Studies of the intermediates produced during peptic digestion of bovine serum albumin, ribonuclease and trypsin

ARC’HIVES

OF

Studies

BIOCHEMISTRY

BIOPHYSICS

92,

of the Intermediates Bovine

MAX

AN,9

Serum

SCHLAMOWITZ,

58-68 (1961)

Produced

Albumin, LINK

during

Ribonuclease

1;. I’l~:TISIW)N

~~19

Peptic

Digestion

and

Trypsin’

I:RXSK

C.

of

WISSJ,ICI1

Received July 9, 1960 The distribution of the fragments formed 1)~ peptic h?-drolysis of t)ovine serum albumin has been studied by rlltmcentrifugal ansl~sis and by fractionation with solutions of trichloroacctic acid in nrea. Int,ermedintes, ranging in molecular weight from abont 69,000 to 5000 were fonntl, and were shown t,o diminish in size in a regnlur manner during the digestion. Evidence for the splitting of peptide bonds, withont separation of the fragmenk, presurnhal~ dur to disnlfide linkages, is also presented. B nonparallel relationship between the loss of enzyme artivity and the formation of trichloroacetic acid-solnble products is described for ribonnclease and tr?-psin, digested with pepsin. These results with pepsin are discussed in the context. of :L general scheme of proteolysis.

The course of action of pepsin on different prot,eins is often different (l-9) and even for a given protein it has been shown to vary with the conditions used (3, 7, 10). The terms “all-or-none” (I), ‘Loue-hy-one” (1 I), or “explosion” (2) have heen applied t,o describe t’he kinetics for cases like ovalbumin, where digestion mixtures were shown t)o contain ‘<. . . unchanged large molecules and fully digested end products, but no appreciable amounts of intermediat’e substances” (1, 4, 10). Peptic hydrolysis of p-lactoglobulin and bovine serum albumin have also been reported to follow “all-or-none” kinetics under defined conditions (2, 5). Under different experimeut,al

conditions,

of ovalbumin

has been found t’o lead to a

preferential

accumulation

peptic

proteolysis

of intcrmediaks

of

multaneously but gradually broken down . . .” and in which “. . . a digestion mixture should contain a number of products which would show a more or less continuous variation in size and ot,her properties bet,wcen those of the original and the end products” (I) has been demonstrated recent,ly for ribonuclease? (Y), a protein of molecular weight 13,683, but definit,ive examplrs of large globular proteins split, by pepsin to give a continuous sp&rum of int)ermediate size particles have not been described. In our previous studies on bovine serum albumin, MA3 (12), a t)ime lag was observed in the appearance of material soluble in trichloroacet,ic acid-urea mixtures during digestion with pepsin at pH’s 1.5 and 3.5, but not, at, pH 3.5 when measurements of the rat,es of reaction were followed with a pHstat.

discrete class sizes (7). Similar st’epmise peptic prot,eolysis has also been described for y-globulin and for horse globin (3, 6). A random process in which “all molecules are si-

These observations

suggested

that, in-

* The anthors wish to thank I)r. Schachman for the opportunity to read his manuscript prior to its publication, while the present investigation was 111progress. 3 The abbreviations wed are: MA, bovine serum albumin; RN&se, ribonnclease; TCA, lrichloroacet,ic acid.

1 This investigation \vas support,ed in part by Grants Xo. A-2799 and C-2585, from the National Inst,itutes of Health, U. S. Public Health Service. 58

trrmedint,es of high molecular weight. w-cre hcing prodwed. The present st,udy dwcrihes the size distrihution of the intermediat,es produced by peptic digestion of bovine serum albumin as a function of digestion time as det,ermined by mca~~s of trichloroacetic acid-urea fract ionat8ioii and ultracent8rifueal analvsis of .somc of these frac*tions. Di‘;;ebon of BSA at pII 1.3-l .7 was found t.o approximntc a raridom prwcss, ~harwt~erixcd by a (bintinumll of intermedint’es ranging in molewlar weights from that, of BSA (about 69,000) tlow11 to pnrticales of ahout 3000. The fate of smaller fragments was not, explored. The formation of a cwntinuously cahunging series of int~rrmediatcs \vas also shown for BSh digested at’ pH 3.5 and for urea-denat,ured B&2 digeskd at pII’s 1.5 and 3.3. The splitting of peptide bonds in BSA wit,hout, asswiat~cd fragrrlellt,at,ioll, attrit)utahle to disulfide bridges, is also presented. Espcrimrnts with trypsin and ribonuclcase (RSase) as suMrates ill which formation of product,s soluble in t~ric~hloroacetic acid and loss of enzyme activit8y were follewd, demo~lstrat~etl that the degradation of these proteins could not, 111:(~hara~tcrizetl its aIt “all-or-iionc” prowss.

I’eriotls of digest ion ;tntl tile ct)nditions l’or I”.ccil)it:ttion with trichloroawtic acid we st:~t~d in cwnnwtion bvik the rrsnlts of racll espcrinwnt It should 1)~:noted tlxtt prccipit:htions of tligwts of BSA with trichlr)roac~c~tic~ acid were all done in the ~~rwenc*c of 1.85 JI IIIYXL, final conwnt ration. For tligcsts of I
t rt,

“P110TEIS”4

,‘ZNALYSIS

.~~ll)--~ilU!J.~ PEPSIS

IXWLIXiLE: l)IGESTS

OF TI(ICHLOltOd(‘E;TIC ~“R.~CTLONS OF

FROM

Bf%i

‘I’richloro:rcet,ic acid-urc:L insolul~le fractious from pepsin tligests were centrifugetl, w:rshcd, tlissolved in ‘I’ris Ijllffer, ant1 tlialyzctl at about 5” against several changes of 0.1 M Tris t)uRer, pH 8.0. I’ortions (us~~nlly 0.40 ml.) of these solution8 bvrre mixed with 0.5 N wet ic acid to give a tot al volume of 1 ml. ‘I’lic optical tlcnsitics acre measurctl in the I
I’ort ions of t ri~hloro:w~tic wid-urea tilt rat cs were mixed Lvith SaOH so that the find so111tions (after correcting for Ilclitr:llizatioIl of the trirhlo-

GO

WHLAMOWITZ, U~LTR.~C~NTI~IFU(;.~L A N.~LTSIS PEPSIN DIGESTS OF BSX

PETERSON AXI) \\-ISSLEI'.

OF

Sedimentation studies were carried out at 20” in a Spinco analytical centrifuge, model 15 at 59,780 r.p.m. with either analytical or sgnt hetic boundary cells. Ditfrtsion tneasttrements tvere carried out with synthetic boundary cells at 12,540 r.p.m. The sedimentation coefficient s was evaluated from t,he slope of the line relating the logarithm of the boundary position as a function of time (16) ; the diffusion coefficient 11 by the “tnasitnlttn ordinate-area” method (17), from the slope of the line obtained 1)~ plotting (are:~)2/(maximum time. Molecttlar weights were caheight)2 versus crtlated frotn the equation

assuming the value 0.73-k (18) for the partial specific volume, 6, of BSA and its digestion products of large molecular weight. The value 1 .OOwas used for the density of the solvent., p (act,ual measured values were 1.002 and 1.00-1). The solttt.ions analyzed are described under Kesulls, in connection with Tal)le I. LVith one esreption, they were first eqttilihrat~ed by dialysis at almat 5” against 0.1 dl Tris btlffer, pH 8.0.

DIGESTI~K

OF I~SASE

BY PEPSIN

The procedure used is lused on that descril)ed I),v Berger et al. (19). R?;ase, 0.33371 m 0.008 dl glycine buffer (pH 1.7), was digested at 3i” with pepsin, using a pepsin to RKase ratio of 1:2500 by bveight. l’ortions (0.75 ml.), were withdrawn periodically, nentralized with equal volumes of 1.5 JI phosphate brtffcr, pH 7, diluted to 250 ml. with cold distilled water, and O.l-ml. aliqttots were then analyzed for RNasc activity (20). At these dilutions the activity of RNase was proportional to its concentration. Corrections were made for tkmks in which RXase was omitted. Sitnilar peptic digests of ItSase were preciptat.ed with trichloroacetic acid (final concentration, (i.2%), filt,ered three Gmes through Whatman Ko. 3 paper, and analyzed for “protein” in the manner described for the trichloroacetic acid-ttrea soluble fractions of BSA digests.

DIGESTIOX

OF TRYPSIN BY PEPHIK

Trypsin solutions, 0.3335, iu 0.008 JZ glycine buffer (pH 2.3), were digested at 37” (pepsin to trypsin ratio, 1:1250 by weight). At chosen intervals (Fig. 6), 0.75.ml. portions were adjusted to pH T.-l with 0.05 dl phosphate httffer (pH 7.7), dilrtted to 50 ml. with water, and analyzed for

residual tryptic activity by the method of Artson (21), except that. the prot)ein-free filtrates were analyzed 1)y measurement in the ultraviolet, at 2(33 mM as described for the trichloroacetic acid-urea soluble fractions of’ BSA digests. Similar peptic digests of t,rypsin cverc direct,ly precipitated with trichIoroarei,ic acid (Fig. 6). and their filtrates atulyzed for “protein” I)y the same tnct hod. Rl5RULTS

Peptic digestion of BSh at pH 1.7 (see I3zprrimental) was allowed to proceed unt.il 12, 40, and 65 % of the BSA became soluble in tjrichloroacetic acid-urea (2.0 %I trichloroacet,ic acid-l.35 31 urea, final concentrations). The insoluble fractions, designated I? II, and III, were centrifuged, washed, dissolved, dialyzed, and t)hen analyzed for sedimentation and diffusion coefficients as described. Anot’her sample of BSA, digested until it,s solubility in 2.0 W trichloroacetjic acid-urea solut’ion first’ became complete, was fractionat’ed wit)h more conccntratcd solutions of trichloroacetic acid-urea. The fraction precipitut,ed by 3. I ‘3s’trichloroacet’ic acid in 1.:35 dl urea (fra&on IV) and the one precipit,ated between 5.1 and 10 0; (fraction \i) were also analyzed in the ult’racentrifuge aft)er washing, dissolut’ion, and dialysis. They represented approximately 61 and 23 % of t,he st,arting protein. l;inally, ultracentrifugal analysis n-as done on a sample of RSA digested until more than 97 :‘5 of the prot,ein had been reduced to products soluble in 10 ‘Z TCh-I 35 dl urea, fraction \‘I, (24 hours at’ 37”, enzyme to substrate ratio 1: 1200). The reaction was stopped by adjustment’ to pH 8.0 wit’h KaOH, and the solut,ion KLS made 0.1 :II with respect to Tris buffer. E’rac+on VI was analyzed in a synthet,ic boundary cell wit’hout prior dialysis t,o avoid loss of dialyzahle fragment,s. However, care was taken to use an overlaying solution of the same pH and elcctrolyte concentrat,ion as t,he “protein” solution. E’ract,ions I-VI thus represent the products formed from BSh, with increasing time of digestion with pepsin.

IKTERMEDL4TES

IN

PEPTIC

As controls, sedimentation data were obtained for untreated H&4 and for samples of undigested BSA treated in the same manner as the fractions described. Except, for t#hose corresponding to fractions III and VI, diffusion voefficsients were also obtained for the con t,rols. X11 solutions were analyzed for “protein” I)y the procedure described for the trichloroacetic acid-urea insoluble fractions (see “Protein” concentrations Erpcrimcntal). ranged from 0.735 to 1. IO 7;. The result#s are summarized in Table I. The molecular weight 69,000 found for the controls agrees reasonably well with other values reported for 13% (17, 18, 23). The average molecular weight, of fraction 1 is very close to that’ of BSA.” With continued digestion (fractions II-VI) there is a continuous decrease in the average molecwlar weight, from 69,000 to a value of about 5000. The sedimentation pat,terns of these fractions show only a single, aymmct~riwl peak (l?g. 1). In no chase was a bimodal distribution found, such as might, characterize a system iu which relatively st,able intermediates or resistant cores accumulate. hlthough i From :rtt:tlysis for KHz-tertnimtl :ttnino acid gtxjrtp, it, nppr:trs that the protein in frwtion 1 is pro\~:t\~ly not native MA. A s:ttnplc of USA bv:ts digest.ed until :tl)out ii;, was soluble in 2.0’; t richlotwtwtit :triti-1.35 :lI urea. The remaining insolul~le fraction wts washed twice to remove wlt~l~le cwmponrnts, and anal>-zed for SHy-termn:tl groups by the phcnylt hiohydntttoitt mrthod of lldm:~ti as dewril)etl hy Ftxenkel-Conrat (23). An rtndigested sample of USA 1~3s similarly :~nalyzcd. In the case of the cwntrol only one spot, wrresportding to usprtic wid, the SH?-terminal group elf BSt\, was found. In the case of the pepsitl-tligested material, t\vo spots were detwtcd, (311~ cc)rresponding to :rsp:trtic acid and the other to v:tIinc or lr,ucitie~isoleucine. By cotnpariso~l with st:ttitl:ud ltno~vn samples, the mnoltnts of :tspnrtir :lGd itt the cwntrol and digested samples were cwmpar:tl~lr :tntl the new NHz-terminnl group was prrqenl in amounts ~ompatxl~le to the nspartic :rc.itl. It appears that in fraction I, rupture of :I pfaptitte ~~otitl~~~ has tnkctr p1:tce without tnajor c*h:tngc in molrcnl:~r weight. This situation could arise if the split tooli place in a region of the tnolecrllc bridged by tlisulfide groups. Additiomtl wtlrwe for splits of this type are descrihrd in :I later hwtion.

DEGHAD.~TIOK

OF

PKOTEISS

TABI,E

I

St-MMARY OF ULTRACENTRIFYGE 1)aT.i OS I'EPTI(' ~)tCESTIO~ OF BOYINE SERL-bt AI,BlWIS

THE

sy. Cl?l./‘SCC.

5.3” 5.2 5.6 6.0 6.X x 3 14.

69 ,000 1 .46 69,000 1.48 56,000, 1.45 48,0001 1.46 33,000 I .47 l!~,OOO, I .4’ 5 ( 200~ 1 23

‘1 Set text for controls. r\vernge of seven vnlue!:; averngr deviat ion from ntean. 10.05 S; ramp, 3.01-4.11 s. h A sma11 amount of a faster moving rotuportetlt was :11x) SPPIL (cf. Fig. 1). Simil:n ol)serv:Ltiotw (16, 17). A s:ttnpI~ with BSh h:tv~ I~rrt reported of Armour and Co., Inc. MA (l,ot Ko. ‘1’68412) did not cant aiti this smxll contponent r Average of two rxprrituents; nverage tirvi;ttion froni mean, O.OT S. Ii See test for controls. Average of three v:ilues; average deviatiotl front mean, ~0.1; range, j.25.5. A vdac of 1) = 4.6 obtained for the caotrtlol corresponding to fractions IV and LT wns trot ittclrtded irl tlw calcul:rtion of the average. e From the nomogrnm of iVym:tn :tnd Itlg:111~ (22).

the sedimentation patterns appear symmetrical it is very likely that they represent polydisperse distributions of weights about a norm rather than a homogeneous substance. In the next experiment it n-ill be seen that material corresponding to fraction V may be subfractionated. The rclutivc constancy of the values for the frictional ratios of RSA and fractions 1-1’ indicates t,hat no explosive opening-up or unfolding took place as digestion progressed. Only in fraction \‘I is there a clca~ difference. This is not altogether unexpected for t,he degradation of a structure like BSX, with it,s numerous disulfide bridges. From the results of the ultracentrifuge analyses, the digestion process seems beet, described as a random one in which a continuum of int’crmediates is produced. It is clearly not “all-or-none.”

Fro. 1. Sedimentation patterns for BSS and for fractions obtained from BS9 after partial digest,ion 1)) pepsin. C”w~tt~11,06 min.; fx~lion I, 72 min.; j’txctions ZZ-T’Z. X0 min. after reaching full speed (59,780 r.p.m.). l’atterns for Control, fractions Z-III t nken with analytical ~11; ftwtions IV-T’Z, with s\-nt,het,ic is from left to right,. SW test for esperiment:tl conditions. boundary cell Scdimentnt,ion

2

4

TRICHLOROACETIC

6

8

IO

ACID (%I

of digestion with pepsin FIG. 2. The influence at pH 1.5 on the preripit,ation of BSA 1)~ solutions of t,richloroacetic acid in 1.35 JZ urea. The points on the steep part. of the slope at the 2.05; tjrichloroacetic acid level represent the average of two experiments. See test) for experiment,al conditions. CHANGES 1x THE DISTRIBUTION OF TMCHLORO;lCETIC r~CID~~RECIPIT.1RLE MATERIAL I)URING DIGESTION OF I!%?% BP PEPSIN

In t’he previous experiment (Table I), it was shown that as digest’ion progressed, the average molecular weight, of mat,erials iw soluble in 2.0 % trichloroaretic: acid-1 35 ~11 urea fell from G!),OOOto bet#ween 33,000 and 48,000. The soluble fra&on was shown to contain materials wit’h molecular weigh& from about 83,000 down t,o about, 5000. The present experiment describes the changes in the di&ribution of subfractions caomprising this soluble fraction, as a function of time of pepsin action. RSA, at, pH 1.5 was digested as described

under E:~perimental for varying periods of time (5 min. to 24 hr.). The reactions were stopped by addition of trichloroucetic acidurea solutions of such concentrat,ions t,hat, the final concentrations were I .35 N urea and 2.0, 3.1, 6.2, or 10% t#richloroacetic acid. These mixtures were filtered three times through What)man So. 3 paper after standing for 15 min. at 37“, and the filtrates were analyzed for “protein.” The results are show-11 in E’ig. 2. As digestion proceeds, the fract,ion soluble in 2.0 ‘3 tjrichloroavetic acid rises rapidly, followed in turn by the fractions soluble in the 3.1, 6.2, and IO’? trichloroacetic acid, till complete solubilit#y is reached. The distribution pattern for the various sizes of intermediates is more clearly shown in the histogram (Fig. 5), constructed from the data of Fig. 2. As the amount of maWia1 precipitated by 2.0’; trichloroacetic acid falls, the concentration of iutermediat’es precipitated by trichloroavetic acid bet,ween 2.0 and 3.1 5; rises, reaches a peak at about, 15 min., and then declines. Intcrmediates, precaipitated by trichloroacetic acid between 3.1 and 5.0 2, representing the next lower size groups reach their peak conccntration in about’ 30 min., etc., until cornplete conversion to products soluble in 10 ‘x tric~hloroacetic acid is achirved. Wit,hin the limits of the experimental data, these results on the subfract’ions of the 2 ‘3 acid-soluble intermediates, trichloroacetic taken together with t’he (Bhanges already dcwribed for t,he insoluble fractions (fractions I-III of Table I) indicate that peptic proteolysis

of RSh

at pH

1.5 is a gradual,

rail-

dam process. Whetjher any bond(s) split in t’he time interval preceding any notjiceable change in molecular weight is (aleaved at a much greater intrinsic rate t,hnn subsequent

DURATION OF PEPSIN DIGESTION (minutes(‘).hours(~~)) FIG. 3. I)ist,ribution of intermediates formed during the digestion of IN.4 by pepsin at pH’s I .5 :~ud 3.5, and of BS.4 in urea (3.6 211) at pH’s 1.5 and 3.5. n . insoluble in 2.0% trichloroacetic acid-l.35 ~11urea; /A, msol~~ble between 2.0 and 3.1y0 trichloroacetir acid-l .35 N urea; Y. •1 , insoluble between 3.1 and 5.OoJ, trichloroacetic ariti-1.35 111urea; &Z, insolul)le het,ween 5.0 and 10% trirhloroacetic acid-l.35 M urea; , solut,le in 10% trirhloroncetic acid-l.35 M urea. See test, for drt:rils.

cleavages, i.e., “zipper” action (II), remains to be est,ablished. Data analogous to those shown in E’ig. 2 were also obtained for BSA digested at pH 3.5 and for HSA in urea (3.6 X) digested at, pH 1.5 and 3.5. The corresponding histograms (l’ig. 3) show that for t’hese conditions too the breakdown of intermediates in the 2.0 ‘;; trichloroacetic acid-urea soluble fraction follow a course roughly like t’hat’ described for the BSA digest#cd at pH 1.5. ~~~~~~~~~~~~~~~~~~OF PARTIALLY DIGERTEI) BSA ~~OI,LOWING RUPTUIW OF DISULFIUE Boxur)s

It was Iloted before that, a new X-t,erminal gronp(s) could be detected before significant changes in molecular weight, were detected. From these observations it was inferred that each rupt’ure of a peptide bond was not necessarily accompanied by fragmentation of the protein molecule. In t’he present experimrnt , evidence bearing directly on this point is submitted, based on comparison of the ultracentrifugal analysis of part,ially digested M=1, with and wit’hout rupture of disulfide bonds. RSAk was digest,ed at pH 1.7 (see Experifrontal) until about) 133’ ; was converted to products soluble in 2.0 7; trichloroacetic

acid-l.35 dl urea. The insoluble fraction n-as washed and dissolved in 8 111urea. A portion of it was t#reated with Swan’s reagent, for 2 hr. at, about, 25”, as described by PechPre et al. (25). The remaining portion served as a cont,rol. The control and treated samples were dialyzed exhaustively in the cold against several changes of 0.1 Af Tris buffer (pH 8.0), and analyzed in the ultracentrifuge for sediment’atJion and diffusion coefficients. E’or the control a single peak with a value, 3.67 8 was observed. The diffusion study gave a value, L) = 5.4 X 1OP sq. cni./sec. The molecular weight calculated from these dat’a, 62,000, is int’ermediate between the values 69,000 and 57,000, obt’ained for fractions I and II (Table I) following 12 and 40 ‘Y digest(ion. Aft,er treatment, wit,11 the Swan reagent, two fractions of lower molecular weight, with sedimentation coefficients, 2.74 and 1.3 A’ were readily discerned (Fig. 4). The diffusion coeficiellt for the treated sample was 4.3 X 1OF sq. cm.,/sec. Facilities for determining the diffusion coefficient for each component were not available, so that precise cstimntcs of the molewlnr \\eight)s of t,he two fractions are not possible. However, rough e&mates of 38,000 and 20,000, respectively, for the fast. and slow components call be got ten using the diffusion

FIG. 4. Sedimentation patterns for partially digested BSA and for partially digest,ed BSA after treatment with S&-an’s reagent. Control, analytical cell, 56 min. after reaching full speed (59,780 r.p.m.); S~Sulfoprotein, synthetic boundary cell, 80 min. after reaching full speed. Sedimentation is from left, to right.

ok0 0

FIG.

products

5. Curves relating during digestion

I

2

coal activity (enzymes, hormones, antibodies). Here. in t’he case of an “all-or-none” process, t)he iate of loss of the biochemical activit,y and the change of some other easily measured parameter; e.g., the formation of trichloroacet’ic acid-soluble product’s, will parallel or mirror each ot’her. Where t,hese condit,ions are not fulfilled, an “all-or-none” interpretation of t’he kinetics of degradation may be ruled out.” Pept,ic digestion of RKase at’ pH 1.7, and of t,rypsin at pH 2.3, was investigated in t,his manner t,o determine whether the process was “all-or-none.” RhTase, digested with pepsin, was analyzed for residual Rsase ac-

3 4 5 6 7 8 TIME (hours)

the loss of RNase activity by pepsin at pH 1.7.

constant of the mixture. It is likely that even smaller fragments were formed by treatment with the Swan reagent, since about a fourth of the “protein” in t’he sample was lost during the dialysis st,ep which preceded the ultracentrifugal analysis. DIGESTION OF RNASE AND TRYPSIS BY PEPSIN

The determination of whether the kinet’ics of enzymic degradation of proteins is “allor-none” is simplified for protein substrates which possess a readily measured biochemi-

9

IO

and the formation

of t,richloroacetic

acid-soluble

tivity and for t8richloroacetir acid-soluble material as described under Experimental. The loss in act,ivity is rapid, i.e., 96 % in the first’ 20 min. and 100% in l-145 hr.7 (Fig. 5). In coutrast’, t,he format8ion of trichloro6 An except,ion to this would be the case where complete biological activity is retained in one of the end products of digestion. Here a differentiation between “all-or-none” and random kinetics could not, be made by this method. 7 Under the same conditions, but in t,he absence of pepsin, t,here was no loss in activity of RNase or trypsin.

60

TIME (hours) l+‘rc:. 6. Curves relxting material during digestion Pepsin to trypsin ratio, I ric*hloronwtic acid). I’rpsin 10 trypsin rztio, acid 1. l’cpsin lo t rypsin ratio, t richloroncctic wid).

the loss in t.rppsin :tci,ivit y ant1 the formation of t~rirhloro:w:t~ic :rcitl-solnl,lc by pepsin :tt pTS 2.3. 1: 1250. f’~o.z~s Q, activity; f’~o.w b, tricahloroawtic ncid-soluble m.xtrri:tl (25% 1 : 110. C’IO.W C, t ri~hloro:wrtic 1:2500. Cum,

tl, wtivit

y; f’urw

:wet,iv aAd-aoluhle products is slow and fairly const.ant~ for the first l-l!,; hr.. Ci“;m in 20 min., 23 “;I in 1 hr., 3-l 5; ill lf i hr.; and !I7 7; complet8c only after 10 lw.e The proteolyt,ic* process is t’hus characterized by tbe formation of int~ermediat~es,and is clearly not ‘L:~ll-or-llolle.” Further studies of the llat,ure of the intermediates were not’ pursued in vie\v of the rwent, detailed study on this aubjevt, 1)~ Ginsburg and Schachman’ (9). These workers reported (CL)the formation of int~ermediat~cs, possessing enzyme wt’ivity, of molecular weight auhstant8inlly less than the original enzyme (molec~ulnr weight, IS,Wi); and (6) loss of the activit)y as digestioll further reduced the pnrtklc weights to about :<700. The molc~ular weights of these pnrticalcs could he further reduced t,o about 2 100 bv treatment wit,h Z-merrapto~t,hanol. It, wnsk~ggcsted that the initial steps in the

witl-solul)lr

nl:rt,eri:ll

e, t ri~hloro:lcel.ic

(25y0 t richloro:~ret,ic

acid-solnl)le

m:~teri:d

(2%

peptic action might lw very rapid; i.e., a “zipper” mechanism. A study analogous to the one for RK:rw was also done with trypsin (see ISxpcrimcnkd) and is shown in Fig. 0. Here too, t’he killet,ics are not those expected for a11 i’all-or-none” process. Thus, employing :I pepsin to trypsin ratio of 1 : 1250, trypsin a(+\-it#y uw completely lost in 2 hr..’ (rurvc a), when only ahout~ :375 of the “protein” had become soluble in ‘25 5 triohloroacetic acid (curve b). Soluble products continued to hr formed at a reduced rate, and were about 67 7; in 24 hr.* The addit,ion of more pclpsiii (pepsill to trypsin ratio, 1 : 110) at, 2 hr. acwlcrntcd t hc process so that, cwnpletth soluhilit~y was obtained by 2-l hr. ((wvc cj. It is interest.iug to note that wndit,ions should he sought which \vill not, ohscure deviations from “all-or-none” kinetics. This is illustrat,cd by the results of an experiment in which an cllzymo to substrat~e ratio of I :2500 was used. IIosscs iit lrypsin act,ivity

66

SCHLARIOWITZ,

PETERSON

were measured as before, but the tjrichloroacetic acid-soluble productIs measured wrt those soluble in 2 % instead of 25 % t8richloroacetic acid. Under these conditions, t,he loss in activity and the appearance of soluble products mirrored each other (curves d,e). DISCUSSION

The results obtained wit,h BSA, RKase, and trypsin in t,he present, study and those of other invest’igators are most easily correlated within the framework of general concepts of proteolytic mechanisms. These concept’s evolved largely under the influence of the Carlsberg school (IO, 11, 26) and elaborated by Green and Neurat’h (27) are expanded here schematically t,o st’ress the importance of substrate st’ructure and the role t,hat the two st’ages (splitDing of peptide bonds, and subsequent unfolding) play in governing the over-all prot’eolytic patt’ern.g N + I, & I,’ + 11 (~2) It>’ --j I,---,, end $ D ---f I, (i$, I,’ + I, @) In’ 4 la---, ~+xoducts

where N, the native prot)ein or its denatured form D, undergoes a series of splitting (-+) and unmasking ((t1:;) steps. I refers to the intermediate products formed by splitting of peptide bonds, and I’ to their corresponding unmasked forms. This scheme is intended to emphasize t,hat) for a mult’istage process like proteolysis, not’ only must, susceptible bonds (those meeting the specificity requirements of t’he enzyme) be present,, t’hey must also be accessible; from which it follows that secondary, t,ertiary, and quaternary (28) struct’ural features assume great importance in governing the over-all degradat,ion pat’tern.‘O The proteo9 The scheme is intended to represent) the process only in a general sense. Thus it is readilv conceived that a fragment split off at any particular stage may itself be an intermediate. Further, as smaller and smaller intermediates approaching simple linear structures are formed, t,he unmasking will become less and less obligatory. lo This view assumes the intrinsic rates of splitting of peptide bonds appears reasonable in over numbers (bonds enzyme) observed for data for the action of

are not rat,e limiting. This view of the very low turnhydrolyzed/min./molecule t,he proteases. Thus, from pepsin on RNase at pH 1.6

AND

WISSLER

lyt’ic process may be visualized as a series of successive splitt,ings of peptide bonds and unmasking of new ones.” Three general patterns of proteolysis emerge from this scheme. First, where susceptible bonds are accessible only in D but not in N, and where the N,:::?D denaturation st’ep is very slow relative to all subsequent] st’eps, the process will be “all-or-none.” Kinet,ics for the breakdown of ovalbumin (1, 4, 6, 26) can be explained on t,his basis without, having to postulabe “cage” effects. Second, where bonds are accessible in N and/or D and where a high degree of structural stability is associated not wit#h N or D but with some intermediate(s), I, farther down the line, the step I,~~~~&.’will be rate limit’ing and the process will be charact’erized by accumulat’ion of select’ed size classes. The observations of Currie and Bull (7) on kinet[Table I in Ref. (9)], a turnover number of about 750 may be estimated. The action of pepsin on ovalbumin at pH 1.65 [Fig. 31 in Ref. (IO)] gives a calculated estimate of about 10. Estimates from our own data give values of about 100 for the peptic digestion of HSA at pH 1.7. Similar low turnover values of 5-50 can be roughly estimat)ed from data on the action of chymotrypsin on ovnlbumin and on p-lactoglobulin [Tables 25 and 27 in Ref. (lo)]. These low turnover numbers for the proteases contrast with those observed for most other errzymes, and points to the operation of steric factors in determining the hydrolysis rat,es. Of special interest in this connection is the influence of urea on the operational turnover number, est,imated from data for the action of pepsin on p-lactoglobw lin [Table 28 and Fig. 33 in Ref. (IO)]. In t,he ahsenre of urea the turnover is estimated to be about 1; in its presence, about 1000. It is by no means certain that the low turnover numbers can be entirely accounted for by steric factors of the above types. There may be other steps in t,he as yet undcfined mechanism of peptic proteolysis that severely limit the intrinsic rates of splitting of peptide bonds. I1 The postulated unmasking of new susceptible bonds in the intermediates (see scheme) may occur via (a) disengagement of a fragment after the weakening of the structure by the split, of a peptide bond(s), or via (b) an intramolecular configurational rearrangement in cases where disengagement of a fragment is prevented by t,he presence of disulfide bridges.

IiXTERMEDIATES

IX

PEPTIC

its of pept,ic prot’eolysis of ovalbumin under somewhat different, conditions from t’he previously cit#ed experiments, and of Petermann (3), with r-globulin appear to fit this t,ype of syst,em, as does the conversion of pepsinogen to pepsin by pepsin. As a special case, if t,he rate-limiting step occurs very near t’he begimling of the process; e.g., I,,:& or I1~~L~~I{,differences between the intermediate and the original protein may escape detection and lead t,o an “all-or-none” interpretation for the over-all process. This possibility cannot, yet be excluded for the “allor-none” interpretations cited for ovalbumin in t,he first, category. Where such early rat)elimit~ing steps have been recognized, the term “zipper mechanism” has somet,imes been applied (11). Third, where bonds are accessible in N and/or D and where t,he relat,ive stabilit)ies of the protein and its intermediates are not, vastly different, t#he process will be seen as a random one, governed chiefly by mass-action considerations. The predominant, molecular species will shift in a more or less cont,inuous manner from ?; and/or D to int,ermediates of lower and lower molecular weigh& until conversion t,o end produ&s is complete. The data for the hydrolysis of BSA by pepsin at, p1-I 151.7 (Table I and Figs. l-3) appear to approximate this type of kinet,iw, subject to the reservation st’at’ed under Results. Those report,ed for RKase (9) also appear to fit, t’his pat8tarn, subject, to the authors’ reservations concerning a “zipper” effect. For RNase and t,rypsin, kinet,ics of the ‘L:dl-or-none” type are also ruled out under tbe condit’ions used in the present! study. A more definit8ive description for t,he course of peptic digestion of trypsin must await st’udies of the weight, dist,ribut8ion of it,s int,ermediates and their rates of appearance. The scheme for prot,eolyt(ic actioii, described in detail for studies involving pepsin, applies as well to the action of other proteases (10, 29-34) and to conversions of zymogens t,o enzymes (27). An analogous role of substrate structure has already been implicated in degradation of nucleiv acids by nucleases (55, 36) and may possibly extend to enzymic: degradat,ion of polymeric substrates; e.g., polysacscharides, etc., in general.

L)EGlt.~I)ATIOS

OF

67

PROTEISS

A., ASD ERIKSSON-QIWSSEL, I. Is., 1. TISELII.S, Riochetn. J. 33, 1752 (1939). u., BND ROBERTS, Ii. hr., J. .lm. 2. HAI:GA~RD, (Them. Sot. 64, 2664 (1942). M. I,., J. Phys. Chew. 46, 183 3. I'ETERMANN, (1942). I., Hiochm. et Biophys. 4. MORISGCLAESSON, Acta 2, 389 (1918). A., ANI) ANFISSEN, C. B., *1. Hid. 5. UELOFF, Chem. 176, 803 (1918). P., RWERY, RI., AND F~ONJOI~R, 6. I~ESNUELLE, C;., Rioch,in~. rt Uiophys. .-1&x 5, 116 (1950). H. T., AND Br-LL, H. B., J. Hid. i. CKRRIE, (‘hem. 193, 29 (1951). 21, 217 (1956). 8. <)I'PEL, v. v., Niobhimiya A.,.4sI) S('HACHMhN. H. Ii., J. Hio(. 9. (+IsSBIIRG, Chew. 235, 115 (19(K)). L. Ii.. f’onlpt. rrnd. O.ur. /ah. 10. CHRISTENSEN, f’nrlsbeu~, Sdr. chim. 28, 37 (1952). Ii., “LaIIe lIedi?:Ll I&c11. I,ISDERSTR~nI-I,.4s(:, tures: Proteins and Enzymes.” Stanford Univ. Press, Stanford, Calif. 1952. M.. .~ND PETERSON. I,. I-.. ./. 12. SWLAMOWITZ, Riol.

(‘hem.

234, 3137

(3959).

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Ph?ysid.

30,

li7

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sot. 80, 3215 (1958). H. K., “Ultr~c:r~ltrifugatiorl l(i. Sc~Acmas, in Biochemistry,” p. 75, 21. Acntlemic I’ress, iVew York, 1!)59. A., -1ctrr (‘hem. &and. 11, 1257 17. I~:HREKBERG, (1957). (H. Seulnth 18. KDSALL, J. T., in “The I’roteins” :tntf I<. C. Bailey, eds.), \‘ol. I, 11, 508, (i17, 721. Academic Press, Xew lTork, 1953. A., xEYW\N, H., .4ND SEI.A, l\I., 19. BERGER, Hiochint. cl Hiophys. dcla 33, 249 (1959). C. R., REDFIELD, I<. R., CIIOATE, 20. .~NFINSE~, \T'. I,.. l'A<'E. J., i\ND C.4RROl.L, \v. Il., J. Hid. (‘hem. 207, 201 (19%). 22, i9 (1938). 21. ASSON, 11. I,., J. Gen. Physid. 22 N’YJIAS, J. K.. .\SD INGALLS, E. N., J. Hid. (Ihem. 147, 297 (1943). H., HARRIS, J. I., ASI) 23 FRAENKEL-COSRAT, I,EVY, -4. I,., dIethods of Miochem. .lnd. 2, 359 (1955). HCGHES. \V. T., in “The Proteins” (H. XVIIrnth and Ii. C. Bailey, eds.), Yol. II, p. 682. Academic Prrss, Kew York, 1954. ct. H., ;~IAYBI:RY, R. 25 ~'EVH&RE, J. E’., I)ISOS, I-I.,

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13M (1958).

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(‘hew.

233,

(23 2Ci. ~,INUERSTRdnl-LhN(:,

SCHLr\MOWITZ,

K.,

A!hstr.

t-‘roc.

PETERSOIS

9th so/-

my (‘ongr. Brussels, Apt+1 6-14. 1953, p. 247. R. Stoops Publ., Brussels, 1953. S. M., AND SEIRATH, H., in “The 27. (;REEN, Prot,eins," (H. h-eurath :md Ii. C. Bdcy, etls.), Vol. II, 1). 1057. Acntlemic Press, Sew York, 1954. J. CT., Fedemlion f’wc. 18, 740 28. KESI)REW, (1959). AI., Biochem. J. 30, 1807 (1936). 29. ANNETTS, M., ~)ESSLXI,LE, P. ASL) BOXJOGR, 30. ROVERI-, G., Biochim. et Biophys. Acta 6, 160 (1950).

.kXD

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31. I\IIH.&I,YI,

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36. I
c., .tm.