The involvement of an arginine residue of trypsin in its interaction with the kunitz soybean trypsin inhibitor

I~iochimica el Biophysica Acta, 303 (L973) 274 283

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA

36374

T H E INVOLVEMENT OF AN A R G I N I N E R E S I D U E OF T R Y P S I N IN ITS I N T E R A C T I O N W I T H T H E K U N I T Z SOYBEAN T R Y P S I N INHIBITOR

J O S E P H E. D E L A R C O * AND I R V I N E. L I E N E R

Department of Biochemistry, College of Biological Sciences, University of 2Vlinnesota, St. Paul, ~3/Iinn. 55rot (U.S.A.) (Received October 23rd, 1972)

SUMMARY

The separate treatment of bovine trypsin and the Kunitz soybean trypsin inhibitor with phenylglyoxal resulted in the modification of both of the arginine residues of trypsin and an average of 5 of the 9 arginine residues of the trypsin inhibitor. When the trypsin-soybean trypsin inhibitor complex was modified under the same conditions, a total of 3 arginine residues had reacted, from which it was concluded that 4 arginine residues had been buried in the complex. Analysis of the trypsin and soybean trypsin inhibitor which were dissociated from the phenylglyoxalated complex revealed that one of the buried arginine residues was derived from trypsin and an average of 2.5 to 3 residues from the trypsin inhibitor. Peptide maps of digests from trypsin which had been phenylglyoxalated in the presence of soybean trypsin inhibitor and from trypsin alone showed one arginine-containing peptide common to both digests. This peptide was isolated by ion-exchange chromatography and two-dimensioned paper chromatography and electrophoresis. From its composition and the known sequence of bovine trypsin, this peptide was shown to contain arginine-55 which is thus presumed to be located at or near the zone of contact in the trypsin-soybean trypsin inhibitor complex.

INTRODUCTION

The stoichiometric interaction of trypsin and the Kunitz soybean trypsin inhibitor provides a unique model system for studying the interaction of two wellcharacterized, biologically active macromolecules. Two aspects of the interaction of trypsin and soybean trypsin inhibitor are now well established (see review by Laskowski and Sealock 1) : (I) chemical modification of the active site of trypsin prevents Present address: National I n s t i t u t e of Child H e a l t h and H u m a n Development, 13ethesda, Md. 20014, U.S.A.

TRYPSIN--TRYPSIN INHIBITOR INTERACTION

275

its combination with soybean trypsin inhibitor* and (2) an arginine-isoleucine bond constitutes the reactive site of the inhibitor. Thus, the combination of trypsin with soybean trypsin inhibitor m a y be viewed as being analogous to the interaction of trypsin with a substrate for which it is specific. Other than an involvement of the catalytic site of trypsin (histidine-46 and serine-I83, based on numbering scheme of trypsinogen) and the arginine-63 and isoleucine-64 residues of soybean trypsin inhibitor, little is known as to what constitutes the full region of contact between these two macromolecules. Based on the kinetics of tritium-hydrogen exchange, Chepyzheva et al. ~ concluded that IO to 35 amino acid residues of trypsin were in contact with the pancreatic inhibitor (mol. wt, 65oo) and 25 to 5o residues with ovomucoid (tool. wt, 28 ooo). Models of the complex of trypsin or chymotrypsin with the pancreatic trypsin inhibitor constructed on the basis of X - r a y crystallographic data have revealed that the contact area, although rather restricted in size, contains m a n y possible sites of interaction a& We have a t t e m p t e d to characterize the contact zone between trypsin and soybean trypsin inhibitor (tool. wt, 22 000) by a chemical approach. The trypsin-soybean trypsin inhibitor complex is subjected to chemical modification with a specific functional group reagent, and each component is then isolated from tile dissociated, modified complex. By comparing peptides derived from each of these components with those derived from components dissociated from the unmodified complex, it should be possible to identify those peptides which are common to both. These peptides should contain those amino acid residues which were not modified in the complex and are therefore presumably located at or near the zone of contact. This paper reports the application of such an approach in which phenylglyoxal has been used as a reagent for the specific modification of arginine residues in trypsin, soybean trypsin inhibitor, and their complex. These studies led to the identification of an arginine residue of bovine trypsin as being located in a region of the trypsin molecule which is at or near its site of binding with soybean trypsin inhibitor. MATERIALS

AND METHODS

Preparation of trypsin, soybean trypsin inhibitor, and trypsin-soybean trypsin inhibitor complex Twice-crystallized bovine trypsin purchased from Worthington Biochemicals was further purified by chromatography on SE-Sephadex 5. No attempt was made to separate tile various forms of trypsin known to be present in commercial preparations of trypsin G. Thrice crystallized soybean trypsin inhibitor was also a Worthington product which was isolated as the F2 component described by Frattali and Steineff. The complex of trypsin and soybean trypsin inhibitor was isolated by chromatography on Sephadex G-75 according to Papaioannou and Liener 8. The complex could then be dissociated and its two components isolated by chromatography on SE-Sephadex as previously described s.

Modification of arginine residues Arginine residues were modified with phenylglyoxal (purchased from K and " Ako et al.2% however, have recently reported t h a t s o y b e a n t r y p s i n inhibitor is capable of combining with a n h y d r o - t r y p s i n in a stoichimetric fashion.

276

J.E.

D E L A R C O , I. E. L I E N E R

K Laboratories, Plainview, N.Y. and recrystallized from hot water) according to the procedure of Takahashi 9. In a typical experiment 50 mg of protein (trypsin, soybean trypsin inhibitor, or trypsin-soybean trypsin inhibitor complex) were dissolved in 5 ml 0.2 M N-ethylmorpholine acetic acid (pH 8), to which was then slowly added 3 ml of the same buffer solution containing 25 nag of phenylglyoxal. After I h at room temperature, the reaction was terminated with I ml glacial acetic acid, and the solution was dialyzed against o.ooi M HC1 at 4 °C and lyophilized. The modified complex could not be dissociated under the conditions used for the unmodified complex (see above). Dissociation of the modified complex could be effected, however, by chromatography on SE-Sephadex using I M propionic acid-o.oI M CaC12 (pH 2.5) in conjunction with a linear salt gradient from o to I M NaC1.

Activity measurements Trypsin activity on N-a-benzoyl-L-arginine ethyl ester was measured spectrophotometrically 1°. For the measurement of antitryptic activity the amount of trypsin activity remaining after the addition of solutions containing soybean trypsin inhibitor, or derivatives thereof, was measured and converted to mg trypsin inhibited per mg of the inhibitor. The concentration (mg/ml) of trypsin and soybean trypsin inhibitor solutions were obtained by multiplying the absorbance readings at 280 nm by the factors 0.64 and 1.o6, respectively n.

Reduction and aminoethylation Prior to enzymatic digestion, the various protein preparations were reduced and aminoethylated as described by Cole 12. The reduced, aminoethylated protein was separated from excess reagents by passage through a column of Sephadex G-25 equilibrated and eluted with 0.2 M acetic acid; fractions containing the protein were pooled and lyophilized. Amino acid analysis 13 revealed that the cystine residues had been quantitatively converted to S-(/~-aminoethyl)cysteine.

Digestion and separation of peptides To a 1% solution of the reduced, aminoethylated protein in o.2 M ammonium acetate buffer (pH 7), containing o.ooi M CaC12, was added 0.02% L-I-chloro-4phenyl-3-tosylamidobutan-2-one-treated trypsin 14. After 24 h at room temperature, glacial acetic acid was added to a final concentration of I M, and the digest was lyophilized. Peptide maps of the digest were made by chromatography in one direction using n-butanol glacial acetic acid-water (4:1:5, by vol.) and eleetrophoresis at right angles in formic acid glacial acetic acid-water (25:87:888, by vol.) (pH 2.1) 15. Arginine-containing peptides were visualized with phenanthrenequinone 16 which produces a highly fluorescent spot when viewed under ultraviolet light. After circling such spots, the chromatograms were then sprayed with ninhydrinlE For the preparation of larger quantities of peptides, the digest was chromatographed first on a column of Dowex 5o-X2 (Aminex 5oW-X2 Bio-Rad Laboratories) using a linear gradient of pyridine acetic acid buffer (0.2 M, pH 3.1 to 2 M, pH 5) 1~. The effluent was monitored with a Technicon autoanalyzer equipped for alkaline hydrolysis and ninhydrin analyses 19. The phenanthrenequinone reagent was used to detect those fractions which contained arginine. The latter were pooled and subjected

TRYPSIN--TRYPSIN INHIBITOR INTERACTION

277

to chromatography on a column of Dowex I-X2 (AG I-X2, Bio-Rad) using a gradient provided by a 4-chambered reservoir containing IOO ml of each of the following buffers in the order shown: (I) 3% pyridine (pH 8.3), (2) 0.5 M pyridine-acetate (pH 6), (3) I.O M pyridine acetate (pH 5.5) and (4) 2.0 M pyridine acetate (pH 5). Final purification of the arginine-containing peptides was achieved by two-dimensional paper chromatography and electrophoresis as described above. After visualization with phenanthrenequinone, the spots were eluted with lO% acetic acid and analyzed for amino acids xa. RESULTS

Measurement of protected arginine residues in trypsin soybean trypsin inhibitor complex D a t a pertaining to the modification of arginine residues before and after for-

/

I ~

smz

°°1 /\

A ,oo

~

I

,so

PG-~ypsin

/X 0

I00 TUBE NUMBE~0

200

Fig. t. D i s s o c i a t i o n of u n m o d i f i e d a n d p h e n y l g l y o x a l a t e d t r y p s i n - s o y b e a n t r y p s i n i n h i b i t o r P G - S T I c o m p l e x b y c h r o m a t o g r a p h y on S E - S e p h a d e x . I n (A) t h e u n m o d i f i e d c o m p l e x w a s diss o c i a t e d a t p H 2.6 u s i n g a l i n e a r g r a d i e n t of o-o. 5 M NaC1 as d e s c r i b e d b y I ' a p a i o a n n o u a n d L i e n e r 8. i n (B) th e p h e n y l g l y o x a l a t e d (PG-) c o m p l e x was d i s s o c i a t e d in I M p r o p i o n i c a c i d us i ng a l i n e a r g r a d i e n t of o i.o M NaC1. I n b o t h cases i o o m g of c o m p l e x were a p p l i e d t o t h e c o l u m n (2.5 cm × i o o cm) a n d 5-ml f r a c t i o n s were collected a t flow r a t e of 3 ° ml / h a t 4 °C. P e a k s d e n o t e d b y d o u b l e - h e a d e d arrows were pooled, d i a l y z e d , a n d lyophi l i z e d.

mation of the trypsin soybean trypsin inhibitor complex (Fig. IA) are shown on Table I. Amino acid analyses of these preparations revealed that only the arginine residues had been affected, although the possibility exists that modifications of other amino acids m a y have been reversed by acid hydrolysis. Both of the arginine residues present in bovine trypsin were reactive towards phenylglyoxal, and, as shown in Table II, modification of these two arginine residues resulted in a complete loss in enzymatic activity. Of the 9 arginine residues present in soybean trypsin inhibitor, only 5 were capable of reacting with phenylglyoxal, and, as shown in Table II, inhibitory activity towards trypsin was completely lost. In order to determine the origin of the arginine residues which m a y have become buried or protected in the formation of the complex, the phenylglyoxalated complex was dissociated into its trypsin and soybean trypsin inhibitor components by chromatography on SE-Sephadex as shown in Fig. IB. Quantitative dissociation was indicated by the virtual absence of any complex in the effluent from the SE-

278

j.E.

TABLE

I ) E L A R C O , I. V;. L I F . N E R

1

ARGININE

RESIDUES

INHIBITOR

COMPLEX

PROTECTED

FROM

PHENYLGLYOXALATION

IN

TRYPSIN--SOYBI~2AN

TRYPSIN

Values shown are moles per mole of protein.

Co~ponent

Total

Trypsin S o y b e a n trypsin inhibitor

2 9

Residues reactive toz~ards phe~ylglyoxal*

l~ejore corrzple.vatio~

After complexation

1.8 5.I

o.9 2.4

Resid~tes prolected** o.9 2.7

* Difference in arginine content before and after phenylglyoxalation. "* Difference in number of reactive arginine residues before and after complexation.

Sephadex column. Analyses of these components for arginine (Table I) revealed that the trypsin now contained only one reactive arginine residue compared to the two obtained for trypsin prior to complexation with the trypsin inhibitor. Soybean trypsin inhibitor from the phenylglyoxalated complex now contained 2. 4 reactive arginine residues as contrasted with 5.1 when this component was modified in the absence of trypsin. These data thus permit the conclusion that one arginine residue from trypsin and an average of 2 to 3 residues of arginine from the trypsin inhibitor had been rendered inaccessible to phenylglyoxal as a result of complexation. It is of interest to note from the activity data in Table II that the components TABLE

II

EFFECT

OF

ACTIVITY

MODIFICATION

OF TRYPSIN

AND

OF

ARGININE

SOYBEAN

Component Belbre complexation : trypsin s o y b e a n trypsin inhibitor After dissociation from modified complex : trypsin s o y b e a n trypsin inhibitor

RESIDUES

TRYPSIN

BEFORE

AND

AFTER

COMPLEXATION

ON

THF

INHIBITOR

Residz~es modified/tolal

Activity" (%)

2/2 5/9

o o

I/2 2-3/9

5l 58

* Compared to activity of lOO% for unmodified trypsin and the trypsin inhibitor.

recovered from tile phenylglyoxalated complex retained only about 50% of ttle activity of unmodified trypsin and soybean trypsin inhibitor. This would suggest that arginine residues of trypsin or soybean trypsin inhibitor which are not directly involved in the contact zone of the complex m a y nevertheless play an important role in the maintenance of the activity of these proteins (see Discussion).

Isolation of arginine peptides from trypsin Fig. 2 shows a comparison of the peptide maps obtained from tryptic digests of reduced, aminoethylated trypsin which had been derived either from the phenyl-

TRYPSIN-TRYPSIN

INHIBITOR

279

INTERACTION

g l y o x a l a t e d or the u n m o d i f i e d t r y p s i n - s o y b e a n t r y p s i n i n h i b i t o r c o m p l e x . O n l y t h o s e p e p t i d e s w h i c h c o n t a i n arginine, a n d h e n c e fluoresce w h e n t r e a t e d w i t h p h e n a n t h r e n e q u i n o n e r e a g e n t , are s h o w n in t h e s e figures. T h e m o s t o b v i o u s difference b e t w e e n t r y p s i n w h i c h h a d b e e n p h e n y l g l y o x a l a t e d while c o m b i n e d w i t h t h e t r y p s i n i n h i b i t o r a n d t r y p s i n d e r i v e d f r o m the u n m o d i f i e d c o m p l e x is t h e a l m o s t c o m p l e t e d i s a p p e a r a n c e of P e p t i d e s I a n d 3 w h i c h w e r e p r e s e n t in u n m o d i f i e d t r y p s i n . T h e p r e s e n c e of d e t e c t a b l e a m o u n t s of P e p t i d e s I a n d 3 in t h e digest o f t r y p s i n f r o m the m o d i f i e d c o m p l e x is n o t s u r p r i s i n g in v i e w o f the r e v e r s i b i l i t y o f the r e a c t i o n of

O8 ± 0

0

(+.1

ELECTROPHORESIS

r

,.3

8~i

T

120

%°0 ×

02

02

B

x

• I 02

~e 5

A

40

(-) origin

ori@n

,~

(+)

.,.~ IJ3 ..2

ELEGTROPHORESIS-~)

1-)

Fig. 2. Peptide maps of tryptic digest of reduced, aminoehtylated trypsin derived from the unmodified trypsin soybean trypsin inhibitor complex (A) and from the phenylglyoxalated complex (B). Only the arginine peptides which were detected as fluorescent spots with phenanthrenequinone 1~ are shown. Weakly reactive spots are denoted with a broken line. Fig. 3. isolation of arginine peptides from tryptic digest of reduced, aminoethylated trypsin. The numbers on the abscissae correspond to fraction numbers, and numbers on the ordinates to absorbance at 57 ° n m due to color produced with ninhydrin after alkaline hydrolysis. The darkened peaks denote the fractions which gave a positive test for arginine with phenanthrenequinone; these fractions were pooled as indicated by the double-headed arrows. Top: chromatography of 75 mg of digest on a o.6 cm × io 3 cm column of Dowex 5o-X2; temperature, 55 °C, flow rate, 3° ml/h; 3 ml per tube. Center: chromatography of Fractions l and [I on o.6 cm × IO() cm column of Dowex I-X2; temp., 37 °C, flow rate, 3° ml/h; 3 ml per tube. Bottom: peptide maps of Fractions I' and l I ' as visualized with phenanthrenequinone reagent; solid spots denote strong fluorescence and weakly fluorescent spots are enclosed with broken lines. p h e n y l g l y o x a l w i t h a r g i n i n e at n e u t r a l p H (ref. 9). P e p t i d e 2 is p r e s e n t in b o t h digests and hence must contain the arginine residue which had not reacted with phenylg l y o x a l while c o m p l e x e d w i t h t h e t r y p s i n i n h i b i t o r . I n t h e p h e n y l g l y o x a l a t e d deriv a t i v e of t r y p s i n , t w o a d d i t i o n a l spots, d e n o t e d as P e p t i d e s 4 a n d 5, m a y be n o t e d . T h e s e t w o p e p t i d e s w e r e a c t u a l l y f l u o r e s c e n t before t r e a t m e n t w i t h p h e n a n t h r e n e q u i n o n e a n d are m o s t l i k e l y d e r i v e d f r o m P e p t i d e s I a n d 3 w h i c h c o n t a i n t h e a r g i n i n e r e s i d u e o f t r y p s i n w h i c h h a d r e a c t e d w i t h p h e n y l g l y o x a l while in t h e c o m p l e x . I n o r d e r to i d e n t i f y t h e a r g i n i n e r e s i d u e of t r y p s i n w h i c h h a d n o t r e a c t e d w i t h

2~0

] . E. I)ELARCO, I. E. L I E N E R

phenylglyoxal, efforts were made to isolate Peptide 2 on a larger scale directly from trypsin itself, care being taken to duplicate as closely as possible the conditions which had led to the results shown in Fig. I. A tryptic digest of reduced, aminoethylated trypsin was first subjected to ion-exchange chromatography on Dowex 5o-X2, and the arginine-containing peaks from this column were re-chromatographed on Dowex I-X2. Final purification of the arginine peptides was achieved by two-dimensional chromatography and electrophoresis. The steps involved in the purification of the arginine are summarized in Fig. 3. Peptides I, 2, and 3 were cut from their respective maps and eluted with lO°6 acetic acid and their composition determined by amino acid analysis. Since treatment with phenanthrenequinone led to partial destruction (about 5o%) of the arginine, a more accurate analysis for arginine was obtained on peptides which were isolated by paper electrophoresis in which guide strips treated with phenanthrenequinone were used to locate the arginine-containing peptidc.~. Amino acid values obtained from hydrolyzates of these peptides are recorded in Table I I I . Based on the composition data of these peptides and the known sequence of bovine trypsin 2°, the most probable sequences for the arginine peptides isolated in this study m a y be deduced as noted in Table I I I . Since Peptide 2 is the only arginine TABLE

11I

AMINO ACID COMPOSITION OF ARGININE PEPTIDES ISOLATED FROM A TRYPTIC DIGEST OF REDUCED, AMINOETHYLATED TRYPSIN V a l u e s s h o w n a r e m o l a r r a t i o s w i t h r e s p e c t t o a r g i n i n e t a k e n as i . o o . F i g u r e s in p a r e n t h e s e s are t h e n e a r e s t w h o l e i n t e g e r s . T h e p r o b a b l e s e q u e n c e is b a s e d o n s e q u e n c e a n d n u m b e r i n g for t r y p s i n o g e n as r e p o r t e d b y W a l s h a n d N e u r a t h 2°.

A m i n o acid

Peptide •

Peptide 2

Peptide 3

Arg Asp Ser Glu Gly Ala Val Ile Leu

i.oo 0.94 2.15 o.15 0.20 2.21 o o o.9o

I.OO o.o 3 0.84 1.o0 1.o8 o.oi o.91 I.OI o

i.oo o 1.12 o.15 o.15 0.02 o o o

Probable sequence

99 Ala-AlaSer-LeuAsn S e r lO 5 Arg

(i) (i) (2) (o) (o) (2) (o) (o) (I)

(i) (o) (i) (i) (i) (o) (I) (I) (o)

5° Ser-GlyIle-Gln V a l - A r g 55

(i) (o) (I) (o) (o) (o) (o) (o) (o)

lO4 lO5 Ser-Arg

peptide to be recovered intact from trypsin which had been phenylglyoxalated while complexed with soybean trypsin inhibitor, the specific arginine residue which had been protected can now be identified as arginine-55 (according to the numbering of bovine trypsinogen 2° which is employed here or arginine-65A according numbering system of chymotrypsinogen A3). The other arginine residue of trypsin, arginine-Io5, is presumably reactive with phenylglyoxal even when complexed with soybean trypsin inhibitor. The finding of

TRYPSIN TRYPSIN INHIBITOR INTERACTION

28I

arginine-Io5 in Peptide 3 in the form of the dipeptide, Ser-Arg, is somewhat surprising. I t must have been derived from the cleavage of the Asn-Ser bond present in Peptide I, a type of linkage not normally split b y trypsin. Walsh et al. ~ have also reported the isolation of Ser Arg from a tryptic digest of S-sulfotrypsinogen. DISCUSSION

The present study represents an a t t e m p t to elucidate the nature of the binding sites between trypsin and soybean trypsin inhibitor by comparing the reactivity of certain amino acid residues before and after complexation. Implicit in this approach is the assumption that any observed differences in reactivity are a direct consequence of the proximity of such groups to the zone of contact between trypsin and soybean trypsin inhibitor. For reasons which have been discussed elsewhere2,22, 2a conformational changes in trypsin or the trypsin inhibitor induced by complex formation are not considered to be of sufficient magnitude to influence the reactivity of amino acid residues to any significant extent. From the data provided by this study, it m a y be concluded that one of the two arginine residues of bovine trypsin is prevented from reacting with phenylglyoxal once it has formed a complex with soybean trypsin inhibitor. A comparison of the arginine-containing peptides derived from trypsin which had been phenylglyoxalated in the presence of the trypsin inhibitor with those obtained from trypsin itself served to identify this particular arginine residue as arginine-55. I f one assumes that the interaction of trypsin with soybean trypsin inhibitor is analogous to its interaction with the basic pancreatic inhibitor, models of the trypsin-pancreatic inhibitor complex~, 4 should provide some clues as to why arginine-55 of trypsin could be involved in the formation of the trypsin-soybean trypsin inhibitor complex. Although these models do not show arginine-55 to lie directly in the zone of contact between trypsin and the pancreatic inhibitor, among the pairs of residues which do make van der Waals' contact are tyrosine-28 of trypsin with isoleucine-I 9 of the inhibitor 4 and tyrosine-I37 of trypsin and arginine-I 7 of the inhibitor (Stroud, R. M., private communication). These same two tyrosine residues of trypsin could very well be the two tyrosines which we previously reported to be present in the contact zone of the trypsin-soybean trypsin inhibitor complex 8. A three-dimensional model of trypsin (Fig. 4) shows arginine-55 to be in very close proximity to tyrosines-28 and -~37 by virtue of a disulfide bridge between half cystine residues 13 and 143. Dlouh~ et al. 24 found that, when the complex of trypsin and the pancreatic inhibitor was subjected to proteolytic digestion, almost half of the trypsin molecule remained bound to the intact inhibitor. They concluded that the regions of the trypsin molecule which were not digested were those which were either located at the zone of interaction or sterically protected by the inhibitor. The bound portion of trypsin which these authors obtained, when placed within the three-dimensional framework of trypsin, just borders arginine-55. I t is entirely possible that, since soybean trypsin inhibitor is over 3 times the size of the pancreatic inhibitor, its zone of contact with trypsin could very well include arginine-55. Alternatively the lack of reactivity of this residue while in the complex could be simply due to steric hindrance as a consequence of its proximity to the actual contact zone. The final answer, of course, must await crystallographic studies of the trypsin-soybean trypsin inhibitor complex.

2~2

J. E. I)ELARCO, [. i£. I.IENI-[~

Arg-55 SS Arg-.O~..5~l~-,45 Trp 40 x-x,~/~_ ~ ~ 2 2 9 C 0 0 - ~

~-Zyr

137

~ T y r

II

~v --~Trp 127 Trp 221- / ~ It~ \\ )~..~/f"f ~ N H f f 7 ,

/ ~ Asp 90

"Trp 199

Fig. 4. T h r e e - d i m e n s i o n a l model of t o s y l - t r y p s i n t a k e n from l ¢e nne r a n d N e u r a t h 'z~. The l oc a t i on of a r g i n i n e resid ues 55 a n d lO 5 has been a d d e d a n d a p p r o p r i a t e l y labeled. Arginine-55 is p o s t u l a t e d to be a t or n e a r t h e c o n t a c t zone w i t h s o y b e a n t r y p s i n i n h i b i t o r . See t e x t for f u r t h e r discussion.

The second and remaining residue of trypsin, which presumably reacts with phenylglyoxal even when complexed with soybean trypsin inhibitor, is arginine-Io5. As can be seen from Fig. 4, this residue is located in an exposed region of the molecule, far removed from the active site and from arginine-55. One might anticipate, therefore, that the trypsin derivative in which only arginine-Io 5 had been modified by phenylglyoxalation would be fully active. Yet a comparison of the activity of trypsin derived from the phenylglyoxalated complex with trypsin derived from the unmodified complex showed that the former had lost about one-half of its activity. This would suggest that arginine-IoS, although remotely situated with respect to the activity site, m a y nevertheless influence the activity of trypsin. This loss in activity could be due to the elimination of the basic nature of the guanidino group and/or the introduction of the bulky hydrophobic groups of phenylglyoxal : in either case a significant effect on the conformation of tile trypsin molecule would not be surprising. Also located at the contact zone of the trypsin-soybean trypsin inhibitor complex are an average of 2 to 3 arginine residues contributed by the inhibitor. Arginine63, the reactive site of tile trypsin inhibitor 1,~6, would of course be expected to be one of these. That this indeed was the case was deduced from the fact ti~at when soybean trypsin inhibitor, which had been isolated from the phenylglyoxalated complex, was modified by a catalytic level of trypsin under acid conditions2L free arginine, o.49 moles/mole of the inhibitor, was released by the action of carboxypeptidase B (ref. 28). The liberation of less than a stoichimetric level of arginine m a y be largely attributed to tile fact that the trypsin inhibitor isolated from the phenylglyoxalated complex had only about 6o}~ of the activity of unmodified inhibitor (see Table II). This reduction in activity could be due to partial modification of arginine-63 of soybean trypsin inhibitor while it was still eomplexed to trypsin; partial modification might also be the consequence of dissociation of the complex during the modification reaction. It is interesting to note that tile sequences surrounding the reactive sites of

T R Y P S I N - T R Y P S I N INHIBITOR INTERACTION

28~

soybean trypsin inhibitor and the basic pancreatic inhibitor, arginine-63 and lysine-I5 respectively, both include nearby arginine residue: 63 64 65 S o y b e a n t r y p s i n inhibitor~°: - A r g - I l e - A r g 15 16 17 Pancreatic inhibitor: -Lys-Ala-Arg-

Since, in the case of the pancreatic inhibitor, arginine-I 7 makes direct contact with trypsin a,4, one would expect arginine-65 of soybean trypsin inhibitor to be similarly involved. Our data would suggest that a third arginine residue of soybean trypsin inhibitor is also buried in the complex, but its identity remains to be established. ACKNOWLEDGMENTS

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