J. Biochem., 80, 1293-1297 (1976)
Masaaki YAMADA,* Misao TASHrRO,** 1 Hiroko YAMAGUCHJ,** Hisao YAMADA,** Fumio IBUKI,** and Masao KANAMORI** •Research Laboratories, The Green Cross Corp., Osaka, Osaka 534, and "Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto Prefectural University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, June 5, 1976
The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-mcdified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed as the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.
Ozawa and Laskowski, Jr. postulated that all naturally occurring trypsin inhibitors have either an Arg-X or a Lys-X trypsin-sensitive bond in their reactive sites ( / ) . A variety of trypsin inhibitors have been classified as arginyl or lysyl inhibitors by reactive site cleavage and by chemical modifications (2). In previous papers, we reported the occurrence of a trypsin inhibitor in eggplant, Solarium melongena L. (5), together with its purification and some properties (4-6). This inhibitor is a low molecular weight protein composed of 57 1
Present address: Department of Food Science, Faculty of Living Science, Kyoto Prefectural University, Sakyo-ku, Kyoto, Kyoto 606. Abbreviations: BAPA, a-N-benzoyl-DL-arginine p-nitroanilide; TAME, ^-tosyl-L-arginine methyl ester; CHD, 1,2-cyclohexanedione; TNBS, 2,4,6-trinitrobenzenesulfonic acid; TNP-, trinitrophenyl-; CPase, carboxypeptidase; DTNB, 5,5'-dithiobis (2-nitrobenzoic acid); SDS, sodium dodecyl sulfate. Vol. 80, No. 6, 1976
1293
amino acid residues, containing 4 lysine, 3 arginine residues, and 4 disulfide bonds. This communication describes the effect of disulfide bond reduction on the inhibitory activity, and the identification of the reactive site peptide bond with respect to trypsin by chemical modifications of lysine or arginine residues, and by limited proteolysis with trypsin. EXPERIMENTAL PROCEDURE Materials—The eggplant inhibitor was prepared from the exocarp of eggplant by the method described previously (6). Trypsin [EC 3. 4. 21. 4] (twice crystallized, from bovine pancreas) and carboxypeptidase B [EC 3.4.12.3] (from hog pancreas) were purchased from Sigma Chemical Co. BAPA, TAME, and DTNB were obtained from Nakarai Chemicals Ltd., Kyoto. All other chemicals were of special or reagent grade.
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The Reactive Site of Eggplant Trypsin Inhibitor
1294
M. YAMADA, M. TASHIRO, H. YAMAGUCHI, H. YAMADA, F. IBUKI, and M. KANAMORI
General Methods—Amino acid analysis was performed with a Hitachi KLA-5 amino acid analyser. Hydrolysis was carried out in an evacuated, sealed tube with 5.7 N HC1 at 110° for 22 hr. Edman degradation was carried out by the method of Iwanaga et al. (7). The phenylthiohydantoin derivative was identified by gas chromatography on a Shimadzu GC-4B gas chromatograph using a Silicon D.C. 560 column according to the method of Pisano and Bronzert (8). Reduction of Bisulfide Bonds—The disulfide bonds in the inhibitor were reduced by the modified method of Kress and Laskowski, Sr. (9). The inhibitor (3 mg/ml in 0.02% EDTA-2Na) in a volume of 1.02 ml was adjusted to pH 8.5 with 0.04 ml of 0.2 N NaOH. Then, 1.06 ml of freshly prepared 0.2 M NaBH4 was added to the inhibitor solution and the mixture was incubated at 2°, flushing with N,. After incubation for 2 min, 0.05 ml of 0.5 N HO was added to 0.1 ml aliquots withdrawn from the reaction mixture at appropriate intervals. From each aliquot, 0.1 ml was used for the determination of disulfide bonds by the procedure of Habeeb (10). The remaining solution (0.05 ml) was diluted 10-fold with distilled watsr and its inhibitory activity was assayed. CHD Modification—Modification of guanidino groups in the inhibitor was done with CHD by the modified method of Haynes and Feeney (11). The inhibitor (1 mg) was dissolved in 1.0 ml of 0.1 M triethylamine buffer, pH 10, containing 0.01 M EDTA-2Na and 0.015 M CHD. The reaction was allowed to proceed in the dark for 12 hr at 25°. An aliquot (0.1 ml) was removed from the reaction mixture, diluted 20-fold with distilled water and its inhibitory activity was determined. The remaining mixture was then desalted by gel filtration on
Sephadex G-25 and lyophilized. The content of arginine was determined by amino acid analysis after acid hydrolysis. Glyoxal Modification—Arginine residues in the inhibitor were modified with glyoxal according to the method of Nakaya et al. (12). The inhibitor (3 mg) was dissolved in 1 ml of 0.5 M bicarbonateNaOH buffer, pH 8.6, and treated with 1 ml of °-4% glyoxal solution for 3 hr at 25°. The reaction mixture was applied to a Sephadex G-25 column to isolate the modified inhibitor. The inhibitory activity was measured with the desalted sample. The amount of modified arginine residues was calculated by amino acid analysis after acid hydrolysis. TNBS Modification—Modification of free amino groups was carried out by the methcd of Haynes et al. (13). To 1 ml of the inhibitor solution (0.28 mg/ml) were added 1 ml of 4% NaHCO,, pH 8.3, and 1 ml of 1 % TNBS in water, and the mixture was incubated at 37°. To determine the number of modified amino groups, the reaction was stopped by the addition of 1 ml of 10% SDS and 0.5 ml of 1 N HC1. The extent of modification was calculated from the absorbance at 344 nm by using the molar extinction coefficient of TNP-amino acid, 1.09x10', given by Haynes et al. (13). For the assay of inhibitory activity, 0.3 ml was removed, diluted with 0.7 ml of cold distilled water, and assayed immediately. Acetic Anhydride Modification—Free amino groups in the inhibitor were acetylated with acetic anhydride according to the method of FraenkelConrat (14). To 0.5 ml of the inhibitor solution (5 mg/ml), 0.5 ml of saturated sodium acetate was added. The mixture was treated with five lots of 0.1 ml of acetic anhydride at 0° during 1 hr. The modified inhibitor was isolated by gel filtration on Sephadex G-25, followed by lyophilization. The lyophilized sample was used for the assay of inhibitory activity. The extent of acetylation was estimated by determining the residual free amino groups by the TNBS method of Haynes, as mentioned above. Limited Proteolysis with Trypsin—Effects of pH and incubation period: One mole % of trypsin was added to the inhibitor dissolved in buffers with pH values from 1.5 to 9.0. After incubation at 25° for 24 hr, an aliquot of each reaction mixture was subjected to disc gel electrophoresis in order to estimate J. Biochem.
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Inhibitory Activity Assay—Inhibitory activity toward trypsin was determined by the spectrophotometric method with BAPA as a substrate, as reported previously (4) or with a pH-stat, RTS622 (Radiometer, Copenhagen). The pHstat assay was carried out at 25° under N t . The incubation mixture consisted of 0.2 ml of 2 M KC1, 100 n\ of trypsin solution (80 /Jg/ml), and an appropriate amount of inhibitor solution in a total volume of 1.80 ml. The mixture was adjusted to pH 8.00 in a vessel, then the reaction was started by the addition of 0.2 ml of 40 mM TAME (pH 8.0) and followed by titration with 0.01 M NaOH.
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M. YAMADA, M. TASHIRO, H. YAMAGUCHI, H. YAMADA, F. IBUKI, and M, KANAMORI 100 u o JJ •H XI
a 4J 3 -H •o >
1
to -U 0) o M (0
" 0 10
Fig. 2. pH Dependence of limited proteolysis of the inhibitor with trypsin. The inhibitor was treated with 1 mole% trypsin at each pH for 24 hr at 25°. The Khji, ratio of modified inhibitor to native inhibitor was estimated densitometrically after disc gel electrophoresis.
2
4 6 8 20" 48 period of incubation(hr)
Fig. 4. Inactivation of trypsin-modified inhibitor by carboxypeptidase B digestion. • , native inhibitor; O, mixture containing modified and native inhibitors.
However, the modification hardly proceeded at pH's below 2. Figure 3 shows the time-dependence of trypsin-modified inhibitor formation. The trypsin-modified inhibitor was detected after incubation for 30 min at pH 2.5. Maximum formation was obtained after incubation for 24 hr and was maintained at an almost constant level up to 144 hr. When the mixture containing modified and native inhibitors was directly treated with CPase B, free arginine was detected by amino acid analysis. The amount of released arginine was 0.52 mole per mole of inhibitor after incubation for 20 hr. The trypsin inhibitory activity fell to 46.8% of the original activity, as shown in Fig. 4. No free amino acid was detected on CPase B Fig. 3. The time-dependence of trypsin-modified in- treatment of the native inhibitor and no effect on hibitor formation. The inhibitor was treated with the inhibitory activity was noted. These results 1 mole% trypsin at pH 2.5. Upper bands in each tube show that a new C-terminal amino acid, arginine, are native and middle bands are modified inhibitor. was exposed. The newly exposed N-terminal Lower bands are BPB. Incubation periods were; tube amino acid residue in the mixture of modified and No. 1, 0 min; No. 2, 5 min; No. 3, 0.5 hr; No. 4, 1 hr; native inhibitors was identified as serine by Edman No. 5, 6 hr; No. 6, 24 hr; No. 7, 48 hr; No. 8, 72 hr; degradation; no N-terminal amino acid residue of No. 9, 144hr. the native inhibitor could be detected by Edman degradation. Consequently, it was concluded that terminal amino acid. Therefore, one amino group the reactive site peptide bond is arginylserine. was not modified. The acitylation of amino groups with ac;tic anhydride was complete, as DISCUSSION judged from the fact that no TNP-amino acids were detected. The loss of inhibitory activity due Full reduction of disulfide bonds in the inhibitor to these amino group modifications was insignifiinducsd complete loss of inhibitory activity. These cant. These results are sum-mrized in Table I. disulfide bonds were thus essential for the activity. Limited Proteolysis with Trypsin—The forma- The loss of activity paralleled the extent of reduction of modified inhibitor was depressed in the pH tion. This suggests that the loss of activity was not region from 4 to 6, and remarkably accelerated at caused by the reduction of a particular disulfide alkaline and more acidic pH's, as shown in Fig. 2. bond, but possibly by conformational changes. J. Biochem.
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•H
REACTIVE SITE OF EGGPLANT TRYPSIN INHIBITOR
RESULTS Effect of Disulfide Bond Reduction on Inhibitory Activity—Figure 1 shows the relationship between the reduction of disulfide bonds and the loss of trypsin inhibitory activity. The inhibitory activity was lost in proportion to the extent of reduction of disulfide bonds. This inhibitor contains 8 half-cystine residues per molecule (6).
>, 100
0
1 2 14 period of incubation(hr)
Fig. 1. Effect of disulfide reduction on trypsin inhibitory activity. • , residual inhibitory activity; O, sulfhydryl groups per mole of inhibitor. Inhibitor solution in 0.02% EDTA-2Na was adjusted to pH 8.5 with 0.2 M NaOH, and treated with an equal volume of 0.2 M NaBH, by incubation at 2° under N,.
No sulfhydryl groups were detected in the intact inhibitor. Seven sulfhydryl froups appeared per mole of inhibitor after reduction for 14 hr. This fully reduced Inhibitor showed no inhibitory activity. Chemical Modifications—The inhibitor contains 3 arginine residues, CHD modification caused a 38% loss of arginine residues, corresponding to the modification of approximately one arginine residue. After this modification, the trypsin inhibitory activity was reduced to 22% of the original activity. Glyoxal reacted with approximately 2.4 moles of arginine residues per mole of inhibitor. The residual inhibitory activity was only 0.8%. The amount of TNP-amino acid groups formed by TNBS modification was 3.9 moles per mole of inhibitor on the basis of a molecular weight of 6,000 (6). The number of free amino groups in the inhibitor was theoretically five; 4 tamino groups and one a-amino group of the N-
TABLE I. Inactivation of inhibitor by chemical modification. Reagent 1,2-Cyclohexanedione Glyoxal Acetic anhydride 2,4,6-Trinitrobenzenesulfonic acid
Vol. 80, No. 6, 1976
Modified residue
Modified
Loss of inhibitory activity (%)
Arginine Arginine Lysine Lysine
38 80 80 80
78 99 8 0
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the extent of modification. Gel electrophoresis was carried out by the method of Davis (15). Staining was performed with Coomassie brilliant blue by the method of Charmbach et al. (16). The ratio of the native and modified inhibitors was estimated with a Yamato-Asuka OZUMOR 82 densitometer. The incubation period required for maximum modification was investigated at pH 2.5 with 1 mole % trypsin at 25° during Incubation for 144 hr. Carboxypeptidase B Treatment of Trypsinmodified Inhibitor—The inhibitor (30 mg) was dissolved in 4.4 ml of 0 . 1 M sodium citrate-HCl buffer, pH 2.0. To this solution was added 0.6 ml of trypsin solution (2 mg/ml). After incubation at 25° for 20 hr, the reaction mixture was immediately applied to a Sephadex G-50 column (2.5 x 39 cm) to remove trypsin. The fractions containing the native and modified inhibitors were pooled and lyophilized. The lyophilized sample (0.8 mg) was dissolved in 0.2 ml of 0.05 M Tris-HCl buffer, pH 8.0. Ten (i\ of CPase B solution (3 mg/ml) was added and the reaction mixture was incubated for 2 days at 37°. An aliquot of the reaction mixture was assayed for trypsin inhibitory activity with a pH-stat. The released amino acid residue was identified by amino acid analysis. The newly exposed N-terminal amino acid was identified by Edman degradation.
1295
REACTrVE SITE OF EGGPLANT TRYPSIN INHIBITOR
1297
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On the basis of the results of chemical modiNo N-terminal amino acid residue of the native fications and sequential treatments with trypsin and eggplant inhibitor was detected by Edman degraCPase B, it was concluded that the eggplant in- dation, and one amino group in the inhibitor was hibitor contained a trypsin-suscsptible arginyl- resistant of TNBS modification. This suggests seryl bond in its reactive site. Modification of the that the N-terminal amino group is blocked. arginine residues was extensive with glyoxal and Studies on this problem are in progress. slight with CHD. However, the inhibitory activity was significantly reduced in both cases. This REFERENCES indicates that the arginine residue in the reactive site was reactive with these reagents. The amount 1. Ozawa, K. & Laskowski, M., Jr. (1966) J. Biol. Chem. 241, 3955-3961 of arginine residue released after the sequential 2. Laskowski, M., Jr. & Sealock, R.W. (1971) in The enzymatic treatments coincided well with the loss Enzymes (Boyer, P.D., ed.) Vol. 3, pp. 375-473, Acaof inhibitory activity. The low yield of released demic Press, New York arginine is due to the co-existence of native in3. Kanamori, M., Ibuki, F., Tashiro, M., Yamada, M., & Miyoshi, M. (1975) /. Nulr. Sci. Vitaminol. 21, hibitor. 421^(28 The reactive site peptide bond of the eggplant 4. Kanamori, M., Ibuki, F., Yamada, M., Tashiro, M., & Miyoshi, M. (1975) /. Nun. Sci. Vitaminol. 21, inhibitor was an arginylseryl bond. It has been 429^436 reported that garden bean (17), and soybean C and 5. Kanamori, M., Ibuki, F., Yamada, M., Tashiro, M., D inhibitors (18) have the same peptide bond in & Miyoshi, M. (1976) Agr. Biol. Chem. 40, 839-844 their reactive sites. Hcchstrasser et al. (19) 6. Kanamori, M., Ibuki, F., Tashiro, M., Yamada, M., & Miyoshi, M. (1976) Biochim. Biophys. Acta showed that the reactive sits psptide bond of the 439, 398-405 peanut inhibitor was an arginylalanyl bond. How7. Iwanaga, S., Wall£n, P., Grondahl, N.J., Henschen, ever, Odani and Ikenaka (20) speculated that an A., & Blomblch, B. (1969) Eur. J. Biochem. 8, 189-199 arginylseryl bond might be the reactive site, on the 8. Pisano, J.J. & Bronzert, T.J. (1969) J. Biol. Chem. basis of the similarity in amino acid sequence 244, 5597-5607 between Bowman-Birk soybean inhibitor and lima 9. Kress, L.F. & Laskowski, M. Sr., (1967) J. Biol. bean inhibitor. As mentioned above, an arginylChem. 242, 4925-4929 seryl reactive site bond has been found in several 10. Habeeb, A.F.S.A. (1972) in Methods in Enzymology (Hirs, C.H.W. & Timasheff, S.N., eds.) Vol. 25, trypsin inhibitors. pp. 457^464 On the basis of comparisons of the amino 11. Haynes, R. & Feeney, R. (1968) Biochemistry 7, 2879-2885 acid sequences of many proteinase inhibitors from materials of different phylogenetic descent, Hokama 12. Nakaya, K., Horinishi, H., & Shibata, K. (1967) J. Biochem. 61, 345-351 et al. (21) suggested that the regions related to 13. Haynes, R., Osuga, D.T., & Feeney, R.E. (1967) their inhibitory functions showed high sequence Biochemistry 6, 541-547 homology, and the other regions reflected their 14. Fraenkel-Conrat, H. (1963) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. phylogenetic relationships. To discuss the phy4, 247-269 Iogeny of the eggplant inhibitor, knowledge of its 15. Davis, B.J. (1964) Ann. New York Acad. Sci. 121, complete amino acid sequence will be required. 404-427 The pH dependence of Kh7i, the equilibrium 16. Charmbach, A., Reisfeld, R.A., Wyckoff, M., & Zaccari, J. (1967) Anal. Biochem. 20, 150-154 constant for limited hydrolysis with trypsin (Fig. 17. Wilson, K.A. & Laskowski, M., Sr. (1975) J. Biol. 2), was similar to that of Kunitz soybean inhibitor Chem. 250, 4261-4267 (22). However, these Khyt values are low in com- 18. Odani, S. & Ikenaka, T. (1975) 26th Tanpakushitsu Kozo Toronkai Yokoshu, pp. 109-112 parison with that of Kunitz soybean inhibitor. The low K^ji value for eggplant inhibitor did not 19. Hochstrasser, K., lllchmann, K., Werle, E., Hoesse', R., & Schwarz, S. (1970) Z. Physiol. Chem. appear to be due to an insufficient incubation 351, 1503-1512 period, on the basis of the results shown in Fig. 3. 20. Odani, S. & Ikenakf, T. (1972) /. Biochzm. 71, 839-848 The A^hyd value decreased markedly upon change of the reaction pH from 2.0 to 1.5. This may be 21. Hokama, Y., Iwanaga, S., Tatsuki, T., & Suzuki, T. (1976) J. Biochem. 79, 559-578 due to inactivation of trypsin rather than changes 22. Mattis, J.A. & Laskowski, M., Jr. (1973) Biochemof the inhibitor. istry 12, 2239-2245 Vol. 80, No. 6, 1976
Masaaki YAMADA,* Misao TASHrRO,** 1 Hiroko YAMAGUCHJ,** Hisao YAMADA,** Fumio IBUKI,** and Masao KANAMORI** •Research Laboratories, The Green Cross Corp., Osaka, Osaka 534, and "Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto Prefectural University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, June 5, 1976
The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-mcdified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed as the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.
Ozawa and Laskowski, Jr. postulated that all naturally occurring trypsin inhibitors have either an Arg-X or a Lys-X trypsin-sensitive bond in their reactive sites ( / ) . A variety of trypsin inhibitors have been classified as arginyl or lysyl inhibitors by reactive site cleavage and by chemical modifications (2). In previous papers, we reported the occurrence of a trypsin inhibitor in eggplant, Solarium melongena L. (5), together with its purification and some properties (4-6). This inhibitor is a low molecular weight protein composed of 57 1
Present address: Department of Food Science, Faculty of Living Science, Kyoto Prefectural University, Sakyo-ku, Kyoto, Kyoto 606. Abbreviations: BAPA, a-N-benzoyl-DL-arginine p-nitroanilide; TAME, ^-tosyl-L-arginine methyl ester; CHD, 1,2-cyclohexanedione; TNBS, 2,4,6-trinitrobenzenesulfonic acid; TNP-, trinitrophenyl-; CPase, carboxypeptidase; DTNB, 5,5'-dithiobis (2-nitrobenzoic acid); SDS, sodium dodecyl sulfate. Vol. 80, No. 6, 1976
1293
amino acid residues, containing 4 lysine, 3 arginine residues, and 4 disulfide bonds. This communication describes the effect of disulfide bond reduction on the inhibitory activity, and the identification of the reactive site peptide bond with respect to trypsin by chemical modifications of lysine or arginine residues, and by limited proteolysis with trypsin. EXPERIMENTAL PROCEDURE Materials—The eggplant inhibitor was prepared from the exocarp of eggplant by the method described previously (6). Trypsin [EC 3. 4. 21. 4] (twice crystallized, from bovine pancreas) and carboxypeptidase B [EC 3.4.12.3] (from hog pancreas) were purchased from Sigma Chemical Co. BAPA, TAME, and DTNB were obtained from Nakarai Chemicals Ltd., Kyoto. All other chemicals were of special or reagent grade.
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The Reactive Site of Eggplant Trypsin Inhibitor
1294
M. YAMADA, M. TASHIRO, H. YAMAGUCHI, H. YAMADA, F. IBUKI, and M. KANAMORI
General Methods—Amino acid analysis was performed with a Hitachi KLA-5 amino acid analyser. Hydrolysis was carried out in an evacuated, sealed tube with 5.7 N HC1 at 110° for 22 hr. Edman degradation was carried out by the method of Iwanaga et al. (7). The phenylthiohydantoin derivative was identified by gas chromatography on a Shimadzu GC-4B gas chromatograph using a Silicon D.C. 560 column according to the method of Pisano and Bronzert (8). Reduction of Bisulfide Bonds—The disulfide bonds in the inhibitor were reduced by the modified method of Kress and Laskowski, Sr. (9). The inhibitor (3 mg/ml in 0.02% EDTA-2Na) in a volume of 1.02 ml was adjusted to pH 8.5 with 0.04 ml of 0.2 N NaOH. Then, 1.06 ml of freshly prepared 0.2 M NaBH4 was added to the inhibitor solution and the mixture was incubated at 2°, flushing with N,. After incubation for 2 min, 0.05 ml of 0.5 N HO was added to 0.1 ml aliquots withdrawn from the reaction mixture at appropriate intervals. From each aliquot, 0.1 ml was used for the determination of disulfide bonds by the procedure of Habeeb (10). The remaining solution (0.05 ml) was diluted 10-fold with distilled watsr and its inhibitory activity was assayed. CHD Modification—Modification of guanidino groups in the inhibitor was done with CHD by the modified method of Haynes and Feeney (11). The inhibitor (1 mg) was dissolved in 1.0 ml of 0.1 M triethylamine buffer, pH 10, containing 0.01 M EDTA-2Na and 0.015 M CHD. The reaction was allowed to proceed in the dark for 12 hr at 25°. An aliquot (0.1 ml) was removed from the reaction mixture, diluted 20-fold with distilled water and its inhibitory activity was determined. The remaining mixture was then desalted by gel filtration on
Sephadex G-25 and lyophilized. The content of arginine was determined by amino acid analysis after acid hydrolysis. Glyoxal Modification—Arginine residues in the inhibitor were modified with glyoxal according to the method of Nakaya et al. (12). The inhibitor (3 mg) was dissolved in 1 ml of 0.5 M bicarbonateNaOH buffer, pH 8.6, and treated with 1 ml of °-4% glyoxal solution for 3 hr at 25°. The reaction mixture was applied to a Sephadex G-25 column to isolate the modified inhibitor. The inhibitory activity was measured with the desalted sample. The amount of modified arginine residues was calculated by amino acid analysis after acid hydrolysis. TNBS Modification—Modification of free amino groups was carried out by the methcd of Haynes et al. (13). To 1 ml of the inhibitor solution (0.28 mg/ml) were added 1 ml of 4% NaHCO,, pH 8.3, and 1 ml of 1 % TNBS in water, and the mixture was incubated at 37°. To determine the number of modified amino groups, the reaction was stopped by the addition of 1 ml of 10% SDS and 0.5 ml of 1 N HC1. The extent of modification was calculated from the absorbance at 344 nm by using the molar extinction coefficient of TNP-amino acid, 1.09x10', given by Haynes et al. (13). For the assay of inhibitory activity, 0.3 ml was removed, diluted with 0.7 ml of cold distilled water, and assayed immediately. Acetic Anhydride Modification—Free amino groups in the inhibitor were acetylated with acetic anhydride according to the method of FraenkelConrat (14). To 0.5 ml of the inhibitor solution (5 mg/ml), 0.5 ml of saturated sodium acetate was added. The mixture was treated with five lots of 0.1 ml of acetic anhydride at 0° during 1 hr. The modified inhibitor was isolated by gel filtration on Sephadex G-25, followed by lyophilization. The lyophilized sample was used for the assay of inhibitory activity. The extent of acetylation was estimated by determining the residual free amino groups by the TNBS method of Haynes, as mentioned above. Limited Proteolysis with Trypsin—Effects of pH and incubation period: One mole % of trypsin was added to the inhibitor dissolved in buffers with pH values from 1.5 to 9.0. After incubation at 25° for 24 hr, an aliquot of each reaction mixture was subjected to disc gel electrophoresis in order to estimate J. Biochem.
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Inhibitory Activity Assay—Inhibitory activity toward trypsin was determined by the spectrophotometric method with BAPA as a substrate, as reported previously (4) or with a pH-stat, RTS622 (Radiometer, Copenhagen). The pHstat assay was carried out at 25° under N t . The incubation mixture consisted of 0.2 ml of 2 M KC1, 100 n\ of trypsin solution (80 /Jg/ml), and an appropriate amount of inhibitor solution in a total volume of 1.80 ml. The mixture was adjusted to pH 8.00 in a vessel, then the reaction was started by the addition of 0.2 ml of 40 mM TAME (pH 8.0) and followed by titration with 0.01 M NaOH.
1296
M. YAMADA, M. TASHIRO, H. YAMAGUCHI, H. YAMADA, F. IBUKI, and M, KANAMORI 100 u o JJ •H XI
a 4J 3 -H •o >
1
to -U 0) o M (0
" 0 10
Fig. 2. pH Dependence of limited proteolysis of the inhibitor with trypsin. The inhibitor was treated with 1 mole% trypsin at each pH for 24 hr at 25°. The Khji, ratio of modified inhibitor to native inhibitor was estimated densitometrically after disc gel electrophoresis.
2
4 6 8 20" 48 period of incubation(hr)
Fig. 4. Inactivation of trypsin-modified inhibitor by carboxypeptidase B digestion. • , native inhibitor; O, mixture containing modified and native inhibitors.
However, the modification hardly proceeded at pH's below 2. Figure 3 shows the time-dependence of trypsin-modified inhibitor formation. The trypsin-modified inhibitor was detected after incubation for 30 min at pH 2.5. Maximum formation was obtained after incubation for 24 hr and was maintained at an almost constant level up to 144 hr. When the mixture containing modified and native inhibitors was directly treated with CPase B, free arginine was detected by amino acid analysis. The amount of released arginine was 0.52 mole per mole of inhibitor after incubation for 20 hr. The trypsin inhibitory activity fell to 46.8% of the original activity, as shown in Fig. 4. No free amino acid was detected on CPase B Fig. 3. The time-dependence of trypsin-modified in- treatment of the native inhibitor and no effect on hibitor formation. The inhibitor was treated with the inhibitory activity was noted. These results 1 mole% trypsin at pH 2.5. Upper bands in each tube show that a new C-terminal amino acid, arginine, are native and middle bands are modified inhibitor. was exposed. The newly exposed N-terminal Lower bands are BPB. Incubation periods were; tube amino acid residue in the mixture of modified and No. 1, 0 min; No. 2, 5 min; No. 3, 0.5 hr; No. 4, 1 hr; native inhibitors was identified as serine by Edman No. 5, 6 hr; No. 6, 24 hr; No. 7, 48 hr; No. 8, 72 hr; degradation; no N-terminal amino acid residue of No. 9, 144hr. the native inhibitor could be detected by Edman degradation. Consequently, it was concluded that terminal amino acid. Therefore, one amino group the reactive site peptide bond is arginylserine. was not modified. The acitylation of amino groups with ac;tic anhydride was complete, as DISCUSSION judged from the fact that no TNP-amino acids were detected. The loss of inhibitory activity due Full reduction of disulfide bonds in the inhibitor to these amino group modifications was insignifiinducsd complete loss of inhibitory activity. These cant. These results are sum-mrized in Table I. disulfide bonds were thus essential for the activity. Limited Proteolysis with Trypsin—The forma- The loss of activity paralleled the extent of reduction of modified inhibitor was depressed in the pH tion. This suggests that the loss of activity was not region from 4 to 6, and remarkably accelerated at caused by the reduction of a particular disulfide alkaline and more acidic pH's, as shown in Fig. 2. bond, but possibly by conformational changes. J. Biochem.
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•H
REACTIVE SITE OF EGGPLANT TRYPSIN INHIBITOR
RESULTS Effect of Disulfide Bond Reduction on Inhibitory Activity—Figure 1 shows the relationship between the reduction of disulfide bonds and the loss of trypsin inhibitory activity. The inhibitory activity was lost in proportion to the extent of reduction of disulfide bonds. This inhibitor contains 8 half-cystine residues per molecule (6).
>, 100
0
1 2 14 period of incubation(hr)
Fig. 1. Effect of disulfide reduction on trypsin inhibitory activity. • , residual inhibitory activity; O, sulfhydryl groups per mole of inhibitor. Inhibitor solution in 0.02% EDTA-2Na was adjusted to pH 8.5 with 0.2 M NaOH, and treated with an equal volume of 0.2 M NaBH, by incubation at 2° under N,.
No sulfhydryl groups were detected in the intact inhibitor. Seven sulfhydryl froups appeared per mole of inhibitor after reduction for 14 hr. This fully reduced Inhibitor showed no inhibitory activity. Chemical Modifications—The inhibitor contains 3 arginine residues, CHD modification caused a 38% loss of arginine residues, corresponding to the modification of approximately one arginine residue. After this modification, the trypsin inhibitory activity was reduced to 22% of the original activity. Glyoxal reacted with approximately 2.4 moles of arginine residues per mole of inhibitor. The residual inhibitory activity was only 0.8%. The amount of TNP-amino acid groups formed by TNBS modification was 3.9 moles per mole of inhibitor on the basis of a molecular weight of 6,000 (6). The number of free amino groups in the inhibitor was theoretically five; 4 tamino groups and one a-amino group of the N-
TABLE I. Inactivation of inhibitor by chemical modification. Reagent 1,2-Cyclohexanedione Glyoxal Acetic anhydride 2,4,6-Trinitrobenzenesulfonic acid
Vol. 80, No. 6, 1976
Modified residue
Modified
Loss of inhibitory activity (%)
Arginine Arginine Lysine Lysine
38 80 80 80
78 99 8 0
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the extent of modification. Gel electrophoresis was carried out by the method of Davis (15). Staining was performed with Coomassie brilliant blue by the method of Charmbach et al. (16). The ratio of the native and modified inhibitors was estimated with a Yamato-Asuka OZUMOR 82 densitometer. The incubation period required for maximum modification was investigated at pH 2.5 with 1 mole % trypsin at 25° during Incubation for 144 hr. Carboxypeptidase B Treatment of Trypsinmodified Inhibitor—The inhibitor (30 mg) was dissolved in 4.4 ml of 0 . 1 M sodium citrate-HCl buffer, pH 2.0. To this solution was added 0.6 ml of trypsin solution (2 mg/ml). After incubation at 25° for 20 hr, the reaction mixture was immediately applied to a Sephadex G-50 column (2.5 x 39 cm) to remove trypsin. The fractions containing the native and modified inhibitors were pooled and lyophilized. The lyophilized sample (0.8 mg) was dissolved in 0.2 ml of 0.05 M Tris-HCl buffer, pH 8.0. Ten (i\ of CPase B solution (3 mg/ml) was added and the reaction mixture was incubated for 2 days at 37°. An aliquot of the reaction mixture was assayed for trypsin inhibitory activity with a pH-stat. The released amino acid residue was identified by amino acid analysis. The newly exposed N-terminal amino acid was identified by Edman degradation.
1295
REACTrVE SITE OF EGGPLANT TRYPSIN INHIBITOR
1297
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On the basis of the results of chemical modiNo N-terminal amino acid residue of the native fications and sequential treatments with trypsin and eggplant inhibitor was detected by Edman degraCPase B, it was concluded that the eggplant in- dation, and one amino group in the inhibitor was hibitor contained a trypsin-suscsptible arginyl- resistant of TNBS modification. This suggests seryl bond in its reactive site. Modification of the that the N-terminal amino group is blocked. arginine residues was extensive with glyoxal and Studies on this problem are in progress. slight with CHD. However, the inhibitory activity was significantly reduced in both cases. This REFERENCES indicates that the arginine residue in the reactive site was reactive with these reagents. The amount 1. Ozawa, K. & Laskowski, M., Jr. (1966) J. Biol. Chem. 241, 3955-3961 of arginine residue released after the sequential 2. Laskowski, M., Jr. & Sealock, R.W. (1971) in The enzymatic treatments coincided well with the loss Enzymes (Boyer, P.D., ed.) Vol. 3, pp. 375-473, Acaof inhibitory activity. The low yield of released demic Press, New York arginine is due to the co-existence of native in3. Kanamori, M., Ibuki, F., Tashiro, M., Yamada, M., & Miyoshi, M. (1975) /. Nulr. Sci. Vitaminol. 21, hibitor. 421^(28 The reactive site peptide bond of the eggplant 4. Kanamori, M., Ibuki, F., Yamada, M., Tashiro, M., & Miyoshi, M. (1975) /. Nun. Sci. Vitaminol. 21, inhibitor was an arginylseryl bond. It has been 429^436 reported that garden bean (17), and soybean C and 5. Kanamori, M., Ibuki, F., Yamada, M., Tashiro, M., D inhibitors (18) have the same peptide bond in & Miyoshi, M. (1976) Agr. Biol. Chem. 40, 839-844 their reactive sites. Hcchstrasser et al. (19) 6. Kanamori, M., Ibuki, F., Tashiro, M., Yamada, M., & Miyoshi, M. (1976) Biochim. Biophys. Acta showed that the reactive sits psptide bond of the 439, 398-405 peanut inhibitor was an arginylalanyl bond. How7. Iwanaga, S., Wall£n, P., Grondahl, N.J., Henschen, ever, Odani and Ikenaka (20) speculated that an A., & Blomblch, B. (1969) Eur. J. Biochem. 8, 189-199 arginylseryl bond might be the reactive site, on the 8. Pisano, J.J. & Bronzert, T.J. (1969) J. Biol. Chem. basis of the similarity in amino acid sequence 244, 5597-5607 between Bowman-Birk soybean inhibitor and lima 9. Kress, L.F. & Laskowski, M. Sr., (1967) J. Biol. bean inhibitor. As mentioned above, an arginylChem. 242, 4925-4929 seryl reactive site bond has been found in several 10. Habeeb, A.F.S.A. (1972) in Methods in Enzymology (Hirs, C.H.W. & Timasheff, S.N., eds.) Vol. 25, trypsin inhibitors. pp. 457^464 On the basis of comparisons of the amino 11. Haynes, R. & Feeney, R. (1968) Biochemistry 7, 2879-2885 acid sequences of many proteinase inhibitors from materials of different phylogenetic descent, Hokama 12. Nakaya, K., Horinishi, H., & Shibata, K. (1967) J. Biochem. 61, 345-351 et al. (21) suggested that the regions related to 13. Haynes, R., Osuga, D.T., & Feeney, R.E. (1967) their inhibitory functions showed high sequence Biochemistry 6, 541-547 homology, and the other regions reflected their 14. Fraenkel-Conrat, H. (1963) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. phylogenetic relationships. To discuss the phy4, 247-269 Iogeny of the eggplant inhibitor, knowledge of its 15. Davis, B.J. (1964) Ann. New York Acad. Sci. 121, complete amino acid sequence will be required. 404-427 The pH dependence of Kh7i, the equilibrium 16. Charmbach, A., Reisfeld, R.A., Wyckoff, M., & Zaccari, J. (1967) Anal. Biochem. 20, 150-154 constant for limited hydrolysis with trypsin (Fig. 17. Wilson, K.A. & Laskowski, M., Sr. (1975) J. Biol. 2), was similar to that of Kunitz soybean inhibitor Chem. 250, 4261-4267 (22). However, these Khyt values are low in com- 18. Odani, S. & Ikenaka, T. (1975) 26th Tanpakushitsu Kozo Toronkai Yokoshu, pp. 109-112 parison with that of Kunitz soybean inhibitor. The low K^ji value for eggplant inhibitor did not 19. Hochstrasser, K., lllchmann, K., Werle, E., Hoesse', R., & Schwarz, S. (1970) Z. Physiol. Chem. appear to be due to an insufficient incubation 351, 1503-1512 period, on the basis of the results shown in Fig. 3. 20. Odani, S. & Ikenakf, T. (1972) /. Biochzm. 71, 839-848 The A^hyd value decreased markedly upon change of the reaction pH from 2.0 to 1.5. This may be 21. Hokama, Y., Iwanaga, S., Tatsuki, T., & Suzuki, T. (1976) J. Biochem. 79, 559-578 due to inactivation of trypsin rather than changes 22. Mattis, J.A. & Laskowski, M., Jr. (1973) Biochemof the inhibitor. istry 12, 2239-2245 Vol. 80, No. 6, 1976
