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The effect of N-bromosuccinimide upon trypsinogen activation and trypsin catalysis
ARCHIVES
OF
BIOCHEMISTRY
The
Effect
AND
(1969)
of N-bromosuccinimide Activation
and
V. W. DANIEL, Department
134, 5066514
BIOPHYSICS
of
Chemislry,
Received
June
III2
upon
Trypsin
Catalysis’
G. G. TROWBRIDGE
AND
Emory
Trypsinogen
University,
3, 1969; accepted
Atlanta, August
Georgia
.%I$22
11, 1969
Modification of trypsinogen with iV-bromosuccinimide (NBS) significantly alters the appearance and the time required for the development of the activation difference spectrum. The kinetic properties of the activated modified trypsinogen (ATGM) were compared with similarly modified trypsin (TM). Neither ATGM nor TM showed significant changes in the binding parameters K...,, and Ki evaluated for mixture of r,-arginine methyl ester (LAM) and benzamidine. There was a sharp decrease in k,,, for ATGM with increasing extent of NBS modification. This apparent decrease in the catalytic efficiency can be explained by a loss in active site concentration, based upon inhibition measurements with soybean trypsin inhibitor. These results were in contrast with those for modified trypsin, where there was a decrease in k,,, with increasing extent of NBS modification, but no loss in active site concentration until the ratio of moles NBS per mole protein had exceeded 2 : 1. Additional kinetic characterization of the ATGM and TM with tosyl-L-arginine methyl ester (L-TAM) and activation of chymotrypsinogen gave results which paralleled those for k.,, with L-AM. Both ATGM and TM exhibited substrate activation with L-TAM.
The chemical and physical events of the autocatalytic activation of trypsinogen have been of interest since Kunitz first explained the acceleration of the enterokinase activation of trypsinogen (1). The loss of the NHz-terminal hexapeptide is accompanied by a change in optical rotation that parallels the appearance of tryptic activity (24). The change in optical rotation observed upon activation indicates that some structural rearrangement has occurred; this change is probably small since no change can be detected in the frictional coefficient (5). Benmouyal and Trowbridge reported a trypsin-trypsinogen difference spectrum be1 This research was supported by the National Institutes of Health Grant GM 13201. A preliminarv renort of this work was m-esented at the Meeting of the American Chemical Society at Atlantic City, September, 1968 (Daniel, V. W. III, and Trowbridge, C. G., Abstracts of the American Chemical Society Meeting, September 1968, Biol., 167). 2 NASA Trainee, 1968-1969.
tween 310 and 250 rnp whose growth paralleled the appearance of tryptic activity (6). Some or all of the tryptophan residues could contribute to the difference spectrum observed. The investigation of the susceptibility of tryptophan to chemical modification in trypsinogen and trypsin and a study of the kinetic properties of the modified species may help to elucidate the role of tryptophan in the activation process and in the enzymic function of the trypsin molecule. N-bromosuccinimide (NBS)3 has been used as an oxidizing reagent for tryptophan (7-14). Extensive use of NBS as an oxidizing agent for tryptophan has been summarized by Witkop (8) and Spande and Witkop (14). They concluded that NBS was specific for tryptophan at pH 4.0 and that little or no reaction with 3 Abbreviations used: NBS, N-bromosuccinimide; TM, NBS-modified trypsin; ATGM, activated NBS-modified trypsinogen; L-AM, L-arginine methyl ester; L-TAM, tosyl-n-arginine methyl ester; IrATrE, acetyl-n-tryptophan ethyl ester; STI, soybean trypsin inhibitor.
506
TRYPSINOGEN
tyrosine and/or histidine occurred under their reaction conditions. Their conclusions were based principally on the following evidence: (1) the amino acid analysis of oxidized trypsin (> 80 % tryptophan oxidized) showed no loss of residues other than tryptophan, with perhaps the loss of 0.5 moles of tyrosine (7) ; (2) the fact that a plot of change in absorbance at 280 rnlc vs. the moles of NBS reacted was linear (14), and (3) that generally oxidations requiring less than 4: 1 mole ratios of NBS to protein are indicative of the loss of tryptophan only (14). They also suggested that no significant peptide bond cleavage would have occurred under these conditions (8, 14). Williams and Laskowski (15) have also concluded that the same reaction conditions used with chymotrypsinogen caused the oxidation of tryptophyls only. Our modification procedures closely followed those set forward by Witkop (7) and were assumed to lead to oxidation of tryptophan with no significant side reactions. In this paper we describe the result,s obtained with trypsinogen modified by NBS and subsequently activated by trypsin, and with trypsin modified by NBS. Activation of the modified trypsinogen was characterized by the appearance and time course of the activation difference spectrum. The modified trypsin (TM) and the activated modified trypsinogen (ATGM) were characterized kinetically by steady state analysis of L-arginine methyl ester-benzamidine mixtures, their efficiency in activating chymotrypsinogen, and their susceptibility to inhibition by soybean trypsin inhibitor. EXPERIMENTAL Materi&. Trypsinogen (lots TG 7A and TG 6402A) and trypsin (salt-free lyophylized lot TRL 6263, and MgSOb lot TR 7FA) were purchased from Worthington Biochemical Corporation, Freehold, New Jersey. Soybean trypsin inhibitor (STI) was obtained from Nutritional Biochemical Company, Cleveland, Ohio. Benzamidine from Aldrich Chemical Company, Milwaukee, Wisconsin was recrystallized from butanol before use. The NBS was reagent grade and was recrystallized from water. All substrates were made in this laboratory and were characterized by melting point and exhaustive hydrolysis. Preparation of solutions. MgSOd was removed from trypsin by chromatography on columns of
ACTIVATION
507
Sephadex G-25 (2 X 50 cm) at pH 2.9 and a flow rate of 1 ml per minute. Salt-free trypsin was chromatographed on a column of Sephadex G-50 (2 X 100 cm) at pH 2.9 and a flow rate of 0.75 to 1 ml per minute; standard solvent (0.2 M KC1 and 0.05 M CaCl2) at pH 2.9 was used as eluent unless otherwise stipulated. Chromatography of trypsinogen on Sephadex G-25, G-50, and Sulfoethyl (SE) C-25 (16) gave a single symmetrical band which exhibited less than 0.3% of its potential tryptic activity. Therefore, trypsinogen was used direct,ly after dissolution and centrifugation at 16,000g to remove undissolved material. Concentrations were determined photometrically using the optical factors 0.65, 1.1, and 0.50 (mg per ml per absorbance unit at 278 mr) for trypsin, soybean trypsin inhibitor, and chymotrypsinogen respectively (Worthington Biochemical Corp., Technical Bulletin). All solutions were stored at 4.0”. Aqueous NBS stock solutions (0.01 M) were prepared as needed and kept from direct light by covering the flask with aluminum foil. All substrate and benzamidine solutions were prepared in “standard solvent.” Modification of proteins. The modification reactions were carried out at pH 4.0 f 0.05 by the addition of lo- to 20-~1 increments of 0.01 M NBS to the protein solution. The pH was maintained at 4.0 by the addition of KOH (0.2 M) from a microburet. The solutions were rapidly stirred during the addition of NBS and for lo-20 min after the final addition. The modified protein was then adjusted to pH 2.9 and stored at 4” until needed. The reaction of NBS with acetyl-L-tryptophan ethyl ester (ATrE) required 1.5 moles of NBS per mole of ATrE oxidized (based on plots of ti280 vs. moles NBS added (7). Trypsinogen and trypsin required 2.0-2.3 and 1.52.0 moles of NBS per moles of tryptophan oxidized respectively. Hachimori et al. (17) found no difference in the ease of oxidation of three tryptophan residues in trypsin and trypsinogen with HzOZ. Oxidized trypsinogen was subjected to ion-exchange chromatography on columns of Sephadex SE C-25 (0.9 X 10 cm) in the manner described by East (16), who has shown that trypsin with peptide bonds cleaved can be separated from intact trypsin by this procedure. Diflerence spectra. The activation difference spectra for chymotrypsinogen and trypsinogen were made at 25” on a Cary 15 spectrophotometer in the manner described by Benmouyal and Trowbridge (6). The zymogen was prepared in “standard solvent” which was buffered with 0.05 M Tris at pH 8.0. The activation wae started by adding 100 pl of the appropriate trypsin solution (2-5.0 X 10-b M) in t,he sample beam and to the solvent cell
508
DANIEL
AND
in the reference beam. The cells were scanned at time intervals between 330 and 250 rnp. In cases where a simultaneous assay for enzymatic activity was desired, 5-50 11 of the activating mixture was removed by Hamilton syringe immediately after the scan and the time recorded. The reaction was quenched by adding the aliquot to 9.0 ml of standard solvent at pH 2.9. If it was necessary to postpone the assay for an extended time the quenched aliquots were refrigerated at 4”. No increase in tryptic activity could be detected between assays made immediately after withdrawal and those made the next day. Rate measurements. All trypsin/substrate rate measurements were determined at 25” by pH stat techniques, using a Radiometer TTTlc. Assays for tryptic activity were performed by adding 1.0 ml of 0.01 M L-TAM to 9.0 ml of “standard solvent” containing the quenched activation aliquot. The pH was adjusted to 8.0 and the rate determined. The assay was reported as VO/PO, where Vo is the initial rate and Pa is the total protein concentration. The trypsin/cAM, benzamidine rates were determined by mixing the appropriate volumes of “standard solvent” and stock L-AM solution to obtain the desired concentration; the trypsin and benzamidine were then added and the pH adjusted to 5.0 for measurement of initial rates. STI inhibition. ST1 and enzyme were mixed with 9.0 ml of solvent at pH 5.0 and the pH adjusted to 8.5. One milliliter of 0.01 M *TAM was added, the pH adjusted to 8.0, and the initial rate determined.
RESULTS Trypsinogen activation The growth of the trypsin-trypsinogen difference spectrum during the autocatalytic activation of trypsinogen parallels the appearance of tryptic activity as measured by the rate of hydrolysis of L-TAM. Figure 1 shows the tryptic activity and magnitude of the difference spectrum (AA) normalized to a single scale plotted versus time. The assay for activity is reported as V,/P,, where PO is the potential molar concentration of trypsin. The AA reported is the sum of ( AA ( at 278 rn@ and 1AA 1 at 293 mp. The figure shows good agreement between the two methods of measuring the extent of activation. After 4-6 hr the difference spectrum begins to develop a larger negative peak at 293 rnp. Since the tryptic activity is stable after 4 hr (V,/P, decreases less than 3 % after 20 hr),
TROWBRIDGE
MINUTES 160
200
FIG. 1. Time course of trypsinogen activation. Open and closed circles represent tryptic activity and difference spectrum development, respectively, normalized to percentage of maximum response. Conditions given in Procedure.
the correlation between AA and tryptic activity is lost. This deviation of the time dependence of AA from a simple autocatalytic curve is not due to activation in the reference beam, since the tryptic activity there has not exceeded 1% of the potential value after 20 hr. Nor does the deviation seem to be caused by autolysis into inactive fragments in the activating mixture, since the activity stabilizes after 4 hr while AA continues to change. Much evidence exists that the autocatalytic activation of trypsinogen produces an enzymatically active form of trypsin with an internal bond cleaved, but with the same amino acid composition and hydrodynamic properties as intact trypsin (16, 18-22). Low pH chromatography of the activation mixture on SE Sephadex C-25 (16) yielded two active components which have been tentatively identified on the basis of kinetic comparison with Shaw’s (Y and p trypsin (16,22). Some kinetically inactive material was also found in small amounts. The formation of these additional products could account for the deviation of the rate of growth of the difference spectrum from the rate of appearance of tryptic activity. Efect of NBS on activation. Trypsinogen was modified as described in the Experimental section at mole ratios of NBS to protein of 1: 1, 2: 1, and 4: 1. These ratios correspond to less than one, approximately oxidized per one, and two tryptophans
TRYPSINOGEN
trypsinogen molecule (based on plots of aAzso rnp vs. moles of NBS added for completely oxidized proteins, seeModification of Proteins). These ratios were well within the linear region of a plot of AAzso against moles of NBS added to trypsinogen solutions. Figure 2 contrasts the activation difference spectra of unmodified and modified trypsinogen. The peak locations of the difference spectrum for all modifications are the same, but the size and time course of the growth of the spectra are quite different. The maximum values of AA/A at 287 rnp are 0.01,0.03,andO.lSforthe1:1,2:1,and4:1 modification ratios, respectively. The difference spectra for 1: 1 and 2: 1 modification experience a maximum change in absorbance at 30 and 55 min, respectively. AA for the 4: 1 modification increased monotonically (Fig. 3). For the 1: 1 and 2: 1 ratios the tryptic activity appeared monotonically and reached a maximum after 2-3 hr of incubation (Fig. 4) ; the activity then remained constant for up to 20 hr. For the 4: 1 modifi-
[
509
ACTIVATION
MINUTES
FIG. 3. Time course of trypsinogen activation difference spectra. (A) unmodified, (B) 4:l and (C) 2:l mole ratio NBS-modified trypsinogen. Left hand ordinate for curves (A) and (C), right hand ordinate for curve (B). Experimental conditions in Procedure. r 20
./Ys
, I
-
3O
.02 I
I
40
I
I
120 MINUTES
I
!
/
I
200
FIG. 4. Appearance of tryptic activity. (A) and (C), 1:l and 4:l mole ratio NBS-modified trypsinogen, respectively; (B) is unmodified trypsinogen. Right ordinate for (B), left ordinate for (A) and (C). Experimental conditions in Procedure.
- .oo PA
--.02
--.04
w 250 I
270 I
290 I
310 I
330 I
FIG. 2. Activation difference spectra for trypsinogen (A), and for trypsinogen reacted with NBS at 2:l mole ratio (B). Experimental conditions in Procedure.
cation ratio (Fig. 4) the activity was low and reached a maximum after about 30 min, with a subsequent gradual decrease in activity. The increase in tryptic activity with time does not parallel the growth of the difference spectra for any of the modifications studied. Kinetic characterization of modified protein. Xixtures of L-arginine methyl ester (~-Ai\l)~ and benzamidine (23) have been shown to provide a convenient system for the charac‘Unpublished
data from this laboratory.
510
DANIEL
AND
terization of trypsin. Initial rate data at pH 5.0 are closely reproduced by Eq. 1. vo -= PO
kS/K* 1 + S/K, + I/&
(1)
Rate data obtained with ATGM and TM preparations were fitted to equation (1) by nonlinear regression analysis. The values of the parameters obtained from the analysis indicate that K, and KC are somewhat affected (Table I) for all modification ratios studied. However, the changes in K, and Kc are not sufliciently large to be kinetically meaningful. On the other hand, the values obtained for k, decrease as the NBS:protein ratio increases for both ATGM and TM (Table I). The decrease is much more marked for ATGM than for TlM (Fig. 5). The benzamidine KC values obtained for the ATGM and TM are in agreement with the dissociation constant determined by East (24) by an equilibrium method, when appropriate corrections are made for pH. The rate of chymotrypsinogen activation by ATGM and TM preparations was measured by observing the development of the activation difference spectrum produced (6, 25). The apparent first-order rate constant for the activation was evaluated and nor-
TROWBRIDGE
malized for differences in the amount of catalyst used. These constants show the same behavior with respect to NBS modification as do the Ic, values for L-AM (Table I). Once again the catalytic “efficiency” of ATGM drops more sharply than does that of TM with increasing extent of NBS modification. The trypsin/L-TAM system has been shown to exhibit substrate activation (26, 27). The ratio of rates of hydrolysis of 0.01 and 0.001 M L-TAM at pH 8.0 are given in Table I and can be taken as an assessment of substrate activation in ATGM, TM, and untreated trypsin; all exhibited a similar degree of substrate activation. Decreases in Vo/Po (0.001 M L-TAM) with increasing extent of NBS modification closely approximate the curves obtained for k, in Fig. 5. All modified trypsin used in making the kinetic measurements in this section was prepared directly from commercially available trypsin with no attempt to separate the two active components present (16, 22). Analysis f the individual components showed that the use of unfractionated trypsin did not Rignificantly alter the kinetic results. STI inhibiticw. The kinetic results sum-
TABLE THE
Proteina T ATG TM TM TM ATGM ATGM ATGM
Mo&;;io, protein 0:l 0:l 1:l 2:l 4:l 1:l 2:l 4:l
VARIATION
OF KINETIC
R,, SK-~
K, x 103, Y
12.92
f
.25
13.50 10.02 8.85 2.91 5.68 2.29 0.50
f f f f f f f
.80 .77 .13 .04 .14 .07 .02
5.53 5.63 5.46 5.45 5.22 4.11 4.32 5.79
f f f f f f f f
I
WITH NBS
PARAMETERS
.40 .75 .75 .33 .34 .45 .52 .92
K< x 100
M
4.92
f
.31
4.95 6.07 5.89 5.20 5.15 5.07 5.46
f f f f f f f
.87 .61 .35 .29 .55 .73 .86
OXIDATION
CTG activationC Substrat.+ activation K @.PP)X ratio, L-TAM 101 Id-1 xc-
6.95 6.95
5.31 3.45 1.16 1.63 .79 -
1.65 1.65 1.62 1.67 1.70 1.54 1.64 1.67
Per cent
activesit& remaining loo 100 100 100 33 29 13 5
0 Protein abbreviations: T, commercial trypsin; ATG, trypsinogen activated in this laboratory; TM, modified trypsin; ATGM, activated modified trypsinogen. * Parameters evaluated by nonlinear regression analysis on Eq. 1; the ranges shown are calculated at 95% confidence level. The substrate and inhibitor are L-AM and benzamidine, respectively. Reaction conditions in Experimental. c Apparent second-order rate constant calculated from the growth of the chymotrypsinogen activation difference spectrum. d The ratio of the L-TAM hydrolysis rate at 0.01 M to that at 0.001 M substrate, pH 8.0. e Based upon 100% for native protein, as inferred from STILL-TAM inhibition study.
TRYPSINOGEN
511
ACTIVATION
up to 2: 1 mole ratio, but that the catalytic efficiency of the site is reduced. The constant value of the So/To intercept has two possible interpretations: (1) To and K change with
5 MOLES
NBS
/ MOLE
PROTEIN
FIG. 5. The effect of the extent of NBS oxidstion upon k, for L-arginine methyl ester at pH 5.0 (Eq. 1). (A), activated modified trypsinogen; (B), modified trypsin. Conditions given in Procedure.
marked in Table I show that, the effect of NBS modification before or after trypsinogen activation is seenprimarily in the values of k,. The decrease in the apparent rate constant could be due to a diminution in the catalytic efficiency of the active sites, to a decrease in the concentration of sites, or to both. In an attempt to resolve this question, FIG. 6. Variation of ST1 inhibition with inthe ST1 inhibition of ATGM- and TMcreasing extent of NBS oxidation, modified trypcatalyzed hydrolysis of L-TAM was investi- sin with L-TAM as substrate at pH 8.0. (A) native gated. If we assume that the reaction of trypsin; (B) l:l, (C) 2:1, (D) 4:l mole ratio respectively. The ordinate is k’ = trypsin with ST1 can be represented by NBS/trypsin, VO/P~, with PO calculated as moles trypsin before T + S @ X, then the concentration of comDlex (X) is given by X = l/,[S, + To + K NBS oxidation* 1 (SO‘+. TO-+ K); - 4 ?&~j112]. SOand To , are the total concentrations of soybean inhibitor and of trypsin, respectively, and K _ 3o is the dissociation constant of X. We find in the limit of small So that dX/dSo = To/ (To + K). This and the fact that T = To X predicts that a plot of T/To vs. So/To will -20 ka be a straight line with slope -To/(To + K) and having an extrapolated intercept on the So/To axis of (To + K)/To. The data in Figs. 6 and 7 are straight lines, and we conclude that they are within the range of validity of the limiting equation for dX/dSo . We assume that the. velocity of L-TAM _ ^ hydrolysis is proportional to free trypsm, so
that I?’ is proportional to T/To . In Fig. 6, the decreasing k’ intercept with a constant So/To intercept is consistent with the view that the concentration of active sites is unchanged by the action of NBS upon trypsin
.05
I
.I5
.25
FIG. 7. Variation of ST1 inhibition with increasing extent of NBS oxidation, activated modified trypsinogen with L-TAM as substrate at pH 8.0. (A) l:l, (B) 2:1, (C) 4:l mole ratio NBS/trypsinogen, respectively. Ordinate as in Fig. 6. Experimental conditions in Procedure.
512
DANIEL
AND TROWBRIDGE
NBS modification in such a way as to keep the intercept constant. This coincidence seemshighly improbable. (2) The remaining possibility is that neither TO nor K have changed. This interpretation, which we advocate, is consistent with the data of Table I which shows that the K values for small ligands were hardly affected by NBS modification. In Fig. 7, corresponding data obtained with ATGM lead to a family of parallel lines. The slopes, which should change with To, are only apparently constant because TO is always much greater than K (Lebowitz and Laskowski estimate K = 10-l’ at pH 8.3 (28)). The decrement in both intercepts is equal, and this is most simply explained by decreasedsite concentration. If the observed velocity (k’) is corrected under the assumption that total concentration of sites is diminished by NBS, a common intercept is obtained on the k’ axis. The slopes -TO/ (To + K) become increasingly negative, as they should if TO is decreasing, and the Xo/To intercepts decrease in proportion to To . We conclude that the reaction of NBS with TG prior to activation causesa decrease in the concentration of sites obtainable upon “activation,” with no significant change in the catalytic properties of the remaining active sites. This is in contrast with the effect of modification of trypsin with NBS, wherein the concentration of sites is unaffected (at least up to 2:l mole ratio), but the catalytic properties of the sites are modified. SE Sephadex C-25 chromatography of ATGM in the manner described by East et al. (16) yielded up to four kinetically inactive species.The ATGM gave up to 95 % (by absorbance) inactive species at 4: 1 modification. The TM at 4: 1 modification gave the expected two active speciesas well as two kinetically inactive species. All the UV spectra for the inactive speciesin ATGM and TM were qualitatively different, but resembled in gross features that of extensively modified trypsin and trypsinogen. The chromatography of ATGM lends important support to the hypothesis that inactive (no active site) trypsin is formed upon activation of NBS modified trypsinogen, as
was put forward in interpretation of the ST1 inhibition data. The two active components found in commercial trypsin or freshly activated trypsinogen were obtained by the chromatography procedure described earlier (16). Both active species were modified separately by NBS in 1: 1, 2: 1, and 4: 1 ratios. The resulting modified trypsin was characterized by the inhibition of L-TAM hydrolysis by soybean trypsin inhibitor. A decreasein k’ with increasing extent of NBS modification was seen with both active components; no decrease in active sites was observed in either caseuntil NBS modification exceeded a 2: 1 ratio. Thus the same results were obtained with either of the two active components or with their mixture. DISCUSSION
We suggest that in trypsinogen there is a tryptophyl residue which is essential to potential tryptic activity, and that this residue is the most reactive with NBS. This is based upon the decrease in active site concentration with increasing NBS oxidation as inferred from Fig. 7, and the result of Fig. 5. The latter shows that all potential activity would be lost at 1 mole of tryptophan oxidized per mole of zymogen, if the efficiency of the NBS reaction (in reducing potential activity) continued undiminished. Viswanatha et al. (7) found that approximately 20% of the four tryptophyl residues in TG could be oxidized with no loss in potential activity, which is in disagreement with our analysis (Table I). The presence of “inert” protein containing readily oxidized tryptophans could explain a lossof absorbancewith no lossin activatable trypsinogen. At 4 moles NBS per mole of trypsinogen, the potential active site concentration (Table I and Fig. 7) is 4 % that of unoxidized trypsinogen. Nonetheless the action of trypsin upon this modified protein still gives rise to a difference spectrum whose growth is completed in lessthan 4 hr (Fig. 3), and thus is comparable in rate to the activation of unmodified trypsinogen. The data, both kinetic and chromatographic, support the conclusion that both active and inactive trypsin molecules are produced during the activation of
TRYPSINOGEN
NBS-modified trypsinogen. The active species produced by the “activation” of modified trypsinogen apparently have kinetic properties unchanged by the NBS reaction. This is concluded from the observation that a correction for active site depletion, calculated from the STI-inhibition data, results in calculated rates of hydrolysis of amino acid esters and of chymotrypsinogen that are equal to those obtained with the unmodiied enzyme. The binding of small ligands is unaffected (Table I). The NBS modification of trypsin does not lead to a decrease in active sites until the ratio of NBS to protein exceeds 2: 1 (Fig. 6). The binding constants for small ligands is hardly affected by NBS modification, but the catalytic efficiency does decrease (Table I). Even for 4: 1 modified trypsin the decrease in catalytic efficiency (k,) cannot be explained wholly by a loss in active sites, although sites are destroyed at this modification ratio (Fig. 6 and Table I). Thus the tryptophan most susceptible to NBS oxidation in trypsin is nonessential to catalytic function. The decreasein active site concentration seenat 4: 1 modification may be due to oxidation of an essential tryptophan in trypsin. The appearance of the activation difference spectrum for 4: 1 modification (Fig. 2) implies a substantial tryptophan contribution; apparently some of the remaining tryptophans still experience an appreciable perturbation when modified trypsinogen is acted upon by trypsin. Since a large contribution to the difference spectrum would not be expected from oxidized tryptophyls, some of the less reactive tryptophyls must underperturbation during activation. %lliIms and Laskowski (15) and Spande and Witkop (14) have considered the possible correlation between NBS reactivity and exposure of the tryptophyls to the medium. If the same essential tryptophyl is detected by NBS oxidation in both trypsinogen and trypsin, then its relative reactivity is less in the latter protein. Hachimori et al. (17) suggested that the susceptibility of individual tryptophyls to Hz02 oxidation may be different in trypsinogen and trypsin. One would then conclude that the degree of ex-
ACTIVATION
513
posure of this essential tryptophyl has changed in the conversion of trypsinogen to trypsin. Oxidized tryptophan residues in active trypsin apparently have no role in “binding” simple substrates and small ligands since no appreciable change can be detected in the binding parameters of L-AM and benzamidine (Table I) for all ATGM and TM preparations tested. The function of these “nonessential” tryptophans in catalysis is not known, but a decreasein catalytic efficiency is observed in TM with no concommitant decrease in active site concentration. The concept of an essential residue is somewhat elusive. If the molecule is progressively “degraded” as succeeding residues are oxidized, with activity finally destroyed with the last tryptophyl, no special role can be assigned with any confidence. If, however, there is a loss of activity when one of the most reactive residues is affected (as with our results) then such a residue is apparently “critical” to normal function and may be considered essential. Investigation into the details of the trypsinogen-trypsin conversion is continuing. ACKNOWLEDGMENT We thank Sharon A. Arihood, who established the validity of Eq. 1, and Emily J. East who developed the chromatographic procedures. REFERENCES J. H., KUNITZ, M., AND HERRIOT, Enzymes,” 2nd Ed., R. M., “Crystalline p. 127. Columbia Univ. Press, New York (1948). DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 515 (1955). PECHERE, J-F., AND NEURATH, H., J. Biol. Chem. 229, 389 (1957). NEURATH, H., RUPLEY, J. A., AND DREYER, W. J., Arch. Biochem. Biophys. 66, 243 (1956). NEURATH, H., Adv. Prot. Chem. 12, 319 (1957). C. G., BENMOUYAL, P., AND TROWBRIDGE, Arch. Biochem. Biophys. 116, 67 (1966). VISWANATHA, T., LAWSON, W. B., AND WITKOP, B., B&him. Biophys. Acta 46, 216 (1960). WITKOP, B., Adv. Prot. Chem. 16, 221 (1961). VISWANATHA, T., AND LAWSON, W. B.. Arch. B&hem. Biophys. 93,128 (1961).
1. NORTHRUP,
2.
3. 4. 5. 6. 7. 8. 9.
514
DANIEL
AND
10. HAYASHI, K., 1~0~0, T., FUNATSU, G., AND FUNATSU, M., J. Biochem. 68, 227 (1965). 11. KRONMAN, M. J., ROBBINS, F. M., AND ADIOTTI, R. E., Biochim. Biophys. Acta 143, 462 (1967). 12. SPANDE, T. F., GREEN, N. M., AND WITKOP, B., Biochemistry 6, 1926 (1966). 13. STEINER, R. F., Biochemistry 6, 1964 (1966). 14. SPANDE, T. F., AND WITKOP, B., Methods in Enzymology 11, 498 (1967). 15. WILLIAMS, E. J., AND LASKOWSKI, M., JR., J. Biol. Chem. 240, 3580 (1965). 16. EAST, E. J., AND TROWBRIDGE, C. G., Abstr. Southeastern ACS Meeting, No. 205, p. 100 (1968). 17. HACHIMORI, Y., HORINISHI, H., KURIHARA, K., AND SHIBATA, K., Biochim. Biophys. Acta 93, 346 (1964). 18. NORD, F. F., AND BIER, M., Biochim. Biophys. Acta 12, 56 (1953). 19. TIMASHEFF, S. N., AND STURTEVANT, J. M., Arch. B&hem. Biophys. 63, 243 (1956).
TROWBRIDGE 20. IACHAN, A., DOMONT, G., DISITZER, L., PEILRONE, J., Nature 203, 43 (1964). 21. MAROUX, S., ROVERY, ;\‘I., AND DESNUELLE, P., Biochim. Biophys. Acta 140, 377 (1967). 22. SCHROEDER, D. D., AND SHAW, E., J. Biol. Chem. 243, 2943 (1967). 23. MARES-GUIA, M., AND SHAW, E., J. Biol. Chem. 240, 1579 (1965). 24. EAST, E. J., AND TROWBRIDGE, C. G., Arch. Biochem. Biophys. 126, 334 (1968). 25. CHERVENU, C. H., Biochim. Biophys. Acta 26, 222 (1957). 26. TROWBRIDGE, C. G., KREHBIEL, A., AND LASKOWSKI, M. JR., Biochemistry 2, 843 (1963). 27. TRENHOLM, H. L., SPOMER, W. E., AND WOOTON, J. F., J. Am. Chem. Sot. 88, 4281 (1966). 28. LEBOWITZ, J., AND LASKOWSKI, M., JR., Biochemistry 1, 1044 (1962).
OF
BIOCHEMISTRY
The
Effect
AND
(1969)
of N-bromosuccinimide Activation
and
V. W. DANIEL, Department
134, 5066514
BIOPHYSICS
of
Chemislry,
Received
June
III2
upon
Trypsin
Catalysis’
G. G. TROWBRIDGE
AND
Emory
Trypsinogen
University,
3, 1969; accepted
Atlanta, August
Georgia
.%I$22
11, 1969
Modification of trypsinogen with iV-bromosuccinimide (NBS) significantly alters the appearance and the time required for the development of the activation difference spectrum. The kinetic properties of the activated modified trypsinogen (ATGM) were compared with similarly modified trypsin (TM). Neither ATGM nor TM showed significant changes in the binding parameters K...,, and Ki evaluated for mixture of r,-arginine methyl ester (LAM) and benzamidine. There was a sharp decrease in k,,, for ATGM with increasing extent of NBS modification. This apparent decrease in the catalytic efficiency can be explained by a loss in active site concentration, based upon inhibition measurements with soybean trypsin inhibitor. These results were in contrast with those for modified trypsin, where there was a decrease in k,,, with increasing extent of NBS modification, but no loss in active site concentration until the ratio of moles NBS per mole protein had exceeded 2 : 1. Additional kinetic characterization of the ATGM and TM with tosyl-L-arginine methyl ester (L-TAM) and activation of chymotrypsinogen gave results which paralleled those for k.,, with L-AM. Both ATGM and TM exhibited substrate activation with L-TAM.
The chemical and physical events of the autocatalytic activation of trypsinogen have been of interest since Kunitz first explained the acceleration of the enterokinase activation of trypsinogen (1). The loss of the NHz-terminal hexapeptide is accompanied by a change in optical rotation that parallels the appearance of tryptic activity (24). The change in optical rotation observed upon activation indicates that some structural rearrangement has occurred; this change is probably small since no change can be detected in the frictional coefficient (5). Benmouyal and Trowbridge reported a trypsin-trypsinogen difference spectrum be1 This research was supported by the National Institutes of Health Grant GM 13201. A preliminarv renort of this work was m-esented at the Meeting of the American Chemical Society at Atlantic City, September, 1968 (Daniel, V. W. III, and Trowbridge, C. G., Abstracts of the American Chemical Society Meeting, September 1968, Biol., 167). 2 NASA Trainee, 1968-1969.
tween 310 and 250 rnp whose growth paralleled the appearance of tryptic activity (6). Some or all of the tryptophan residues could contribute to the difference spectrum observed. The investigation of the susceptibility of tryptophan to chemical modification in trypsinogen and trypsin and a study of the kinetic properties of the modified species may help to elucidate the role of tryptophan in the activation process and in the enzymic function of the trypsin molecule. N-bromosuccinimide (NBS)3 has been used as an oxidizing reagent for tryptophan (7-14). Extensive use of NBS as an oxidizing agent for tryptophan has been summarized by Witkop (8) and Spande and Witkop (14). They concluded that NBS was specific for tryptophan at pH 4.0 and that little or no reaction with 3 Abbreviations used: NBS, N-bromosuccinimide; TM, NBS-modified trypsin; ATGM, activated NBS-modified trypsinogen; L-AM, L-arginine methyl ester; L-TAM, tosyl-n-arginine methyl ester; IrATrE, acetyl-n-tryptophan ethyl ester; STI, soybean trypsin inhibitor.
506
TRYPSINOGEN
tyrosine and/or histidine occurred under their reaction conditions. Their conclusions were based principally on the following evidence: (1) the amino acid analysis of oxidized trypsin (> 80 % tryptophan oxidized) showed no loss of residues other than tryptophan, with perhaps the loss of 0.5 moles of tyrosine (7) ; (2) the fact that a plot of change in absorbance at 280 rnlc vs. the moles of NBS reacted was linear (14), and (3) that generally oxidations requiring less than 4: 1 mole ratios of NBS to protein are indicative of the loss of tryptophan only (14). They also suggested that no significant peptide bond cleavage would have occurred under these conditions (8, 14). Williams and Laskowski (15) have also concluded that the same reaction conditions used with chymotrypsinogen caused the oxidation of tryptophyls only. Our modification procedures closely followed those set forward by Witkop (7) and were assumed to lead to oxidation of tryptophan with no significant side reactions. In this paper we describe the result,s obtained with trypsinogen modified by NBS and subsequently activated by trypsin, and with trypsin modified by NBS. Activation of the modified trypsinogen was characterized by the appearance and time course of the activation difference spectrum. The modified trypsin (TM) and the activated modified trypsinogen (ATGM) were characterized kinetically by steady state analysis of L-arginine methyl ester-benzamidine mixtures, their efficiency in activating chymotrypsinogen, and their susceptibility to inhibition by soybean trypsin inhibitor. EXPERIMENTAL Materi&. Trypsinogen (lots TG 7A and TG 6402A) and trypsin (salt-free lyophylized lot TRL 6263, and MgSOb lot TR 7FA) were purchased from Worthington Biochemical Corporation, Freehold, New Jersey. Soybean trypsin inhibitor (STI) was obtained from Nutritional Biochemical Company, Cleveland, Ohio. Benzamidine from Aldrich Chemical Company, Milwaukee, Wisconsin was recrystallized from butanol before use. The NBS was reagent grade and was recrystallized from water. All substrates were made in this laboratory and were characterized by melting point and exhaustive hydrolysis. Preparation of solutions. MgSOd was removed from trypsin by chromatography on columns of
ACTIVATION
507
Sephadex G-25 (2 X 50 cm) at pH 2.9 and a flow rate of 1 ml per minute. Salt-free trypsin was chromatographed on a column of Sephadex G-50 (2 X 100 cm) at pH 2.9 and a flow rate of 0.75 to 1 ml per minute; standard solvent (0.2 M KC1 and 0.05 M CaCl2) at pH 2.9 was used as eluent unless otherwise stipulated. Chromatography of trypsinogen on Sephadex G-25, G-50, and Sulfoethyl (SE) C-25 (16) gave a single symmetrical band which exhibited less than 0.3% of its potential tryptic activity. Therefore, trypsinogen was used direct,ly after dissolution and centrifugation at 16,000g to remove undissolved material. Concentrations were determined photometrically using the optical factors 0.65, 1.1, and 0.50 (mg per ml per absorbance unit at 278 mr) for trypsin, soybean trypsin inhibitor, and chymotrypsinogen respectively (Worthington Biochemical Corp., Technical Bulletin). All solutions were stored at 4.0”. Aqueous NBS stock solutions (0.01 M) were prepared as needed and kept from direct light by covering the flask with aluminum foil. All substrate and benzamidine solutions were prepared in “standard solvent.” Modification of proteins. The modification reactions were carried out at pH 4.0 f 0.05 by the addition of lo- to 20-~1 increments of 0.01 M NBS to the protein solution. The pH was maintained at 4.0 by the addition of KOH (0.2 M) from a microburet. The solutions were rapidly stirred during the addition of NBS and for lo-20 min after the final addition. The modified protein was then adjusted to pH 2.9 and stored at 4” until needed. The reaction of NBS with acetyl-L-tryptophan ethyl ester (ATrE) required 1.5 moles of NBS per mole of ATrE oxidized (based on plots of ti280 vs. moles NBS added (7). Trypsinogen and trypsin required 2.0-2.3 and 1.52.0 moles of NBS per moles of tryptophan oxidized respectively. Hachimori et al. (17) found no difference in the ease of oxidation of three tryptophan residues in trypsin and trypsinogen with HzOZ. Oxidized trypsinogen was subjected to ion-exchange chromatography on columns of Sephadex SE C-25 (0.9 X 10 cm) in the manner described by East (16), who has shown that trypsin with peptide bonds cleaved can be separated from intact trypsin by this procedure. Diflerence spectra. The activation difference spectra for chymotrypsinogen and trypsinogen were made at 25” on a Cary 15 spectrophotometer in the manner described by Benmouyal and Trowbridge (6). The zymogen was prepared in “standard solvent” which was buffered with 0.05 M Tris at pH 8.0. The activation wae started by adding 100 pl of the appropriate trypsin solution (2-5.0 X 10-b M) in t,he sample beam and to the solvent cell
508
DANIEL
AND
in the reference beam. The cells were scanned at time intervals between 330 and 250 rnp. In cases where a simultaneous assay for enzymatic activity was desired, 5-50 11 of the activating mixture was removed by Hamilton syringe immediately after the scan and the time recorded. The reaction was quenched by adding the aliquot to 9.0 ml of standard solvent at pH 2.9. If it was necessary to postpone the assay for an extended time the quenched aliquots were refrigerated at 4”. No increase in tryptic activity could be detected between assays made immediately after withdrawal and those made the next day. Rate measurements. All trypsin/substrate rate measurements were determined at 25” by pH stat techniques, using a Radiometer TTTlc. Assays for tryptic activity were performed by adding 1.0 ml of 0.01 M L-TAM to 9.0 ml of “standard solvent” containing the quenched activation aliquot. The pH was adjusted to 8.0 and the rate determined. The assay was reported as VO/PO, where Vo is the initial rate and Pa is the total protein concentration. The trypsin/cAM, benzamidine rates were determined by mixing the appropriate volumes of “standard solvent” and stock L-AM solution to obtain the desired concentration; the trypsin and benzamidine were then added and the pH adjusted to 5.0 for measurement of initial rates. STI inhibition. ST1 and enzyme were mixed with 9.0 ml of solvent at pH 5.0 and the pH adjusted to 8.5. One milliliter of 0.01 M *TAM was added, the pH adjusted to 8.0, and the initial rate determined.
RESULTS Trypsinogen activation The growth of the trypsin-trypsinogen difference spectrum during the autocatalytic activation of trypsinogen parallels the appearance of tryptic activity as measured by the rate of hydrolysis of L-TAM. Figure 1 shows the tryptic activity and magnitude of the difference spectrum (AA) normalized to a single scale plotted versus time. The assay for activity is reported as V,/P,, where PO is the potential molar concentration of trypsin. The AA reported is the sum of ( AA ( at 278 rn@ and 1AA 1 at 293 mp. The figure shows good agreement between the two methods of measuring the extent of activation. After 4-6 hr the difference spectrum begins to develop a larger negative peak at 293 rnp. Since the tryptic activity is stable after 4 hr (V,/P, decreases less than 3 % after 20 hr),
TROWBRIDGE
MINUTES 160
200
FIG. 1. Time course of trypsinogen activation. Open and closed circles represent tryptic activity and difference spectrum development, respectively, normalized to percentage of maximum response. Conditions given in Procedure.
the correlation between AA and tryptic activity is lost. This deviation of the time dependence of AA from a simple autocatalytic curve is not due to activation in the reference beam, since the tryptic activity there has not exceeded 1% of the potential value after 20 hr. Nor does the deviation seem to be caused by autolysis into inactive fragments in the activating mixture, since the activity stabilizes after 4 hr while AA continues to change. Much evidence exists that the autocatalytic activation of trypsinogen produces an enzymatically active form of trypsin with an internal bond cleaved, but with the same amino acid composition and hydrodynamic properties as intact trypsin (16, 18-22). Low pH chromatography of the activation mixture on SE Sephadex C-25 (16) yielded two active components which have been tentatively identified on the basis of kinetic comparison with Shaw’s (Y and p trypsin (16,22). Some kinetically inactive material was also found in small amounts. The formation of these additional products could account for the deviation of the rate of growth of the difference spectrum from the rate of appearance of tryptic activity. Efect of NBS on activation. Trypsinogen was modified as described in the Experimental section at mole ratios of NBS to protein of 1: 1, 2: 1, and 4: 1. These ratios correspond to less than one, approximately oxidized per one, and two tryptophans
TRYPSINOGEN
trypsinogen molecule (based on plots of aAzso rnp vs. moles of NBS added for completely oxidized proteins, seeModification of Proteins). These ratios were well within the linear region of a plot of AAzso against moles of NBS added to trypsinogen solutions. Figure 2 contrasts the activation difference spectra of unmodified and modified trypsinogen. The peak locations of the difference spectrum for all modifications are the same, but the size and time course of the growth of the spectra are quite different. The maximum values of AA/A at 287 rnp are 0.01,0.03,andO.lSforthe1:1,2:1,and4:1 modification ratios, respectively. The difference spectra for 1: 1 and 2: 1 modification experience a maximum change in absorbance at 30 and 55 min, respectively. AA for the 4: 1 modification increased monotonically (Fig. 3). For the 1: 1 and 2: 1 ratios the tryptic activity appeared monotonically and reached a maximum after 2-3 hr of incubation (Fig. 4) ; the activity then remained constant for up to 20 hr. For the 4: 1 modifi-
[
509
ACTIVATION
MINUTES
FIG. 3. Time course of trypsinogen activation difference spectra. (A) unmodified, (B) 4:l and (C) 2:l mole ratio NBS-modified trypsinogen. Left hand ordinate for curves (A) and (C), right hand ordinate for curve (B). Experimental conditions in Procedure. r 20
./Ys
, I
-
3O
.02 I
I
40
I
I
120 MINUTES
I
!
/
I
200
FIG. 4. Appearance of tryptic activity. (A) and (C), 1:l and 4:l mole ratio NBS-modified trypsinogen, respectively; (B) is unmodified trypsinogen. Right ordinate for (B), left ordinate for (A) and (C). Experimental conditions in Procedure.
- .oo PA
--.02
--.04
w 250 I
270 I
290 I
310 I
330 I
FIG. 2. Activation difference spectra for trypsinogen (A), and for trypsinogen reacted with NBS at 2:l mole ratio (B). Experimental conditions in Procedure.
cation ratio (Fig. 4) the activity was low and reached a maximum after about 30 min, with a subsequent gradual decrease in activity. The increase in tryptic activity with time does not parallel the growth of the difference spectra for any of the modifications studied. Kinetic characterization of modified protein. Xixtures of L-arginine methyl ester (~-Ai\l)~ and benzamidine (23) have been shown to provide a convenient system for the charac‘Unpublished
data from this laboratory.
510
DANIEL
AND
terization of trypsin. Initial rate data at pH 5.0 are closely reproduced by Eq. 1. vo -= PO
kS/K* 1 + S/K, + I/&
(1)
Rate data obtained with ATGM and TM preparations were fitted to equation (1) by nonlinear regression analysis. The values of the parameters obtained from the analysis indicate that K, and KC are somewhat affected (Table I) for all modification ratios studied. However, the changes in K, and Kc are not sufliciently large to be kinetically meaningful. On the other hand, the values obtained for k, decrease as the NBS:protein ratio increases for both ATGM and TM (Table I). The decrease is much more marked for ATGM than for TlM (Fig. 5). The benzamidine KC values obtained for the ATGM and TM are in agreement with the dissociation constant determined by East (24) by an equilibrium method, when appropriate corrections are made for pH. The rate of chymotrypsinogen activation by ATGM and TM preparations was measured by observing the development of the activation difference spectrum produced (6, 25). The apparent first-order rate constant for the activation was evaluated and nor-
TROWBRIDGE
malized for differences in the amount of catalyst used. These constants show the same behavior with respect to NBS modification as do the Ic, values for L-AM (Table I). Once again the catalytic “efficiency” of ATGM drops more sharply than does that of TM with increasing extent of NBS modification. The trypsin/L-TAM system has been shown to exhibit substrate activation (26, 27). The ratio of rates of hydrolysis of 0.01 and 0.001 M L-TAM at pH 8.0 are given in Table I and can be taken as an assessment of substrate activation in ATGM, TM, and untreated trypsin; all exhibited a similar degree of substrate activation. Decreases in Vo/Po (0.001 M L-TAM) with increasing extent of NBS modification closely approximate the curves obtained for k, in Fig. 5. All modified trypsin used in making the kinetic measurements in this section was prepared directly from commercially available trypsin with no attempt to separate the two active components present (16, 22). Analysis f the individual components showed that the use of unfractionated trypsin did not Rignificantly alter the kinetic results. STI inhibiticw. The kinetic results sum-
TABLE THE
Proteina T ATG TM TM TM ATGM ATGM ATGM
Mo&;;io, protein 0:l 0:l 1:l 2:l 4:l 1:l 2:l 4:l
VARIATION
OF KINETIC
R,, SK-~
K, x 103, Y
12.92
f
.25
13.50 10.02 8.85 2.91 5.68 2.29 0.50
f f f f f f f
.80 .77 .13 .04 .14 .07 .02
5.53 5.63 5.46 5.45 5.22 4.11 4.32 5.79
f f f f f f f f
I
WITH NBS
PARAMETERS
.40 .75 .75 .33 .34 .45 .52 .92
K< x 100
M
4.92
f
.31
4.95 6.07 5.89 5.20 5.15 5.07 5.46
f f f f f f f
.87 .61 .35 .29 .55 .73 .86
OXIDATION
CTG activationC Substrat.+ activation K @.PP)X ratio, L-TAM 101 Id-1 xc-
6.95 6.95
5.31 3.45 1.16 1.63 .79 -
1.65 1.65 1.62 1.67 1.70 1.54 1.64 1.67
Per cent
activesit& remaining loo 100 100 100 33 29 13 5
0 Protein abbreviations: T, commercial trypsin; ATG, trypsinogen activated in this laboratory; TM, modified trypsin; ATGM, activated modified trypsinogen. * Parameters evaluated by nonlinear regression analysis on Eq. 1; the ranges shown are calculated at 95% confidence level. The substrate and inhibitor are L-AM and benzamidine, respectively. Reaction conditions in Experimental. c Apparent second-order rate constant calculated from the growth of the chymotrypsinogen activation difference spectrum. d The ratio of the L-TAM hydrolysis rate at 0.01 M to that at 0.001 M substrate, pH 8.0. e Based upon 100% for native protein, as inferred from STILL-TAM inhibition study.
TRYPSINOGEN
511
ACTIVATION
up to 2: 1 mole ratio, but that the catalytic efficiency of the site is reduced. The constant value of the So/To intercept has two possible interpretations: (1) To and K change with
5 MOLES
NBS
/ MOLE
PROTEIN
FIG. 5. The effect of the extent of NBS oxidstion upon k, for L-arginine methyl ester at pH 5.0 (Eq. 1). (A), activated modified trypsinogen; (B), modified trypsin. Conditions given in Procedure.
marked in Table I show that, the effect of NBS modification before or after trypsinogen activation is seenprimarily in the values of k,. The decrease in the apparent rate constant could be due to a diminution in the catalytic efficiency of the active sites, to a decrease in the concentration of sites, or to both. In an attempt to resolve this question, FIG. 6. Variation of ST1 inhibition with inthe ST1 inhibition of ATGM- and TMcreasing extent of NBS oxidation, modified trypcatalyzed hydrolysis of L-TAM was investi- sin with L-TAM as substrate at pH 8.0. (A) native gated. If we assume that the reaction of trypsin; (B) l:l, (C) 2:1, (D) 4:l mole ratio respectively. The ordinate is k’ = trypsin with ST1 can be represented by NBS/trypsin, VO/P~, with PO calculated as moles trypsin before T + S @ X, then the concentration of comDlex (X) is given by X = l/,[S, + To + K NBS oxidation* 1 (SO‘+. TO-+ K); - 4 ?&~j112]. SOand To , are the total concentrations of soybean inhibitor and of trypsin, respectively, and K _ 3o is the dissociation constant of X. We find in the limit of small So that dX/dSo = To/ (To + K). This and the fact that T = To X predicts that a plot of T/To vs. So/To will -20 ka be a straight line with slope -To/(To + K) and having an extrapolated intercept on the So/To axis of (To + K)/To. The data in Figs. 6 and 7 are straight lines, and we conclude that they are within the range of validity of the limiting equation for dX/dSo . We assume that the. velocity of L-TAM _ ^ hydrolysis is proportional to free trypsm, so
that I?’ is proportional to T/To . In Fig. 6, the decreasing k’ intercept with a constant So/To intercept is consistent with the view that the concentration of active sites is unchanged by the action of NBS upon trypsin
.05
I
.I5
.25
FIG. 7. Variation of ST1 inhibition with increasing extent of NBS oxidation, activated modified trypsinogen with L-TAM as substrate at pH 8.0. (A) l:l, (B) 2:1, (C) 4:l mole ratio NBS/trypsinogen, respectively. Ordinate as in Fig. 6. Experimental conditions in Procedure.
512
DANIEL
AND TROWBRIDGE
NBS modification in such a way as to keep the intercept constant. This coincidence seemshighly improbable. (2) The remaining possibility is that neither TO nor K have changed. This interpretation, which we advocate, is consistent with the data of Table I which shows that the K values for small ligands were hardly affected by NBS modification. In Fig. 7, corresponding data obtained with ATGM lead to a family of parallel lines. The slopes, which should change with To, are only apparently constant because TO is always much greater than K (Lebowitz and Laskowski estimate K = 10-l’ at pH 8.3 (28)). The decrement in both intercepts is equal, and this is most simply explained by decreasedsite concentration. If the observed velocity (k’) is corrected under the assumption that total concentration of sites is diminished by NBS, a common intercept is obtained on the k’ axis. The slopes -TO/ (To + K) become increasingly negative, as they should if TO is decreasing, and the Xo/To intercepts decrease in proportion to To . We conclude that the reaction of NBS with TG prior to activation causesa decrease in the concentration of sites obtainable upon “activation,” with no significant change in the catalytic properties of the remaining active sites. This is in contrast with the effect of modification of trypsin with NBS, wherein the concentration of sites is unaffected (at least up to 2:l mole ratio), but the catalytic properties of the sites are modified. SE Sephadex C-25 chromatography of ATGM in the manner described by East et al. (16) yielded up to four kinetically inactive species.The ATGM gave up to 95 % (by absorbance) inactive species at 4: 1 modification. The TM at 4: 1 modification gave the expected two active speciesas well as two kinetically inactive species. All the UV spectra for the inactive speciesin ATGM and TM were qualitatively different, but resembled in gross features that of extensively modified trypsin and trypsinogen. The chromatography of ATGM lends important support to the hypothesis that inactive (no active site) trypsin is formed upon activation of NBS modified trypsinogen, as
was put forward in interpretation of the ST1 inhibition data. The two active components found in commercial trypsin or freshly activated trypsinogen were obtained by the chromatography procedure described earlier (16). Both active species were modified separately by NBS in 1: 1, 2: 1, and 4: 1 ratios. The resulting modified trypsin was characterized by the inhibition of L-TAM hydrolysis by soybean trypsin inhibitor. A decreasein k’ with increasing extent of NBS modification was seen with both active components; no decrease in active sites was observed in either caseuntil NBS modification exceeded a 2: 1 ratio. Thus the same results were obtained with either of the two active components or with their mixture. DISCUSSION
We suggest that in trypsinogen there is a tryptophyl residue which is essential to potential tryptic activity, and that this residue is the most reactive with NBS. This is based upon the decrease in active site concentration with increasing NBS oxidation as inferred from Fig. 7, and the result of Fig. 5. The latter shows that all potential activity would be lost at 1 mole of tryptophan oxidized per mole of zymogen, if the efficiency of the NBS reaction (in reducing potential activity) continued undiminished. Viswanatha et al. (7) found that approximately 20% of the four tryptophyl residues in TG could be oxidized with no loss in potential activity, which is in disagreement with our analysis (Table I). The presence of “inert” protein containing readily oxidized tryptophans could explain a lossof absorbancewith no lossin activatable trypsinogen. At 4 moles NBS per mole of trypsinogen, the potential active site concentration (Table I and Fig. 7) is 4 % that of unoxidized trypsinogen. Nonetheless the action of trypsin upon this modified protein still gives rise to a difference spectrum whose growth is completed in lessthan 4 hr (Fig. 3), and thus is comparable in rate to the activation of unmodified trypsinogen. The data, both kinetic and chromatographic, support the conclusion that both active and inactive trypsin molecules are produced during the activation of
TRYPSINOGEN
NBS-modified trypsinogen. The active species produced by the “activation” of modified trypsinogen apparently have kinetic properties unchanged by the NBS reaction. This is concluded from the observation that a correction for active site depletion, calculated from the STI-inhibition data, results in calculated rates of hydrolysis of amino acid esters and of chymotrypsinogen that are equal to those obtained with the unmodiied enzyme. The binding of small ligands is unaffected (Table I). The NBS modification of trypsin does not lead to a decrease in active sites until the ratio of NBS to protein exceeds 2: 1 (Fig. 6). The binding constants for small ligands is hardly affected by NBS modification, but the catalytic efficiency does decrease (Table I). Even for 4: 1 modified trypsin the decrease in catalytic efficiency (k,) cannot be explained wholly by a loss in active sites, although sites are destroyed at this modification ratio (Fig. 6 and Table I). Thus the tryptophan most susceptible to NBS oxidation in trypsin is nonessential to catalytic function. The decreasein active site concentration seenat 4: 1 modification may be due to oxidation of an essential tryptophan in trypsin. The appearance of the activation difference spectrum for 4: 1 modification (Fig. 2) implies a substantial tryptophan contribution; apparently some of the remaining tryptophans still experience an appreciable perturbation when modified trypsinogen is acted upon by trypsin. Since a large contribution to the difference spectrum would not be expected from oxidized tryptophyls, some of the less reactive tryptophyls must underperturbation during activation. %lliIms and Laskowski (15) and Spande and Witkop (14) have considered the possible correlation between NBS reactivity and exposure of the tryptophyls to the medium. If the same essential tryptophyl is detected by NBS oxidation in both trypsinogen and trypsin, then its relative reactivity is less in the latter protein. Hachimori et al. (17) suggested that the susceptibility of individual tryptophyls to Hz02 oxidation may be different in trypsinogen and trypsin. One would then conclude that the degree of ex-
ACTIVATION
513
posure of this essential tryptophyl has changed in the conversion of trypsinogen to trypsin. Oxidized tryptophan residues in active trypsin apparently have no role in “binding” simple substrates and small ligands since no appreciable change can be detected in the binding parameters of L-AM and benzamidine (Table I) for all ATGM and TM preparations tested. The function of these “nonessential” tryptophans in catalysis is not known, but a decreasein catalytic efficiency is observed in TM with no concommitant decrease in active site concentration. The concept of an essential residue is somewhat elusive. If the molecule is progressively “degraded” as succeeding residues are oxidized, with activity finally destroyed with the last tryptophyl, no special role can be assigned with any confidence. If, however, there is a loss of activity when one of the most reactive residues is affected (as with our results) then such a residue is apparently “critical” to normal function and may be considered essential. Investigation into the details of the trypsinogen-trypsin conversion is continuing. ACKNOWLEDGMENT We thank Sharon A. Arihood, who established the validity of Eq. 1, and Emily J. East who developed the chromatographic procedures. REFERENCES J. H., KUNITZ, M., AND HERRIOT, Enzymes,” 2nd Ed., R. M., “Crystalline p. 127. Columbia Univ. Press, New York (1948). DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 515 (1955). PECHERE, J-F., AND NEURATH, H., J. Biol. Chem. 229, 389 (1957). NEURATH, H., RUPLEY, J. A., AND DREYER, W. J., Arch. Biochem. Biophys. 66, 243 (1956). NEURATH, H., Adv. Prot. Chem. 12, 319 (1957). C. G., BENMOUYAL, P., AND TROWBRIDGE, Arch. Biochem. Biophys. 116, 67 (1966). VISWANATHA, T., LAWSON, W. B., AND WITKOP, B., B&him. Biophys. Acta 46, 216 (1960). WITKOP, B., Adv. Prot. Chem. 16, 221 (1961). VISWANATHA, T., AND LAWSON, W. B.. Arch. B&hem. Biophys. 93,128 (1961).
1. NORTHRUP,
2.
3. 4. 5. 6. 7. 8. 9.
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DANIEL
AND
10. HAYASHI, K., 1~0~0, T., FUNATSU, G., AND FUNATSU, M., J. Biochem. 68, 227 (1965). 11. KRONMAN, M. J., ROBBINS, F. M., AND ADIOTTI, R. E., Biochim. Biophys. Acta 143, 462 (1967). 12. SPANDE, T. F., GREEN, N. M., AND WITKOP, B., Biochemistry 6, 1926 (1966). 13. STEINER, R. F., Biochemistry 6, 1964 (1966). 14. SPANDE, T. F., AND WITKOP, B., Methods in Enzymology 11, 498 (1967). 15. WILLIAMS, E. J., AND LASKOWSKI, M., JR., J. Biol. Chem. 240, 3580 (1965). 16. EAST, E. J., AND TROWBRIDGE, C. G., Abstr. Southeastern ACS Meeting, No. 205, p. 100 (1968). 17. HACHIMORI, Y., HORINISHI, H., KURIHARA, K., AND SHIBATA, K., Biochim. Biophys. Acta 93, 346 (1964). 18. NORD, F. F., AND BIER, M., Biochim. Biophys. Acta 12, 56 (1953). 19. TIMASHEFF, S. N., AND STURTEVANT, J. M., Arch. B&hem. Biophys. 63, 243 (1956).
TROWBRIDGE 20. IACHAN, A., DOMONT, G., DISITZER, L., PEILRONE, J., Nature 203, 43 (1964). 21. MAROUX, S., ROVERY, ;\‘I., AND DESNUELLE, P., Biochim. Biophys. Acta 140, 377 (1967). 22. SCHROEDER, D. D., AND SHAW, E., J. Biol. Chem. 243, 2943 (1967). 23. MARES-GUIA, M., AND SHAW, E., J. Biol. Chem. 240, 1579 (1965). 24. EAST, E. J., AND TROWBRIDGE, C. G., Arch. Biochem. Biophys. 126, 334 (1968). 25. CHERVENU, C. H., Biochim. Biophys. Acta 26, 222 (1957). 26. TROWBRIDGE, C. G., KREHBIEL, A., AND LASKOWSKI, M. JR., Biochemistry 2, 843 (1963). 27. TRENHOLM, H. L., SPOMER, W. E., AND WOOTON, J. F., J. Am. Chem. Sot. 88, 4281 (1966). 28. LEBOWITZ, J., AND LASKOWSKI, M., JR., Biochemistry 1, 1044 (1962).