid alne. The lwest cncentatin at which the ate is appeciably affected by the pesence f ddecylsulfnate is stichimetically equivalent t the amunt f stngly basic gups in the ptein.s The cncentatin at which the uppe limiting ate is eached cespnds ughly t the sum f the amin gups (.8 millimle pe g) plus the amide gups (.8 t.9 millimle pe g) f the wl. The ate f peptide hydlysis, as measued by the evlutin f nnammnia nitgen (ight-hand fame f fig. 2), is affected by the same ange f ddecylsulfnate cncentatins in a diffeent way. Lw cncentatins (.1 t.2 M) cause nly a small incease in the ate f libeatin f nnammnia nitgen as cmpaed with thei effect n the ate f evlutin f ammnia. Thus the additin f small amunts f this anin t slutins f hydchlic acid selectively favs the hydlysis f amide linkages. With lage amunts the ate f pductin f nnammnia nitgen steadily inceases (withut a cespnding incease in ate f amide hydlysis). N limit is eached within the ange f cncentatins studied, and the selective effect disappeas. The ates f libeatin f disslved nitgen ae nt pptinal in a simple manne t the cncentatins f ddecylsulfnate. nstead, the esults ughly fllw Schlitz's ule, which states that the squae t f the time equied t pduce a given enzymatic hydlysis is pptinal t the cncentatin f enzyme [2]. 3. DEPENDENCE OF REACTON RATE ON CONCENTRATON OF HYDROGEN ONS Althugh the ate f amide hydlysis eaches an uppe limit as the cncentatin f effective anins is inceased, this limiting ate may be aised by inceasing the cncentatin f hydgen ins. Expeiments in which the cncentatin f hydchlic acid was vaied at tw diffeent cncentatins f sdium ddecylsulfnate (fig. 3) shw that the dependence f the ate f hydlysis n hydgen-in cncentatin is als limited. Thus the ate becmes independent f the acid cncentatin at a cncentatin five t seven times as high as the cncentatin f anin at which a limit is eached. The cuves epesenting the evlutin f ammnia at bth anin cncentatins studied ae pactically identical. The ate f peptde hydlysis, indicated by the pductin f nnammnia disslved nitgen, als ises as the hydgen-in cncenta1 The slight diminutin in ate at.5 M is due t the slightly lwe h ydgen-in cneentatin f t his slutin, in which decmpsitin pducts me quickly accumulate. Abut.8 millimle pe g, cespnding t abut.8 M at the ati f wl t slutin in these expeiments. (

7 Oatalyzed Hydly.sis f Pteins 321 tin is inceased. Extaplatin f the data appeas t indicate that a limiting ate is eached at cncentatins::f acid smewhat highe than thse studied. NdD'.3 M NdD.3 M t f------h4.. 6 Nd DODECYLSULFONATE z PLUS HYDROCHLORC ACD 2. ::; ; a:.25 l!i a. ().15 M <n e.1 M :::E 3 ;; w " :; w.6 NdD '.2 M M Z." ; " ":n.3 4. " z in <n < ";= ;: <n 2. J 3: TME - HOURS FGU RE 3.- Relative ates f anin-catalyzed hydlysis f wl at 65 C, as affected by the cncentatin f acid. Unlike figues 2 and 4, t.he cuves epesent the esults btained with diffeent cncentatins f acid in stead f diffeent cncentatins f salt. Results with tw diffeent cncentatins f ddccylsllifnate ae gi ven. Bth cncentatins ae abve the level at which the limiting ate f amide hydlysis in.5 M acid is attained. 4. DEPENDENCE OF REACTON RATE ON CONCENTRATON OF EFFECTVE ANONS AND ON TEMPERATURE AT HGHER CONCENTRATONS OF ACD The cuse f hydlysis with espect t time, shwn in figues 1 and 2, is affected by changes in ph and in cncentatin f effective anins, as the pducts f hydlysis accumulate. These effects may be educed by wking with highe cncentatins f acid. The esults f expeiments made with thee times the cncentatin f acid epesented in figue 2 ae shwn in figue 4. Data ae given f tw tempeatues. Owing t the high initial cncentatin, the change in ph in these expeiments neve exceeded.4 unit. This esults in smewhat simple chemical kinetic elatins. The ate f libeatin f nnammnia disshed nitgen is diectly pptinal t the cncentatin f catalytically effective anins, with gd appximatin. Hweve, the time cuse f the hydlysis f amide bnds emains cmplex. A apid inital evlutin f ammnia (abut.6 millimle pe g) is

8 322 Junal f Resem'ch f the Natinal Bueau f Standads fllwed by a much me gadual eactin, which is hadly faste than when n ddecylsulfnate is pesent. The cuse f the eactin in slutins f hydchlic acid alne appeas t be simple; if the amide cntent f wl is abut.84 millimle pe g,9 the eactin is f the fist de t cmpletin. The eactin catalyzed by ddecylsulfnate fllws fist-de kinetics nly in the peid f the initial apid evlutin f ammnia (.6 millimle pe g). Tbus it appeas that nly pat f the amide bnds may be susceptible t this catalyst. 1 The effect f tempeatue n the ate f hydlysis is smalle in the pesence f the added salt (Ql=2.3) than when acid alne is.6 ".3 z "' i5 a. " in...j ;;; ":; "..J ;:: ;= ;;:.6 ;li...j < " z.. ;::.3 " " =< : TME - HOURS FGURE 4.-Hydlysis f wl in.15 M hydchlic acid, as affected by the cncentatin f ddecylsulfnate in and by the tempeatue. used (Q!=3.). This diffeence agees with the cmmn ule that the activatin enegies f catalyzed eactins ae lwe than thse f the same eactins in the absence f a catalyst. Because f this diffeence, the catalytic cefficients f the anin (the fact by which it inceases the ates) is lwe at the highe tempeatue. The catalytic cefficient is als lwe at.15 M acid than at.5 M. Thus, at still highe cncentatins, such as thse nmally used f ptein hydlysis, the catalytic effect f ddecylsulfnate ins may be entiely negligible (cf. sectin V). Tbis value (1.18%) is clse t thes btained in this labaty by a numbe f diffeent metbds. 1 Of tbe tw amin-acids in wl wbicb may be pesent in tbe m f amides, glutamic acid is und in a quantity which cespnds clsely with tbe apidly evlved ptin f tbe ammnia.

9 Oatalyzed Hydlysis f Pteins RESULTS OBTANED WTH A SOLUBLE PROTEN t is f geat inteest t detemine whethe the specific hydlytic effects f anins which manifest themselves with wl als affect the pteins. Egg: albumin has been chsen f investigatin as an example f a sluble ptein, pincipally because the caeful wk f She, Wilsn, and Stucck [6J pvides a clse estimate f its amide cntent. The esults btained in a seies f expeiments analgus t thse pefmed with wl (fig. 2) ae shwn in figue 5. n mst imptant O.75F"'=====,;,,===,;===..sl--f-Jl':;"''----t----t EGG ALBUMN 65 C.7% ::;; <t ffi n: w CL 1.5 (f) w...j ::;; :J...J EVOLUTON OF AMMONA.5 M Hel PLUS SODUM DODECYLSULFONATE.3 M.16 M -fh-<--+---j-.13 M.1 M 2. ;:: j= (f) -u :u G :u J ;;: NON PRECPTABLE NTROGEN T ME - _--- He HOURS FGURE 5.-Hydlysis f fu times cystallized egg albumin in.5 M hydchlic acid at 65 C., as affected by the cncentatin f ddecylwlfnate in. The hizntal bken line in the uppe cft hand fame epesents the amide cntent f egg albumin [fij. The lwe 1ft hand fame epesents the change in the fml tite as the esult f libeatin f cabxyl gups in the ptein. The bken line in this fame epesents the hydlysis f peptide bnds at the highest cncentatin f ddecylsulfnate epesented. t was btained by subtacting the apppiate Cuve f ammnia evlved fm the tp cuve in this figue. paticulas they ae entiely paallel with thse btained with wl. The ate f evlutin f ammnia becmes independent f the ddecyisulfllate cncentatin at a lwe cncentatin (.11 M) than with wl, cespnding t stichimetic equivalence t the sum f the basic gups plus amide gups f these ptein slutins. ll Unlike wl, egg albumin libeates pactically all f its amide nitgen vey quickly; the expeimental values thus sn appach thse peviusly epted [6]. Althugh the state f dispesin f the tw pteins is entiely diffeent, the initial ate f ammnia evlutin is nly slightly highe with egg albumin than with wl. 11 The titatin data f Kekwick and Cannan [5J indicate that egg albumin cntains pe gam abut.9 millimle f smiu and guanidin gups. The amide cntent (6) is abut.7 millimle pe g. The sum, 1.6 millimles pe g, cespnds t a cncentatin f abut.11 M at the cncentatin f ptein used n these expeiments.

10 324 Junal f Reseach f the Natinal Bueau f Standads With this sluble ptein, the fml titatin gives a diect measue f the ttal numbe f bnds hydlyzed. Data btained by this methd, with seveal cncentatins f ddecylsulfnate, ae shwn in figue 5. The ate f hydlysis inceases withut limit within the ange f cncentatins studied. At the lwest cncentatin the hydlysis indicated by the fml titatin is hadly lage than that due t beaking amide linkages nly, but at highe cncentatins the inceased ate is bviusly due t nceased peptide hydlysis. The selective effect f lw cncentatins f anins, indiectly indicated by the wk with wl, is hee diectly shwn. 12 The de f effectiveness f the anins tested with wl (table 1 and fig. 1) is the same when egg albumin is the test ptein. Althugh the mst effective f the acids tested (cetylsulfnic) has a catalytic cefficient (with espect t hydchlic acid) f abut 2, nne f these catalysts appaches clsely t the catalytic activity f t.he ptelytic enzymes, which ae substances f much highe mlecula weight. Weight f weight, cystalline pepsin at 35.5 is abut 3, times me effective than cetylsulfnate at 65 in libeating cabxyl gups in egg albumin. V. MECHANSM OF THE CAT AL YSS The existence f an uppe limit t the anin-catalyzed ate f hydlysis f amide bnds, and the absence f a simila limit t the ate f hydlysis f the me numeus peptde bnds, within the ange f anin cncentatins studied, suggest a mechanism f hydlysis which invlves cmbinatin f the effective anins wth the gups hydlized. This suggeston is suppted by the stichimet.ic cespndence between the amunts f ddecylsulfnate which suffice t give the maximum ate and the numbe f stngly basic plus amide gups in the pteins tested, which indicates that small amunts f anins cmbine pefeentally with the amide gups. The stichimetic intepetatin f this limit has been suppted by shwing that, unde a given set f cnditins, the amunt f ddecylsulfnate pesent, elative t the amunt f wl, athe than its cncentatin in slutin, detemines the hydlytic ate. One f the slut.ins"'epesented 'in' figue 2- (.2 M in 'sdium ddecylsulfnate, and.5 M in hydchlic acid) was diluted with an equal vlume f.5 M hydchlic acid. The cncentatin f effective anin thus fell belw that equied f the attainment f the maximum ate f amide hydlysis in the ealie expeiments, althugh the ttal amunt emained unchanged. The ate f pductin f ammnia emained as high as if the ddecylsulfnate had nt been diluted. The esult f the expeiment just descibed depends n the fact that ddecylsnlfnate in is almst quantitatively tansfeed fm the slutin t the fibes [9J. With anins having a lwe affinity f wl, the cncentatin at which a limiting at.e is attained shuld be highe than with ddecylsulfnate, and shuld nt be assigned stichimetic significance. Data btained with an anin f nly mdeate affinity f wl, ctylsulfate, ae shwn in figue The fml data als indicate that nly abut 1 pecent f the peptide bnds in the ptein wee hy dlyzed when tw-thid s f the ptein was nt pecipitated with tichlacetic acid.

11 Oatalyzed Hydly,sis f Pteins 325 The cncentaton f ctylsulfate at which a maximum ate f amide hydlysis is attained is at least five times as geat as in the expeiments with the in f highe affinity. Hweve, the maximum ate btained with ctylsulfate is nly slightly lwe than the maximum attained wth ddecylsulfnate. Thus, the tw anins, when cmbined with the ptein, ae almst equally effective. 13 Since the maximum anin-catalyzed ates attained at a given acid cncentatin may be inceased by aising the cncentatin f acid, it is appaent that hydgen ins, as well as anins, paticipate in the " <t : C : W l.!/l W..J :J "..J :E w..j W.6.4 WOOL- 65 C / <t Z.2 J--.'cf--cf '''' l " <t.15 M Hel PLUS SODUM OCTYLSULFATE.15 M.1 M (j).5 M e.3m.15m z 3. G) z in " ;: ". ;1l ; G) ; l TME - HOURS 4 FGURE 6.-Hydlysis f wl at 65 C, as affected by the cncentatin f ctylsulfate in. The cncentatin f acid (.15 M) is highe tban in tbe expeiments epesented in figue 2. Tbe bken lines, included [ cmpaisn, epesent data btained witb the same cncentatin f acid, and.3 Al sdinm dctecylsulcnate, a cncentatin well in excess f tbe amunt equied t attain the maximum ate [ amide hydlysis witb tbis anin. eactin. Hweve, the fact that the effect f vaying the cncentatin f acid is als limited t elatively lw cncentatins «.1 M) shws that its influence is nt a cmmn case f smple hydgen-in catalysis. n the latte, nly an exceedingly small factin f the hydgen ins cmbine with the substate mlecules. n the pesent case, a vey cnsideable pat must cmbine since satuatin f the cmbining gups in the ptein is evidently attained at elatively lw cncentatins. Unde dinay cnditins, amide and peptide gups ae t weakly basc t cmbine t any appeciable extent with hydgen ins at these cncentatons f acid, but thei 11 The small esidual diffeences bctween tbe limiting effects f vaius anins may be attibuted t a kinetic salt effect f tbe uncmbined ins.

12 326 Junal f Reseaph f the Natinal Bueau f Standaas eadiness t cmbine with hydgen ins may be geatly inceased by acquiing a negative chage by cmbinatin with an anin. Smila effects f chage n the stengths f acids and bases have been discussed by the pesent auths [8, 9] and have been teated theetically by many thes. The fact that much me hydgen in than ddecylsulfnate in must be pesent t attain the maximum ate f amide hydlysis mdicates that the pteins cm bine with hydgen in less eadily than with ddecylsulfnate. This indicatin is cnsistent with the elative affinities f wl f these tw ins, as peviusly epted [91. f insufficient hydgen in is pesent, the amide gups may be fully cmbined with anins f high affilllty withut attaining the maximum ate f hydlysis (fig. 3). This maxmum is attained with full cmbinatin with hydgen ins. Accdingly, the influence Lthe catalytically active anins n bth amide and peptde gups is pimaily exeted by pmting the cmbinatin f hydgen ins with these weakly basic gups in dilute acid slutins. The agent which actively induces the liability f the C-N bnds is the cmbined hydgen in.14 n a sufficiently acid envinment the catalytic influence f the anins shuld disappea (cf. p. 322). Unlike the geneal basic catalysis defined by BS'lnsted, the catalytic activity f the anin depends n its affinity f ptein athe than n its inheent stength as a base. Whethe the mechanism descibed is applicable t the amides, and whethe the actin f anins f high mlecula weight gives a clue t the still me effective actin f ptelytic enzymes, cannt be detemined withut futhe wk. V. POTENTAL FELDS OF APPLCATON The pactical cnsequences f these phenmena aise pat.v fm the pssibility they affd f hydlyzing pteins unde milde cnditins than thse which ae habitually emplyed, bth with espect t cncentatin f acid and t tempeatue. The selective actin f lw cncentatins f the catalysts in hydlyzing amide bnds als may be useful in analysis, in the pductin f mdified pteins. This selective effect is faved by (a) use f anin cncentatins nly slightly geate than ae equied f cmbinatin with the amm and guanidin gups f the ptem, (b) lw acidities, such as.5 M, (c) elatvely lw tempeatues, such as 65 C. The esult is als influenced by the chice f anin, a highe selective actin esulting fm the use f cetylsulfnate diphenylbenzenesulfnate than fm the use f any the ins tested. Mild methds f ptein hydlysis ae f value in the pepaatin f hydlysates f intavenus 1'[11 use in medicine [3], b ecause they ae less likely t impai the nutitive value f the digested mateial. Likewise, it is smetimes desiable t disslve desty ptein mateials withut affecting the mateials which may be assciated with them. The emval f glues and albuminus catings fm cellulsic mateials is ne instance; the puificatin f enzymes and the bilgical pducts is anthe. "The eactive intemediate may be eithe /OH R-J'-NH+a R-C=NH+,.

13 Oatalyzed Hydlysis f Pteins 327 V. REFERENCES [1] M. L. Ansn, J. Gen. Physi!. 23, 239 (1939). [2] S. Ahenius, Quantitative Laws in Bilgical Chemisty (Hacut Bace, nc., N ew Yk, 1915.) [3] R. Elman, Pc. Sc. Exp. Bi. & Med. 36, 867 (1937); 43, 14 (194); A. White and R. Elman, J. Bi!. Chen. 143, 797 (1942). [4] J. H. Nthp, J. Gen. Physi!. 16,41 (1932). [5] R. A. Kekwick, and R. K. Cannan, Bichem. J. 3, 227 (1936). [61 A. She, H. Wilsn, and G. Stueck, J. Bi. Chen. 112, 47 (1935). [7] J. Steinhadt, J. Bi. Chen. 141, 995 (1941); Fedeatin Pc. 1, 136 (1942). [8] J. Steinhadt, C. H. Fugitt, and M. Hais, J. Reseach NBS 26, 293 (1941) RP1377; Am. Dyestuff Rpt. 3, 223 (1941); Textile Reseach 11, 259 (1941). [9] J. Steinhadt, C. H. Fugitt, and M. Hais, J. Reseach NBS 28, 21 (1942) RP1453; Am. Dyestuff Rpt. 31, 77 (1942). [1] P. van Rysselbeghe, J. Phys. Chen. 43, 149 (1939). WASHNGTON, Septembe 7,1942.