Int. J. Peptide Protein Res. 8, 1976, 167-175 Published by Munksgaard. Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s
O N T H E REACTION O F A C E T A M I D O M E T H A N O L WITH N A T I V E A N D R E D U C E D BOVINE P A N C R E A T I C T R Y P S I N INHIBITOR ( K U N I T Z I N H I B I T 0 R) RANIERO ROCCHI,CARLOA. BENASSI,ROBERTO TOMATIS, ROBERTO FERRONI and ENEAMENEGATTI
Istituto di Chimica Farmaceutica e Tossicologica dell’ Universita di Ferrara, Ferrara, Italy Received 21 April 1975 The stability of native and reduced bovinepancreatic trypsin inhibitor (Kunitz inhibitor) in anhydrous hydrogen fluoride and their reaction with acetamidomethanol, in the same solvent, have been investigated. The bovine Kunitz inhibitor appears to be stable in liquid hydrogen fluoride but the reduced molecule loses about 50% of its ability to regain inhibitory power, upon air oxidation, by exposure to this solvent. Tyrosine residues appear to be aflected by acetamidomethylation of the native protein to give a modified inhibitor which is still highly active in inhibiting trypsin. The extent of correct refolding, upon reoxidation, of the reduced tyrosine modified-inhibitor is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in the presence of excess anisole. The stability constants and the standard jree energies of binding of the complexes between trypsin and the native and the tyrosine modified-inhibitor have been determined. The bovine pancreatic trypsin inhibitor (1) (BPTI, Kunitz inhibitor), whose sequence has been determined in several laboratories (2-5), consists of a single chain of 58 amino acid residues. It contains three disulfide bonds which are very important in stabilizing its native conformation (2.5). It has been shown that the reduced inhibitor, in which the three disulfide bonds have been broken (R-BFTI), may be renatured to varying extents upon air oxidation (5-8). The three-dimensional structure of BPTI is known with a resolution of 1.5 A (9). Because of its interest as a model of protein-protein interaction, association of BPTI with trypsin has been extensively investigated in recent years (for references see 10 and 11). We are presently engaged in studies aimed at the identification of the role of different amino acid residues in the biological function of BPTI and we have examined the possibility of preparing some BPTI
analogs. Many attempts have been made to use naturally obtained fragments of proteins and peptides as ready-made intermediates in the synthesis of the entire molecule. A crucial point in the semisynthetic approach is the reversible protection of the different chemical functions present in a protein molecule. The acetamidomethyl (Acm) group (12,13) is promising for the reversible protection of sulfhydryl groups, but indications of side reactions involving tyrosine residues have been reported (13,14) during reactions of proteins with acetamidomethanol in anhydrous hydrogen fluoride. Moreover, the solvent itself may cause some undesired changes in the protein molecule. The present paper is an investigation of this react ion. A schematic representation of the different experiments is shown in Fig. 1. Native BPTI is the standard reference in our measurements. Different types of experinental approaches have 167
RANIERO ROCCHI, CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI A N D ENEA MENEGATTI
diluted to 1 ml with a suitable buffer and tested for inhibitory activity. The H F treatment of BPTI derivatives and the reactions with Acm-OH in liquid hydrogen fluoride were carried out in the Sakakibara HF-apparatus (24). A sample of the protein (50-100 mg) was dissolved in anhydrous hydrogen fluoride (1-2 ml) at -78", the solution was warmed in an ice-bath and stirred for 30 min at 0". The hydrogen fluoride was removed by distillation, the reactor was tc). flushed with nitrogen and finally high vacuum EXPERIMENTAL PROCEDURES was applied for 10 min. The residue was lyophilized from water. In the acetamidomethylation Materials BPTI was a gift from Ormoterapia Richter and reactions, Acm-OH was added to the protein was purified according to the literature (15). before dissolving in liquid hydrogen fluoride Nu-Benzoyl-DL-arginine-4-nitroanilidehydro- and the reaction was carried out as described. The chloride (BAPA) was obtained from Merck residue was freed from the reagents by gel methyl filtration on Sephadex G 25 using 2.5% acetic AG and Na-p-toluensulfonyl-L-arginine ester hydrochloride (TAME) from Fluka AG. acid as the eluent, followed by lyophilization. Dithiothreitol (DTT, Cleland's reagent) was Routine amino acid analyses were done according obtained from Calbiochem. Liquid hydrogen to the method of Moore et al. (25) with a Carlo fluoride was purchased from J. T. Baker Chemical Erba mod. 3,427 amino acid analyzer, following Co. Bovine trypsin (TRL) was obtained from hydrolyses, for 22 h at IIO", in thoroughly evaWorthington Biochemical Co. Acetamidomethan- cuated sealed tubes, in constant boiling hydro01 (Acm-OH) was prepared according to the chloric acid containing 0.1 % reagent grade literature (16,17). Doubly distilled, deaerated phenol (26). Solutions of BPTI or BPTI* (at M to 6.0 x and nitrogen gassed water was used for the concentrations from 1.0 x preparation of all buffers and solutions. Sephadex M) were prepared by dissolving the dry, saltfree powders in water. Concentration of trypsin G 25 (fine) was obtained from Pharmacia. solution in 0.001 M HCl was determined by measuring the optical density-at 280 nm using Methods The assay methods used for determining the value of 0.65 as the absorbance of 1 mglml activity of BPTI or BPTI derivatives were the solution (27). The concentration of BPTI or BPTI* in the inhibition of tryptic hydrolysis of BAPA and of TAME, measured by following the change in stock solutions was determined by measuring the absorbance at 405 nm (18) and at 247 nm (19) number of micromoles of amino acids resulting respectively. BPTI and BPTI* were denatured in from the acid hydrolysis of an aliquot of the 8 M urea and reduced with DTT, under a barrier protein aqueous solution (28). For the spectroof nitrogen, according to the literature (20,21). photometric measurements and for the activity The thiol contents of the reduced proteins assays against BAPA, the aqueous solutions of (R-BPTI and R-BPTI*) were determined accord- BPTI, or BPTI*, were diluted with known ing to the method of Ellman (22). Renaturation of volumes of 0.2 M triethanolamine hydrochloride R-BPTI and R-BPTI* was accomplished by air buffer, pH 7.8. For the activity assays, against oxidation ( 5 ) . A 5 x lo-' M protein solution TAME, 0.046 M tris hydrochloride buffer, pH in 0.01 M phosphate buffer, pH 7.8,was kept in 8.1, containing 0.0115 M CaCL was used. contact with air, at O", for 48 h and then at room The pH was measured with a Radiometer pH temperature until the thiol reaction was negative meter 26, equipped with a 7-9 scale expander. (22) (about 10 days)t. Aliquots of 0.1 ml were The absorption spectra, and their first derivatives, were recorded on the Cary model 118c recording t A faster reoxidation procedure has been recently spectrophotometer. Derivative spectra were taken proposed (23). with slit widths below 0.3 mm. The selected speed been utilized to compare the properties of the tyrosine-modified BPTI (BPTI*) with those of BPTI, namely, (a) U.V. spectroscopy, (b) difference spectroscopy, (c) determination of the inhibitory capacity against trypsin using different substrates. The stability constants and the standard free energies of binding of the trypsinBPTI and trypsin-BPTI* adducts have been evaluated from measurements of types (b) and
168
ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTI
of scan was 0.5 nmlsec (chart 10 nmiinch). Spectrophotometric titrations were performed with the same recording spectrophotometer, equipped with a 0 to 0.1 optical density unit slide wire at 20", with tandem-mix fused quartz cuvets (238 QS) obtained from Hellma GmbH and Co. The two chambers of these cuvets contain 1.5 ml of solution and have identical light path of 4.375 mm. Samples of 1.0 ml of trypsin solution (concentration varied from 0.4 x to 1.0 x M) were placed in one compartment of both experimental and reference cuvets, and 1.O ml of the solution containing the desired amount of BPTI, or BPTI*, was placed in the other compartment of each cuvet. The absorption cells were placed in the compartments of the spectrophotometer, thermostated at 20°, and 15 min were allowed for the equilibration. The base line was then checked and adjusted if necessary, the contents of the experimental cuvet were mixed by inversion and the difference spectrum was recorded after 5 min and 25 min. No time dependence was observed. Since variation of pH from 3 to 7.8 causes a positive difference spectrum in a solution of trypsin alone in the region considered, the maximum absorbance changes determined from the reported curves have been corrected for the difference spectrum of trypsin itself.
RESULTS A N D DISCUSSION
Acetamidomethylation of sulfhydryl groups is carried out by reacting the reduced protein with Acm-OH in anhydrous hydrogen fluoride (23) and we examined first the effect of this solvent on both native and reduced BPTI (Fig. 1). As found for other proteins (29-32), BPTI appeared to be remarkably stable in liquid hydrogen fluoride, and exposure to this solvent did not affect its ability to inhibit the tryptic hydrolysis of BAPA or TAME. The preparation of a solution of R-BPTI (5.8 k0.15 thiol groups per molecule) in anhydrous hydrogen fluoride, followed by lyophilization and air oxidation, gave a renatured protein that was only 50% active in inhibiting trypsin. The same renaturation conditions used with a sample of R-BPTI, not previously exposed to hydrogen fluoride, regenerated 100% of the BPTI inhibitory activity.
\ R-Pcrn-BPTt AcmOH
FIGURE 1
Schematic representation of the different reactions carried out on native BPTI and BPTI derivatives. * Tyrosines are absent in the acid hydrolysate; % indicates the percentage of native BPTI inhibitory capacity; Ox, oxidation by air: Hg+ +, mercuric acetate treatment. See text for details. When BPTI was allowed to react in anhydrous hydrogen fluoride with Acm-OH (10-fold molar excess) and freed of the reagent by gel filtration, the isolated material (BPTI*) was about 80% active, at 1 :1 inhibitor-trypsin molar ratio. Tyrosines were absent in the BPTI* acid hydrolysate indicating that they had been modified by acetamidomethylation under the indicated experimental conditions. Reduction and reoxidation of this material gave an approximately 15% active inhibitor. When the R-BPTI was treated in liquid hydrogen fluoride with the same molar excess of Acm-OH and allowed to reoxidize after cleavage of the Acm groups by mercuric acetate (13), less than 15% of the BPTI activity was recovered and no tyrosines were detected in the acid hydrolysate. Similar results have been reported by Sealy et al. (14) for the reaction of reduced insulin with Acm-OH in liquid hydrogen fluoride. These authors also demqnstrated that tyrosine itself is largely modified under the same experimental conditions yielding two new products which are separated on the long column of the amino acid analyzer. It was assumed (14) that they arise from tyrosine by acetamidomethylation in one or both positions ortho to the hydroxyl group.
169
RANIERO ROCCHI, CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI AND ENEA MENEGATTI
We carried out, independently, the same experiments and our results and conclusions agreed perfectly with those previously reported (14). We found that by reacting an equimolar mixture of cysteine and tyrosine with Acm-OH (2-fold molar excess with respect to cysteine) in liquid hydrogen fluoride in the presence of anisole (4-foid molar excess with respect to tyrosine), cysteine was totally S-alkylated, but the tyrosine modification was prevented. The acetamidomethylation reaction was therefore carried out on R-BFTI in the presence of Idfold molar excess of anisole as scavenger. After removal of the hydrogen fluoride the mixture was chromatographed on Sephadex G25 and the fractions containing the modified inhibitor were pooled,
and lyophilized. No free thiol groups were present in the protein, and amino acid analysis of the acid hydrolysate showed the presence of the expected number of tyrosine residues. Removal of the Acm groups (13) followed by gel filtration and reoxidation by air, yielded a renatured inhibitor possessing about 50% of the native inhibitory activity. Comparison of this result with the inhibitory ability of a renatured sample of R-BPTI, previously exposed to liquid hydrogen fluoride, suggests that the activity decrease is due to contact with this solvent. Moreover, comparison of the trypsin inhibitory capacity of a sample of BPTI* with the activity which is recovered when a sample of R-Acm-BPTI is oxidized by air after
w
16
FIGURE 2
1
I 170
260
270
280 290 300 WaveWqth (nm)
310
320
Comparison of the absorption and derivative spectra of BPTI (-) and BPTP (- - - -) in 0.1 M triethanolamine HCI buffer, pH 7.8, 20". BPTI concentration 7.77x 10- s M, BPTP concentration 6.82 x lo-' M. The left-hand ordinate is for the normal absorption curves (1 and 2) while the right-hand ordinate is for the derivative spectra (3 and 4).
ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTI
cleavage of the Acm protecting groups (13), demonstrated that modification of the tyrosine residues, which lie far from the contact site with bovine trypsin, as shown by X-ray structural analysis of the trypsin-inhibitor complex (33), does not affect drastically the inhibitory capacity of BPTI but strongly influences the refolding of the reduced molecule. The ultraviolet absorption and derivative spectra of BPTI and BPTI* in triethanolamine HCI buffer, pH 7.8, are shown in Fig. 2. The peak at 276 nm in the BPTI absorption spectrum is shifted to 279 nm in the BPTI* and the shoulder at 282.5 nm has disappeared. In the derivative spectrum of BPTI, which is closely similar to that reported by Brandts & Kaplan (341, two main peaks are present, the major one in the 287-288 nm region and the smaller one in the 279-281 nm region. The BPTI* spectrum is further to the red by about 2-3 nm, considerably broader, and the peak at 279.5 nm has disappeared. Only a small shoulder is present at 280.5 nm. Essentially, the same results were obtained when the absorption and derivative spectra of Fig. 2 were recorded in water. The ultraviolet difference spectra, resulting from combination of trypsin with increasing amounts of BPTI, or BPTI* (Figs. 3 and 4), were qualitatively similar for the two proteins. In both cases, a positive difference spectrum was developed in the region from 292 to 300 nm arising from perturbation of the tryptophyl residues as already observed by other authors (35). Since the inhibitor does not contain tryptophan, the environment of tryptophyl residue@) in trypsin must be affected. Two peaks are present at 286 and 280 nm, which mainly reflect modifications on the tyrosyl groups. Tyrosyl groups in either trypsin or the inhibitor may contribute to these bands but, as already pointed out (36), it is likely to be the trypsin tyrosyls which are affected in view of the equal accessibility of the tyrosyls of BPTI to nitration in the complex and in the free inhibitor (37). Also it was found that the difference spectrum in this region is apparently not affected by modification of the tyrosyl residues of BPTI. The total Ac in the 297 nm region, where the optical density difference exhibits a maximum, was estimated to be about 660 M-' cm-l from the Ae vs [BPTI] plot. If the maximum absorbance changes in the 297 nm region, in
experiments similar to those shown in Figs. 3 and 4, are plotted as molar extinction changes against the inhibitor-trypsin molar ratio, the curves of Fig. 5 are obtained. The curve obtained for BPTI reached a maximum at an inhibitor-trypsin molar ratio near unity, but that obtained foi BPTI* reached a maximum at a molar ratio between 1.5 and 2. In agreement with these findings, full inhibition of trypsin was obtained at a BPTI*-trypsin molar ratio higher than one, using BAPA as the substrate (Fig. 6). Essentially the same results were obtained using TAME as the substrate. The dissociation constants for the BPTI- and BPTI*-trypsin complex were calculated from the inhibitory assays (38) and from the spectrophotometric titration curves. No significant differences were found in the spectrophotometric values by using different procedures of calculation (39-42). The dissociation constants (Kd), the binding constants (KJ and the standard free energies of binding (- AFo = RT In Kb) of the different inhibitor-trypsin adducts are reported in Table 1. The extremely low value of the dissociation constant of the BPTI-trypsin complex makes the use of difference spectroscopy for the Kd determination unsuitable. Nevertheless, within the limits of the method, we found that the spectrophotometric Kd value of the BPTI*trypsin complex is distinctly larger than that calculated for the BPTI-trypsin complex by the same technique. The Kd values calculated for the BPTI-trypsin complex, from activity assays, are of the same order of magnitude as those found by other authors (38,43,44) and distinctly smaller than those calculated for the BPTI*-trypsin complex. A dissociation constant 6.0 x 10- l 4 M, pH 8.0,25", for the BPTI-trypsin association has been reported (10). Although this value is more generally accepted, the dissociation constant values calculated according to the Green & Work method (38) are still useful for comparing native and modified inhibitors. As has been observed (45,46) in the S peptide-S protein system, the dissociation constant values of the adducts in the presence of the substrate are some orders of magnitude smaller than the spectrophotometric ones. In conclusion, the Kunitz inhibitor is stable in anhydrous hydrogen fluoride but exposure of R-BPTI to this solvent reduces its ability to 171
RANIERO ROCCHI, CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI AND ENEA MENEGA'ITI
3
O
.
O
k
0.02-
0
c
a
f
4
0.01-
a
0.00-
FIGURE 3 difference spectra developed by trypsin upon interaction with BPTI. Curve 1 is for the base line; curve 2 is for trypsin; curve 3 is for a molar ratio of BPTI to trypsin of 0.10; curve 4,0.26; curve 5, 0.66; curve 6, 1.06. The concentration of trypsin before mixing is 0 . 4 7 6 ~lo-' M. See text for conditions. U.V.
1 2jo
2k
290
Wavelength
300
1
1
310
320
(nm)
I
I
270
200
290
300
Wavrlength (nm)
172
310
320
FIGURE 4 U.V. difference spectra developed by trypsin upon interaction with BPTP. Curve 1 is for the base line; curve 2 is for trypsin; curve 3 is for a molar ratio of BFTI* to trypsin of 0.20; curve 4, 0.51 ; curve 5, 1.30; curve 6, 2.08. The concentration of trypsin before mixing is 0 . 4 7 6 ~lo-* M. See text for conditions.
ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTI
FIGURE 5 Spectrophotometric titration 800. curves for the titration of trypsin 600 with BPTI (0-0) and BPTI* (0-0). The maximum absorb- f ance changes in the 297 nm 24 400. region, in experiments similar to those shown in Figs. 3 and 4, 2ooare expressed as molar extinction changes as a function of the
f--
..'
.-.
100
.-
c L
0,
FIGURE 6 Inhibition of trypsin by BPTI (0-0) and by BPTI* (0-0) as a function of the inhibitor-trypsin molar ratio, r. Substrate: BAPA.
U
2 1.0
3.0
2.0
r
TABLE 1 Dissociation constants ( K d ) ,binding constants (4) and standard free energy of binding (-AFo) of different inhibitor-trypsin adducts calculated: ( A ) by spectrophotometric technique, pH 7.8; ( B )from activity measurements using BAPA, pH 7.8, or TAME, pH 8.1, as the substrate. The reported figures are the average of at least three independent experiments. The standard free energies of binding are calculated at room temperature (-AFo = 1.4 log Kb)(39).See text for details
A
B
Inhibitor (M)
(M-l)
-AFo (kcal mole-')
BPTI
i.ox 10-7
1 . o 1~07
9.8
BPTI*
1.ox 10-5 1.0~105
7.0
Kd
Kb
K* (M)
Kb (M-')
:.Ox lo-" 1.4~ 10'O 3.7 x 10-l 1 2.7 x 1O'O 1.1 x 10-9 o . 9 1 ~09 o . 9 10-9 ~ 1.1 x 109
-AFo (kcal mole-')
Substrate
14.20 14.60 12.54 12.66
BAPA TAME BAPA TAME
173
RANIERO ROCCHI. CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI AND ENEA MENEGATIl
regain inhibitory power upon reoxidation by about 50%. Acetamidomethylation of R-BPTI yields an S-acetamidomethylated protein in which all four tyrosines appear to be modified. Acetamidomethylation of the tyrosine residues of the BPTI results in a nearly fully active (80%) modified inhibitor even at a molar ratio of 1 :1 with trypsin, although the binding constant of the inhibitor-trypsin complex is lowered, but the extent of correct refolding of the reduced molecule is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in anhydrous hydrogen fluoride in the presence of excess anisole. Comparison of the difference spectra of the BPTI- and BPTI*-trypsin adducts suggests, in agreement with previous observations (36), that only the environment of trypsin tyrosyls is affected by complex formation. The possible use of the Acm group for the reversible blocking of the thiol functions in the preparation of fragments, chemically or enzymically cleaved from proteins, suitable for attempting a semi-synthetic route appears to be handicapped by the effects induced by the anhydrous hydrogen fluoride during the S-alkylation of the reduced protein. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Mr. L. Morini for skilful technical assistance and to the Italian C.N.R. and Lepctit S.p.A. Milan for financial support. We also thank Professor B. Kassel for reading the manuscript. REFERENCES
1. KUNITZ, M. & NORTHROP, J. H. (1936) J . Gen. Physiol. 19, 991-1007. B. & LASKOWSKI, M. (1965) Biochem. 2. KASSELL, Biophys. Res. Commun. 20,463468. G. & ACHER,R. (1966) 3. CHAWET,J., NOUVEL. Biochim. Biophys. Actu 115, 121-129. V., POSPISILOVA, D., MELOUN, B. & 4. DLOUHA, SORM, F. (1966) Coll. Czech. Chem. Commun. 31, 346-352. F. A. & HORNLE, S. (1966) J . Biol. 5. ANDERER, Chem. 241, 1568-1572. J. & ACHER, R.(1966) Bull. Soc. Chim. 6. CHAUVET, Biol. 48, 1284-1286. 7. AVINERI-GOLDMAN, R.,SNIR,I., BLAUER, G. &
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RIGBI,M. (1967) Arch. Biochem. Biophys. 121, 107-1 16. 8. POSPISILOVA, D., MELOUN, B., FRIC,I. & SORM, F. (1967) Coll. Czech. Chem. Commun.32,410841 17. 9. DEISENHOFER, H. & STEIGEMANN, W. (1974) in
Proteinase Inhibitors (Fritz, H., Tschesche. H.. Greene, L. J. & Truxheit, E., eds.). pp. 484496, Springer-Verlag, Berlin, Heidelberg, New York. 10. LAZDUNSKI. H.. . M... VINCENT.J. P.. SCHWEITZ. PERON-RENNER, M. & ~ J D L & , J. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J. & Truscheit, E., eds.), pp. 420-431. Springer-Verlag, Berlin, Heidelberg, New York. 1 1 HUBER,R., KUKLA,D., STEIGEMANN, W., DEISENHOFER, J. & JONES, A. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J. &Truscheit, E., eds.), pp. 497-512, SpringerVerlag, Berlin, Heidelberg, New York. J. D., DENKEWALTER, 12. VEBER,D.F.,MILKOWSKI, R. G. & HIRSCHMANN, R. (1968) Tetrahedrorr Letters 3057-3058. J. D., VARGA,S. L.. 13. VEBER,D. F., MILKOWSKI, DENKEWALTER, R. G.& HIRSCHMANN, R. (1972) J. Am. Chem. SOC.94, 5456-5461. J. H., RUEGG,U. & RUDINGER, J. (1973) 14. SEALY, in Peptides 1972 (Hanson, H. & Jakubke, H. J., eds.), pp, 8 6 8 9 , North Holland-American Elsevier, Amsterdam-New York. 15. SACH,E., THELY, M. & CHOAY, J. (1965) C. R: Acad. Sci. Paris 260,3491-3493. 16. EINHORN, A. (1905) Justus Liebigs Ann. Chem. 343,207-222. A. 8c LADISCH, C. (1905) Justus Liebigs 17. EINHORN, A m . Chem. 343,264-266. 18. KASSELL,3. (1970) in Methoak in Enzymobgy 19, 844-852, Academic Press, New York. 19. HUMMEL, B. C. W. (1959) Canad. J. Biochem. Physiol. 37, 1393-1399. H. & HAENISCH, G. (1970) FEBS 20. TSCHESCHE, Letters 11,209-212. A. M., MOORE, S. & STEIN,W. H. 21. CRESTFIELD, (1963) J. Biol. Chem. 238, 622-627. G. L. (1959) Arch. Biochem. Biophys. 22. ELLMAN, 82, 70-77. T.E.(1974) J. Mol. Biol. 87,563-577. 23. CREIGHTON, S., SHIMONISHI, Y., KISHIDA, Y., 24. SAKAKIBARA, OKADA, M. & SUGIHARA, H. (1967) Bull. Chem. SOC.Jap. 40,21642167. S., SPACKMANN, D. H. & STEIN, W. H. 25. MOORE, (1958) Anal. Chem. 30, 1185-1 190. F.&THOMPSON, E.D. P. (1963) Biochid 26. SANGER, Biophys. Acta, 71, 468471. M. & LASKOWSKI, M., JR.(1954) in 21. LASKOWSKI, Adv. in Protein Chemistry 9, 203-242.
ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTl
28. SPACKMANN, D. H., STEIN,W. H. & MOORE,S. (1958) Anal. Chem. 30, 1190-1205. 29. KATZ,J. J. (1954) Nafure (Lond.) 173, 265. 30. KATZ, J. J. (1954) Arch. Biochem. Biophys. 51,293-305. 31. ANFINSEN, C. B., ONTJES,D., OHNO,M., CORLEY, L. & EASTLAKE, A. (1967) Proc. Nafl. Acad. Sci. US.58, 1806-1811. 32. KOCH, A. L., LAMONT,W. A. & KATZ, J. J. (1956) Arch. Biochem. Biophys. 63, 106-117. 33. RUHLMANN, A., KUKLA,D., SCHWAGER, P., BARTELS, K. & HUBER, R. (1973) J. Mol. Biol. 77, 417436. 34. BRANDTS,J. F. & KAPLAN,L. J. (1973) Biochemistry 10,201 1-2024. 35. EDELHOCH, H. & STEINER, R. F. (1965) J. Biol. Chem. 240,2877-2882. 36. LASKOWSKI, M., JR. & SEALOCK, R. W. (1971) in The Enzymes (Boyer, P. D., ed.), 3rd edn., vol. 111, pp. 375473, Academic Press, New York and London. 37. MELOUN,B., FRIC,I. & SORM,F. (1969) Coll. Czech. Chem. Commun. 34, 3127-3135. 38. GREEN,N. M. & WORK,E. (1953) Biochem. J. 54,347-352. 39. BERGER, A. & LEVIT,S . (1973) in Peptides 1971
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Address : Prof. Raniero Rocchi Istituto di Chimica Farmaceutica e Tossicologica Via Scandiana, 21 44100 Ferrara Italy
175
O N T H E REACTION O F A C E T A M I D O M E T H A N O L WITH N A T I V E A N D R E D U C E D BOVINE P A N C R E A T I C T R Y P S I N INHIBITOR ( K U N I T Z I N H I B I T 0 R) RANIERO ROCCHI,CARLOA. BENASSI,ROBERTO TOMATIS, ROBERTO FERRONI and ENEAMENEGATTI
Istituto di Chimica Farmaceutica e Tossicologica dell’ Universita di Ferrara, Ferrara, Italy Received 21 April 1975 The stability of native and reduced bovinepancreatic trypsin inhibitor (Kunitz inhibitor) in anhydrous hydrogen fluoride and their reaction with acetamidomethanol, in the same solvent, have been investigated. The bovine Kunitz inhibitor appears to be stable in liquid hydrogen fluoride but the reduced molecule loses about 50% of its ability to regain inhibitory power, upon air oxidation, by exposure to this solvent. Tyrosine residues appear to be aflected by acetamidomethylation of the native protein to give a modified inhibitor which is still highly active in inhibiting trypsin. The extent of correct refolding, upon reoxidation, of the reduced tyrosine modified-inhibitor is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in the presence of excess anisole. The stability constants and the standard jree energies of binding of the complexes between trypsin and the native and the tyrosine modified-inhibitor have been determined. The bovine pancreatic trypsin inhibitor (1) (BPTI, Kunitz inhibitor), whose sequence has been determined in several laboratories (2-5), consists of a single chain of 58 amino acid residues. It contains three disulfide bonds which are very important in stabilizing its native conformation (2.5). It has been shown that the reduced inhibitor, in which the three disulfide bonds have been broken (R-BFTI), may be renatured to varying extents upon air oxidation (5-8). The three-dimensional structure of BPTI is known with a resolution of 1.5 A (9). Because of its interest as a model of protein-protein interaction, association of BPTI with trypsin has been extensively investigated in recent years (for references see 10 and 11). We are presently engaged in studies aimed at the identification of the role of different amino acid residues in the biological function of BPTI and we have examined the possibility of preparing some BPTI
analogs. Many attempts have been made to use naturally obtained fragments of proteins and peptides as ready-made intermediates in the synthesis of the entire molecule. A crucial point in the semisynthetic approach is the reversible protection of the different chemical functions present in a protein molecule. The acetamidomethyl (Acm) group (12,13) is promising for the reversible protection of sulfhydryl groups, but indications of side reactions involving tyrosine residues have been reported (13,14) during reactions of proteins with acetamidomethanol in anhydrous hydrogen fluoride. Moreover, the solvent itself may cause some undesired changes in the protein molecule. The present paper is an investigation of this react ion. A schematic representation of the different experiments is shown in Fig. 1. Native BPTI is the standard reference in our measurements. Different types of experinental approaches have 167
RANIERO ROCCHI, CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI A N D ENEA MENEGATTI
diluted to 1 ml with a suitable buffer and tested for inhibitory activity. The H F treatment of BPTI derivatives and the reactions with Acm-OH in liquid hydrogen fluoride were carried out in the Sakakibara HF-apparatus (24). A sample of the protein (50-100 mg) was dissolved in anhydrous hydrogen fluoride (1-2 ml) at -78", the solution was warmed in an ice-bath and stirred for 30 min at 0". The hydrogen fluoride was removed by distillation, the reactor was tc). flushed with nitrogen and finally high vacuum EXPERIMENTAL PROCEDURES was applied for 10 min. The residue was lyophilized from water. In the acetamidomethylation Materials BPTI was a gift from Ormoterapia Richter and reactions, Acm-OH was added to the protein was purified according to the literature (15). before dissolving in liquid hydrogen fluoride Nu-Benzoyl-DL-arginine-4-nitroanilidehydro- and the reaction was carried out as described. The chloride (BAPA) was obtained from Merck residue was freed from the reagents by gel methyl filtration on Sephadex G 25 using 2.5% acetic AG and Na-p-toluensulfonyl-L-arginine ester hydrochloride (TAME) from Fluka AG. acid as the eluent, followed by lyophilization. Dithiothreitol (DTT, Cleland's reagent) was Routine amino acid analyses were done according obtained from Calbiochem. Liquid hydrogen to the method of Moore et al. (25) with a Carlo fluoride was purchased from J. T. Baker Chemical Erba mod. 3,427 amino acid analyzer, following Co. Bovine trypsin (TRL) was obtained from hydrolyses, for 22 h at IIO", in thoroughly evaWorthington Biochemical Co. Acetamidomethan- cuated sealed tubes, in constant boiling hydro01 (Acm-OH) was prepared according to the chloric acid containing 0.1 % reagent grade literature (16,17). Doubly distilled, deaerated phenol (26). Solutions of BPTI or BPTI* (at M to 6.0 x and nitrogen gassed water was used for the concentrations from 1.0 x preparation of all buffers and solutions. Sephadex M) were prepared by dissolving the dry, saltfree powders in water. Concentration of trypsin G 25 (fine) was obtained from Pharmacia. solution in 0.001 M HCl was determined by measuring the optical density-at 280 nm using Methods The assay methods used for determining the value of 0.65 as the absorbance of 1 mglml activity of BPTI or BPTI derivatives were the solution (27). The concentration of BPTI or BPTI* in the inhibition of tryptic hydrolysis of BAPA and of TAME, measured by following the change in stock solutions was determined by measuring the absorbance at 405 nm (18) and at 247 nm (19) number of micromoles of amino acids resulting respectively. BPTI and BPTI* were denatured in from the acid hydrolysis of an aliquot of the 8 M urea and reduced with DTT, under a barrier protein aqueous solution (28). For the spectroof nitrogen, according to the literature (20,21). photometric measurements and for the activity The thiol contents of the reduced proteins assays against BAPA, the aqueous solutions of (R-BPTI and R-BPTI*) were determined accord- BPTI, or BPTI*, were diluted with known ing to the method of Ellman (22). Renaturation of volumes of 0.2 M triethanolamine hydrochloride R-BPTI and R-BPTI* was accomplished by air buffer, pH 7.8. For the activity assays, against oxidation ( 5 ) . A 5 x lo-' M protein solution TAME, 0.046 M tris hydrochloride buffer, pH in 0.01 M phosphate buffer, pH 7.8,was kept in 8.1, containing 0.0115 M CaCL was used. contact with air, at O", for 48 h and then at room The pH was measured with a Radiometer pH temperature until the thiol reaction was negative meter 26, equipped with a 7-9 scale expander. (22) (about 10 days)t. Aliquots of 0.1 ml were The absorption spectra, and their first derivatives, were recorded on the Cary model 118c recording t A faster reoxidation procedure has been recently spectrophotometer. Derivative spectra were taken proposed (23). with slit widths below 0.3 mm. The selected speed been utilized to compare the properties of the tyrosine-modified BPTI (BPTI*) with those of BPTI, namely, (a) U.V. spectroscopy, (b) difference spectroscopy, (c) determination of the inhibitory capacity against trypsin using different substrates. The stability constants and the standard free energies of binding of the trypsinBPTI and trypsin-BPTI* adducts have been evaluated from measurements of types (b) and
168
ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTI
of scan was 0.5 nmlsec (chart 10 nmiinch). Spectrophotometric titrations were performed with the same recording spectrophotometer, equipped with a 0 to 0.1 optical density unit slide wire at 20", with tandem-mix fused quartz cuvets (238 QS) obtained from Hellma GmbH and Co. The two chambers of these cuvets contain 1.5 ml of solution and have identical light path of 4.375 mm. Samples of 1.0 ml of trypsin solution (concentration varied from 0.4 x to 1.0 x M) were placed in one compartment of both experimental and reference cuvets, and 1.O ml of the solution containing the desired amount of BPTI, or BPTI*, was placed in the other compartment of each cuvet. The absorption cells were placed in the compartments of the spectrophotometer, thermostated at 20°, and 15 min were allowed for the equilibration. The base line was then checked and adjusted if necessary, the contents of the experimental cuvet were mixed by inversion and the difference spectrum was recorded after 5 min and 25 min. No time dependence was observed. Since variation of pH from 3 to 7.8 causes a positive difference spectrum in a solution of trypsin alone in the region considered, the maximum absorbance changes determined from the reported curves have been corrected for the difference spectrum of trypsin itself.
RESULTS A N D DISCUSSION
Acetamidomethylation of sulfhydryl groups is carried out by reacting the reduced protein with Acm-OH in anhydrous hydrogen fluoride (23) and we examined first the effect of this solvent on both native and reduced BPTI (Fig. 1). As found for other proteins (29-32), BPTI appeared to be remarkably stable in liquid hydrogen fluoride, and exposure to this solvent did not affect its ability to inhibit the tryptic hydrolysis of BAPA or TAME. The preparation of a solution of R-BPTI (5.8 k0.15 thiol groups per molecule) in anhydrous hydrogen fluoride, followed by lyophilization and air oxidation, gave a renatured protein that was only 50% active in inhibiting trypsin. The same renaturation conditions used with a sample of R-BPTI, not previously exposed to hydrogen fluoride, regenerated 100% of the BPTI inhibitory activity.
\ R-Pcrn-BPTt AcmOH
FIGURE 1
Schematic representation of the different reactions carried out on native BPTI and BPTI derivatives. * Tyrosines are absent in the acid hydrolysate; % indicates the percentage of native BPTI inhibitory capacity; Ox, oxidation by air: Hg+ +, mercuric acetate treatment. See text for details. When BPTI was allowed to react in anhydrous hydrogen fluoride with Acm-OH (10-fold molar excess) and freed of the reagent by gel filtration, the isolated material (BPTI*) was about 80% active, at 1 :1 inhibitor-trypsin molar ratio. Tyrosines were absent in the BPTI* acid hydrolysate indicating that they had been modified by acetamidomethylation under the indicated experimental conditions. Reduction and reoxidation of this material gave an approximately 15% active inhibitor. When the R-BPTI was treated in liquid hydrogen fluoride with the same molar excess of Acm-OH and allowed to reoxidize after cleavage of the Acm groups by mercuric acetate (13), less than 15% of the BPTI activity was recovered and no tyrosines were detected in the acid hydrolysate. Similar results have been reported by Sealy et al. (14) for the reaction of reduced insulin with Acm-OH in liquid hydrogen fluoride. These authors also demqnstrated that tyrosine itself is largely modified under the same experimental conditions yielding two new products which are separated on the long column of the amino acid analyzer. It was assumed (14) that they arise from tyrosine by acetamidomethylation in one or both positions ortho to the hydroxyl group.
169
RANIERO ROCCHI, CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI AND ENEA MENEGATTI
We carried out, independently, the same experiments and our results and conclusions agreed perfectly with those previously reported (14). We found that by reacting an equimolar mixture of cysteine and tyrosine with Acm-OH (2-fold molar excess with respect to cysteine) in liquid hydrogen fluoride in the presence of anisole (4-foid molar excess with respect to tyrosine), cysteine was totally S-alkylated, but the tyrosine modification was prevented. The acetamidomethylation reaction was therefore carried out on R-BFTI in the presence of Idfold molar excess of anisole as scavenger. After removal of the hydrogen fluoride the mixture was chromatographed on Sephadex G25 and the fractions containing the modified inhibitor were pooled,
and lyophilized. No free thiol groups were present in the protein, and amino acid analysis of the acid hydrolysate showed the presence of the expected number of tyrosine residues. Removal of the Acm groups (13) followed by gel filtration and reoxidation by air, yielded a renatured inhibitor possessing about 50% of the native inhibitory activity. Comparison of this result with the inhibitory ability of a renatured sample of R-BPTI, previously exposed to liquid hydrogen fluoride, suggests that the activity decrease is due to contact with this solvent. Moreover, comparison of the trypsin inhibitory capacity of a sample of BPTI* with the activity which is recovered when a sample of R-Acm-BPTI is oxidized by air after
w
16
FIGURE 2
1
I 170
260
270
280 290 300 WaveWqth (nm)
310
320
Comparison of the absorption and derivative spectra of BPTI (-) and BPTP (- - - -) in 0.1 M triethanolamine HCI buffer, pH 7.8, 20". BPTI concentration 7.77x 10- s M, BPTP concentration 6.82 x lo-' M. The left-hand ordinate is for the normal absorption curves (1 and 2) while the right-hand ordinate is for the derivative spectra (3 and 4).
ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTI
cleavage of the Acm protecting groups (13), demonstrated that modification of the tyrosine residues, which lie far from the contact site with bovine trypsin, as shown by X-ray structural analysis of the trypsin-inhibitor complex (33), does not affect drastically the inhibitory capacity of BPTI but strongly influences the refolding of the reduced molecule. The ultraviolet absorption and derivative spectra of BPTI and BPTI* in triethanolamine HCI buffer, pH 7.8, are shown in Fig. 2. The peak at 276 nm in the BPTI absorption spectrum is shifted to 279 nm in the BPTI* and the shoulder at 282.5 nm has disappeared. In the derivative spectrum of BPTI, which is closely similar to that reported by Brandts & Kaplan (341, two main peaks are present, the major one in the 287-288 nm region and the smaller one in the 279-281 nm region. The BPTI* spectrum is further to the red by about 2-3 nm, considerably broader, and the peak at 279.5 nm has disappeared. Only a small shoulder is present at 280.5 nm. Essentially, the same results were obtained when the absorption and derivative spectra of Fig. 2 were recorded in water. The ultraviolet difference spectra, resulting from combination of trypsin with increasing amounts of BPTI, or BPTI* (Figs. 3 and 4), were qualitatively similar for the two proteins. In both cases, a positive difference spectrum was developed in the region from 292 to 300 nm arising from perturbation of the tryptophyl residues as already observed by other authors (35). Since the inhibitor does not contain tryptophan, the environment of tryptophyl residue@) in trypsin must be affected. Two peaks are present at 286 and 280 nm, which mainly reflect modifications on the tyrosyl groups. Tyrosyl groups in either trypsin or the inhibitor may contribute to these bands but, as already pointed out (36), it is likely to be the trypsin tyrosyls which are affected in view of the equal accessibility of the tyrosyls of BPTI to nitration in the complex and in the free inhibitor (37). Also it was found that the difference spectrum in this region is apparently not affected by modification of the tyrosyl residues of BPTI. The total Ac in the 297 nm region, where the optical density difference exhibits a maximum, was estimated to be about 660 M-' cm-l from the Ae vs [BPTI] plot. If the maximum absorbance changes in the 297 nm region, in
experiments similar to those shown in Figs. 3 and 4, are plotted as molar extinction changes against the inhibitor-trypsin molar ratio, the curves of Fig. 5 are obtained. The curve obtained for BPTI reached a maximum at an inhibitor-trypsin molar ratio near unity, but that obtained foi BPTI* reached a maximum at a molar ratio between 1.5 and 2. In agreement with these findings, full inhibition of trypsin was obtained at a BPTI*-trypsin molar ratio higher than one, using BAPA as the substrate (Fig. 6). Essentially the same results were obtained using TAME as the substrate. The dissociation constants for the BPTI- and BPTI*-trypsin complex were calculated from the inhibitory assays (38) and from the spectrophotometric titration curves. No significant differences were found in the spectrophotometric values by using different procedures of calculation (39-42). The dissociation constants (Kd), the binding constants (KJ and the standard free energies of binding (- AFo = RT In Kb) of the different inhibitor-trypsin adducts are reported in Table 1. The extremely low value of the dissociation constant of the BPTI-trypsin complex makes the use of difference spectroscopy for the Kd determination unsuitable. Nevertheless, within the limits of the method, we found that the spectrophotometric Kd value of the BPTI*trypsin complex is distinctly larger than that calculated for the BPTI-trypsin complex by the same technique. The Kd values calculated for the BPTI-trypsin complex, from activity assays, are of the same order of magnitude as those found by other authors (38,43,44) and distinctly smaller than those calculated for the BPTI*-trypsin complex. A dissociation constant 6.0 x 10- l 4 M, pH 8.0,25", for the BPTI-trypsin association has been reported (10). Although this value is more generally accepted, the dissociation constant values calculated according to the Green & Work method (38) are still useful for comparing native and modified inhibitors. As has been observed (45,46) in the S peptide-S protein system, the dissociation constant values of the adducts in the presence of the substrate are some orders of magnitude smaller than the spectrophotometric ones. In conclusion, the Kunitz inhibitor is stable in anhydrous hydrogen fluoride but exposure of R-BPTI to this solvent reduces its ability to 171
RANIERO ROCCHI, CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI AND ENEA MENEGA'ITI
3
O
.
O
k
0.02-
0
c
a
f
4
0.01-
a
0.00-
FIGURE 3 difference spectra developed by trypsin upon interaction with BPTI. Curve 1 is for the base line; curve 2 is for trypsin; curve 3 is for a molar ratio of BPTI to trypsin of 0.10; curve 4,0.26; curve 5, 0.66; curve 6, 1.06. The concentration of trypsin before mixing is 0 . 4 7 6 ~lo-' M. See text for conditions. U.V.
1 2jo
2k
290
Wavelength
300
1
1
310
320
(nm)
I
I
270
200
290
300
Wavrlength (nm)
172
310
320
FIGURE 4 U.V. difference spectra developed by trypsin upon interaction with BPTP. Curve 1 is for the base line; curve 2 is for trypsin; curve 3 is for a molar ratio of BFTI* to trypsin of 0.20; curve 4, 0.51 ; curve 5, 1.30; curve 6, 2.08. The concentration of trypsin before mixing is 0 . 4 7 6 ~lo-* M. See text for conditions.
ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTI
FIGURE 5 Spectrophotometric titration 800. curves for the titration of trypsin 600 with BPTI (0-0) and BPTI* (0-0). The maximum absorb- f ance changes in the 297 nm 24 400. region, in experiments similar to those shown in Figs. 3 and 4, 2ooare expressed as molar extinction changes as a function of the
f--
..'
.-.
100
.-
c L
0,
FIGURE 6 Inhibition of trypsin by BPTI (0-0) and by BPTI* (0-0) as a function of the inhibitor-trypsin molar ratio, r. Substrate: BAPA.
U
2 1.0
3.0
2.0
r
TABLE 1 Dissociation constants ( K d ) ,binding constants (4) and standard free energy of binding (-AFo) of different inhibitor-trypsin adducts calculated: ( A ) by spectrophotometric technique, pH 7.8; ( B )from activity measurements using BAPA, pH 7.8, or TAME, pH 8.1, as the substrate. The reported figures are the average of at least three independent experiments. The standard free energies of binding are calculated at room temperature (-AFo = 1.4 log Kb)(39).See text for details
A
B
Inhibitor (M)
(M-l)
-AFo (kcal mole-')
BPTI
i.ox 10-7
1 . o 1~07
9.8
BPTI*
1.ox 10-5 1.0~105
7.0
Kd
Kb
K* (M)
Kb (M-')
:.Ox lo-" 1.4~ 10'O 3.7 x 10-l 1 2.7 x 1O'O 1.1 x 10-9 o . 9 1 ~09 o . 9 10-9 ~ 1.1 x 109
-AFo (kcal mole-')
Substrate
14.20 14.60 12.54 12.66
BAPA TAME BAPA TAME
173
RANIERO ROCCHI. CARL0 A. BENASSI, ROBERTO TOMATIS, ROBERTO FERRONI AND ENEA MENEGATIl
regain inhibitory power upon reoxidation by about 50%. Acetamidomethylation of R-BPTI yields an S-acetamidomethylated protein in which all four tyrosines appear to be modified. Acetamidomethylation of the tyrosine residues of the BPTI results in a nearly fully active (80%) modified inhibitor even at a molar ratio of 1 :1 with trypsin, although the binding constant of the inhibitor-trypsin complex is lowered, but the extent of correct refolding of the reduced molecule is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in anhydrous hydrogen fluoride in the presence of excess anisole. Comparison of the difference spectra of the BPTI- and BPTI*-trypsin adducts suggests, in agreement with previous observations (36), that only the environment of trypsin tyrosyls is affected by complex formation. The possible use of the Acm group for the reversible blocking of the thiol functions in the preparation of fragments, chemically or enzymically cleaved from proteins, suitable for attempting a semi-synthetic route appears to be handicapped by the effects induced by the anhydrous hydrogen fluoride during the S-alkylation of the reduced protein. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Mr. L. Morini for skilful technical assistance and to the Italian C.N.R. and Lepctit S.p.A. Milan for financial support. We also thank Professor B. Kassel for reading the manuscript. REFERENCES
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ACETAMIDOMETHYLATION OF NATIVE AND REDUCED BOVINE BPTl
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Address : Prof. Raniero Rocchi Istituto di Chimica Farmaceutica e Tossicologica Via Scandiana, 21 44100 Ferrara Italy
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