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Inhibition of the thrombin-like principle of Agkistrodon acutus venom by group-specific enzyme inhibitors
Toxtcon, 1974, Vol. 12, pp. 449-433 . Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION INHIBITION OF THE THROMBIN-LIKE PRINCIPLE OF AGKISTRODON ACUTUS VENOM BY GROUP-SPECIFIC ENZYME INHIBITORS CHAOHO OUYANG
and
SAU-SHYONG HONG
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (.lccepted jor publication 27 February 1974)
thrombin-hke principles isolated from the venoms of Agkistrodon (FSNOUF and TUNNAH, 1967) and Agkistrodon acutus (OUYANG et al., 1971) are glycoproteins that resemble thrombin in moleculâr weight and DFP-inhibited esterase nature . But they differ from thombin in several respects. They are more heat-stable than thrombin (EsxouF and TUNNAH, 1967 ; OUYANG et al., 1971). Both peptides A and B are removed from fibrinogen during the formation of fibrin by thrombin, while only peptide A is removed from Reptilase, the thrombin-like principle of Bothrops jararaca venom (B1.oMSF~cIC, 1958). Unlike thrombin, they are not inhibited by heparin (FrsNOUF and TUNNAH, 1967 ; OUYANG et al., 1971). No clot retraction nor factor XIII (fibrin stabilizing factor) activation is found with the thrombin-like principle (OUYANG et al., 1971). Hence the active center of the thrombin-like principle of snake venom might be quite different from that of thrombin . Inthepresent study we studied theessential groups) of the thrombinlike principle of Agkistrodon acutus venom in comparison with those of bovine thrombin using various group-specific enzyme inhibitors . Thrombin-like principle of Agkistrodon acutus venom was purified by the procedure of OUYANG et al. (1971) . This principle was homogeneous as judged by electrophoresis on cellulose acetate membrane, disc electrophoresis on polyacrylamide gel and ultracentrifugal analysis. The effects of the enryme inhibitors on the thrombin or the thrombin-like activity of the venom was measured by the method of CALDWELL and $EEGERS (196 modified as follows : 0~5 ml of the thrombin or the venom solution (1 mg/ml) was incubated with 0~5 ml of the enzyme inhibitors (pH 7~0, 002 M, except as indicated) at 28°C for 2 hr, and then the residual thrombin or thrombin-like activity was determined . At zero time, immediately upon mixing thrombin or the venom solution and the enzyme inhibitors, an aliquot was removed and activity determined . This value was considered to be the 100 per cent activity, and also served as a control. Controls were also set up which contained one part protein and one part distilled water in order to measure any spontaneous deterioration of the protein during the 2-hr incubation period . The quantitative determination of the thrombin or thrombin-Gke activity of the venom was made according to the method of $BBGERS and ShuTH (1942) . Fibrinogen solution (0~1 ml of 1 per cent) was mined with 0~3 ml of titration mixture, then 0~1 ml of the test solution was blown into the fibrinogen mixture THE HOMOGENEOUS
rhodostoma
TOXlCON 197f Yot. I?
449
C. OUYANG and J.-S . HONG
450
and the clotting time was determined . The number of units, equivalent to the activity of thrombin required to produce a clot in the standard 15 sec interval at 28°C, was calculated from the dilution factors . Table 1 summarizes the results with the various group-specific reagents. TABLE 1 . THE EFFECTS OF VARIOUS GROUP-SP6CQ:IC REAGEN78 ON THE C1 .OTI1N0 ACTIVITIHS OF THROAfHiN AND THE THROMBIN-LIKE PRIIVCIPLS OF Apkistrodon acutus vENOSI
Inhibition Reagent Diaulflde group reag~ta : Cysteine Thioglyoolic acid Ascorbic acid Sodium sulfite Sulfhydryl group reagents : Cys6ne PheyLmercuric acetate 4-Cliloro-mercuric benzoic acid N-Ethyl-makimide Iodoacetamide Amino group Teagents : ~Naphthoquinone-4sulfonic acid Chloro-fluoro-p-benzoquinone Carbonyl group reagents Phenylhydrazine Hydroxylanûne Imidazok group (histidine) reagents : Diazontum-l-H-tetrazide N-a-tasyl-l-lysine cJilormethyl-ketone Guanidyl group (arginine) reagent : Glyoxal Pheaol group (tyrosine) reagent : N-Aryl-inûdazok Hydroxyl group (serine) reagent : Diiaopropylfluorophosphate Sulfonation : Phenyl-methane-sulfonyl-fluoride
Thrombin
The thrombin-like principle of Agktrtrodor acorns venom
lOt3 2f 2 0 22f4
7~3 5 f 4 0 15f8
0 >99 >99 lOf2 9f3
0 >99 > 99 7f4 Sf3
67f8 94f6
28f4 20f5
31f5 3f2
28f5 7f3
64f8 76f7
73f12 8f2
lOf2
27f5
14f4
6f3
> 99 65f6
>99 SOf4
The final concentration of the enzyme inhibitors was 0-01 M, except that the final concentration of phenylmercuric acetate and 4-c61oro-mercuric benzoic acid was 0001 M and that of diisopropylfluorophosphate was 003 M. Each exp~irr~ent was performed in triplicate, and the results are presented as means f standard errors . Reducing agents act on unmodified proteins to reduce disulfide bonds to sulfhydryl groups . Those reducing reagents containing sulfhydryl groups are very specific in their action on disulfide bonds. Sulfites and bisulfites add to disulfide bonds to give a sulfhydryl and thiosulfite (Ci AKP~ 1932). Both thrombin (Park-D~vis Co., U.S.A.) and the thrombinlike principle of Agkistrodon acutus venom were unaffected by reducing agents having SH groups such as cysteine and thioglycolic acid and ascorbic acid. Both of them wen only slightly affected by sodium sulfite . From these results we may say that the disulfide group is not essential in thrombin or the thrombin-like principle of Agkistrodon acutus venom. Our results with thrombin agree with the finding of CALDWELL and S>?t:Gmts (196 . TOXlCON 197~ Yd. 12
Thrombin-like principle of Agkistrodon acutus venom
431
Sulfhydryl groups can be oxidized by cystine to form disulfide bonds while phenylmercuric acetate and 4-chloromercuric benzoic acid combine with sulfhydryl groups and form mercaptides, and N-ethylmaleimide or iodoacttamide induce alkylation. 4Chloromercuric benzoic acid and phenylmercuric acetate inhibited the activities of both thrombin and the thrombin-like principle of Agkistrodon acutus venom, while N-ethyl-maleimide and iodoacetamide were inactive . According to Fxf xxa.-CoxxnT (1957), phenylmercuric acetate and 4-chloro-mercuric benzoic acid have relatively strong actions on masked SH groups, while N-ethyl-maleimide and iodoacetamide act only on free SH group . Cystine, N-ethyl-maleimide and iodoacetamide possibly did not inhibit activity due to their weak action on masked SH groups . From our results we may conclude that SH groups are essential in both thrombin and the thrombin-like principle of Agkistrodon acutus venom. M~~rsustna~ and St~ATA (1967) and NARAYA and SHIHATA (1967) state that ß-naphthoquinone-4-sulfonic acid ($eikagaku Co .) and monochlorotrißuoroquinone (Seikagaku Co .) act on free (unburied) amino groups specifically (chiefly lysine). These two enzyme inhibitors inhibited the activity of thrombin markedly, while they showed only a slight inhibition on the thrombin-like activity of Agkistrodvn acutus venom. Hence we know that a free amino group is an essential group in the thrombin molecule, but not important for the thrombin-like activity of Agkistrodon acutus venom. Our result on thrombin agrees with the observations of Cu.nw~-r- and SE6GERS (1%~ . Hydrazine and hydroxylamine react with aldehyde and ketone groups . Both thrombin and the thrombin-like principle of Agkistrodon acutus venom retained full activity following incubation with hydroxylamine, and decreased only slightly with phenylhydrazine. These results indicate that the carbonyl group is not important for the activities of thrombin or the thrombin-like principle of Agkistrodon acutus venom. HIROO and SHIBATA (1964) stated that although diazonium-l-H-tetrazide acted on histidine, the action was not very specific since it also reacts with tyrosine and lysine . Diazonium-l-H-tetrazide markedly inhibited the activities of both thrombin and the throm bin-like principle of Agkistrodon acwtus venom. As lysine and tyrosine are not the essential group in the molecule of the thrombin-like principle of Agkistrodon acutus venom (see Table 1), the effect of diazonium-l-H-tetrazide on the thrombin-like principle of Agkistrodon acutus venom may be due to an action on histidine. Our results on thrombin agrees with the statement of M~cirn~x and $EEGERS (1966) that histidine is an essential group in the thrombin molecule . Stow (1%~ reported that N-ap-tosyl-L-lysine chloromethyl ketone (TLCK) (Sigma Co .) inactivates trypsin stoichiometrically by alkylation of a histidine residue. This action of TLCK is very specific . The molecule of this reagent contains lysine, trypsin can break the peptide linkage. Hence TLCK can inhibit trypsin, while it has no action on chymotrypsin . Tosyl-amide-phénylalanine chloromethyl-ketone (TPCK) (Sigma Co .) contains phenylalanine, and chymotrypsin can break its peptide linkage, hence TPCK can inhibit chymotrypsin, while it has no action on trypsin (SCHOII.LMANN and Stow, 1%3). The steric effects of TLCK and TPCK on the molecular structure are highly specific . Mwttcixtwx and Sa~fiRS (1966) and Swats et al. (196 reported that the activity of thrombin was inhibited by TLCK, but not by TPCK . TLCK could inhibit the activity of thrombin, but not the thrombin-like activity of Agkistrodon acutus venom . As the water solubility of TPCK is very small, organic solvents such as methanol should be used . However, all of the organic solvents we used precipitated the venom, thus we could not study the effects of TPCK . N~tuY~ et al. (1967) reported that glyoxal acted on the guanidyl group of arginine . 7YJXlCON 1971 Yot. 1 2
45 2
C. OUYANG and J.-S. HONG
Glyoxal had no action on thrombin, and only a slight action on the thrombin-like action of
Agkistrodon status venom. This indicated that arginine is not a part of the active centers of thrombin or the thrombin-like principle of Agkistrodon status venom. Jnlrtss et al. (1965) reported that N-acetyl-imidazole could acetylate free (unburied)
tyrosine in a relatively specific manner. N-Acetyl-imidazole showed only a slight inhibitory action on thrombin, and no significant action on the thrombin-like action of Agkistrodon status venom. Hence we know that a free tyrosine residue is not a part of the active centers of thrombin or the thrombin-like principle of Agkistrodon status venom. GLADNER and Lexl (195 and MILLER and VAN VwlvuKts (1956) found that düsopropylfluorophosphate destroyed the activity of thrombin, and the combination of düsopropylfluorophosphate with a single serine amino acid residue was demonstrated (GLADNF-R and LAx1,1958) . This indicates that serine is a part of the active center of the thrombin molecule. In our study the activities of both thrombin and the thrombin-like principle of the venom were inhibited completely by düsopropylfluorophosphate, hence serine is a part of the active centers of both thrombin and the thrombin-like principle of Agkistrodon status venom. FAHRNSY and GOLD (1963) reported that phenyl-methane sulfonyl fluoride (Sigma Co.) inhibits the enzymatic activities of acetylcholinesterase, a-chymotrypsin and trypsin. CALDWm-t- and S$ecExs (196 stated that the clotting activity of thrombin was also in hibited by phenyl-methane sulfonyl fluoride. Phenyhnethane-sulfonyl fluoride inhibited the activities of both thrombin and the thrombin-like principle of Agkistrodon status venom markedly, although the effect on the former was greater than on the latter . Sulfonation can thus affect the activities of both thrombin and the thrombin-like principle of Agkistrodon status venom. From the above results, we may say that the sulfhydryl group, histidine and serine are essential for the activities of both thrombin and the thrombin-like principle of Agkistrodon status venom, while the carbonyl group, disulfide group, arginine and tyrosine are not important for their activities . A free amino group is essential for the activity of thrombin, but not for the thrombin-like activity of Agkistrodon status venom. Acknowledgements-This work was supported by the National Science Council Research Grant of the Republic of China. The authors express their sincere appt+xiation to Prof. C. Y. Lee, Dean of the College of Medicine, National Taiwan University for his invaluable advice. REFERENCES Bt oMaxcs:, H. (1958) Studies on the action of thrombic enzymes on bovine fibrinogen as measured by N-terminal analysis . Ark. Kem. 12, 321. Cwi.nwet,t., M. J. and Seeceas, W. H. (1965) Inhibition of prothrombin, thrombin and autoprothrombin with enzyme inhibitors. 79rromb. Dlath. haemorrh . 132, 373. CLwKe, H. T. (1932) The action of sulfite upon cystine. J. blot. Chem . 97, 235. ESNOUF, M . P . and Tutvwwx, G. W. (1967) The isolation and properties of the thrombin-like activity from Ancistrodon rhodartoma venom. Br. J. Hasmat . 13, 581. FAHRNEY, D. E. and Gow, A. M. (1963) Sulfonyl-fluoride as inhibitor of esterase. J. Am . them . Soc. 85, 997 . FRANREIrCONRAT, H. (1957) Methods for investigating the essential groups for enzyme activity. Meth . Enzymology 4, 256. Giwnnex, J. A. and Lwxt, K. (1956) The inhibition of thrombin by düsopropylphosphomfluoridate . .lrchs Bioohem. Biophys. 62, SOI . Gt .wnxea, J. A. and Lwtcr, K. (1958) The active site of thrombin . J. .Im. chsm. Soc. 80, 1263 . Hntoo, H. and Swtawrw, K. (1964) States of amino acid residues in proteins. III. Histidine residues in insulin, lysozyme, albumin and proteinases determined with a new reagent of diazonium-l-H-tetrazok . Biochlm. biophys. .lcta 86, 475. 7~OXJCON 197~ Yol. J2
Thrombin-like principle of Agkistrodon status venom
453
Jas, F. R., WNexe, E. C. W. and B©tT, L. V. (1965) A reagent for determination of `free tyrosyl' residues of protein. Biochemistryy, 4,1758. Mwxcmaex, E. and S~oERS, W. H. (19C~ Characteristics of the prethrombin subunit of prothrombin . ?fuomb. Diatlr . lulemorrb .l5, 633. M~~ravsfmu, A. and Sz~Tly K. (1967) Amino groups with different reactivities toward naphthoquinone sulfonic acid . l. Biochtm. 63, 1328 . Mn.l~e, K. D. and vl r~ Venvms, H. (1956) The effect of diisopropyl-fiuorophosphate on the proteinase and esterase activities of thrombin and on prothrombin and its activators. l, bioi. Clam. 223, 227. NAICAYA, K., HonnvlsHa, H. and Sluenre, K. (1967) Glyoxal as a reagent for discrimination of arginine residues . l. Biochem. 61, 345. NAiCAYA, K, and S1m~Te, K. (1967) Monochlorotrifluoroquinone as a new reagent for discrimination of amino groups . I. Biocbem. 61, 337. t7uverao, C., Hor~ct, J. S. and Tr~a, C, M. (1971) Purification and properties of the thrombin-like principle of Agkistrodon acutru venom and its comparison with bovine thrombin . 7lrromb. Diath. haemorrh. 26, 224. Scxoiz raurrN, G. and Sll~w, E. (1963) Direct evidence for the presence of histidine in the active center of chymotrypain . Biochumistr 2, 252. S~asRS, W. H., Hr~re, D., MNtcavl~tc, E., Iwuvovtc, N, and CALDWELL, M. J. (1965) Sensitivity of thrombin and autoprothrombin C to selected enzyme inhibitors . L{fe Sct. 4, 425. S~omes, W. H, and ~rx, H. P, (1942) Factors which influence the activity of purified thrombin . l. PhysioJ. 137, 348. Sxsw, E. (1965) Evidenz for an active center histidine in trypsin through use of a specific reagent l~hloro3-toaylamido-7-amino-2-heptanone, the cliloromethylketone derived from N-artoayl-l-lysine. Biochemistry 4, 2219.
TOXICON 1971 Yol. 11
SHORT COMMUNICATION INHIBITION OF THE THROMBIN-LIKE PRINCIPLE OF AGKISTRODON ACUTUS VENOM BY GROUP-SPECIFIC ENZYME INHIBITORS CHAOHO OUYANG
and
SAU-SHYONG HONG
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (.lccepted jor publication 27 February 1974)
thrombin-hke principles isolated from the venoms of Agkistrodon (FSNOUF and TUNNAH, 1967) and Agkistrodon acutus (OUYANG et al., 1971) are glycoproteins that resemble thrombin in moleculâr weight and DFP-inhibited esterase nature . But they differ from thombin in several respects. They are more heat-stable than thrombin (EsxouF and TUNNAH, 1967 ; OUYANG et al., 1971). Both peptides A and B are removed from fibrinogen during the formation of fibrin by thrombin, while only peptide A is removed from Reptilase, the thrombin-like principle of Bothrops jararaca venom (B1.oMSF~cIC, 1958). Unlike thrombin, they are not inhibited by heparin (FrsNOUF and TUNNAH, 1967 ; OUYANG et al., 1971). No clot retraction nor factor XIII (fibrin stabilizing factor) activation is found with the thrombin-like principle (OUYANG et al., 1971). Hence the active center of the thrombin-like principle of snake venom might be quite different from that of thrombin . Inthepresent study we studied theessential groups) of the thrombinlike principle of Agkistrodon acutus venom in comparison with those of bovine thrombin using various group-specific enzyme inhibitors . Thrombin-like principle of Agkistrodon acutus venom was purified by the procedure of OUYANG et al. (1971) . This principle was homogeneous as judged by electrophoresis on cellulose acetate membrane, disc electrophoresis on polyacrylamide gel and ultracentrifugal analysis. The effects of the enryme inhibitors on the thrombin or the thrombin-like activity of the venom was measured by the method of CALDWELL and $EEGERS (196 modified as follows : 0~5 ml of the thrombin or the venom solution (1 mg/ml) was incubated with 0~5 ml of the enzyme inhibitors (pH 7~0, 002 M, except as indicated) at 28°C for 2 hr, and then the residual thrombin or thrombin-like activity was determined . At zero time, immediately upon mixing thrombin or the venom solution and the enzyme inhibitors, an aliquot was removed and activity determined . This value was considered to be the 100 per cent activity, and also served as a control. Controls were also set up which contained one part protein and one part distilled water in order to measure any spontaneous deterioration of the protein during the 2-hr incubation period . The quantitative determination of the thrombin or thrombin-Gke activity of the venom was made according to the method of $BBGERS and ShuTH (1942) . Fibrinogen solution (0~1 ml of 1 per cent) was mined with 0~3 ml of titration mixture, then 0~1 ml of the test solution was blown into the fibrinogen mixture THE HOMOGENEOUS
rhodostoma
TOXlCON 197f Yot. I?
449
C. OUYANG and J.-S . HONG
450
and the clotting time was determined . The number of units, equivalent to the activity of thrombin required to produce a clot in the standard 15 sec interval at 28°C, was calculated from the dilution factors . Table 1 summarizes the results with the various group-specific reagents. TABLE 1 . THE EFFECTS OF VARIOUS GROUP-SP6CQ:IC REAGEN78 ON THE C1 .OTI1N0 ACTIVITIHS OF THROAfHiN AND THE THROMBIN-LIKE PRIIVCIPLS OF Apkistrodon acutus vENOSI
Inhibition Reagent Diaulflde group reag~ta : Cysteine Thioglyoolic acid Ascorbic acid Sodium sulfite Sulfhydryl group reagents : Cys6ne PheyLmercuric acetate 4-Cliloro-mercuric benzoic acid N-Ethyl-makimide Iodoacetamide Amino group Teagents : ~Naphthoquinone-4sulfonic acid Chloro-fluoro-p-benzoquinone Carbonyl group reagents Phenylhydrazine Hydroxylanûne Imidazok group (histidine) reagents : Diazontum-l-H-tetrazide N-a-tasyl-l-lysine cJilormethyl-ketone Guanidyl group (arginine) reagent : Glyoxal Pheaol group (tyrosine) reagent : N-Aryl-inûdazok Hydroxyl group (serine) reagent : Diiaopropylfluorophosphate Sulfonation : Phenyl-methane-sulfonyl-fluoride
Thrombin
The thrombin-like principle of Agktrtrodor acorns venom
lOt3 2f 2 0 22f4
7~3 5 f 4 0 15f8
0 >99 >99 lOf2 9f3
0 >99 > 99 7f4 Sf3
67f8 94f6
28f4 20f5
31f5 3f2
28f5 7f3
64f8 76f7
73f12 8f2
lOf2
27f5
14f4
6f3
> 99 65f6
>99 SOf4
The final concentration of the enzyme inhibitors was 0-01 M, except that the final concentration of phenylmercuric acetate and 4-c61oro-mercuric benzoic acid was 0001 M and that of diisopropylfluorophosphate was 003 M. Each exp~irr~ent was performed in triplicate, and the results are presented as means f standard errors . Reducing agents act on unmodified proteins to reduce disulfide bonds to sulfhydryl groups . Those reducing reagents containing sulfhydryl groups are very specific in their action on disulfide bonds. Sulfites and bisulfites add to disulfide bonds to give a sulfhydryl and thiosulfite (Ci AKP~ 1932). Both thrombin (Park-D~vis Co., U.S.A.) and the thrombinlike principle of Agkistrodon acutus venom were unaffected by reducing agents having SH groups such as cysteine and thioglycolic acid and ascorbic acid. Both of them wen only slightly affected by sodium sulfite . From these results we may say that the disulfide group is not essential in thrombin or the thrombin-like principle of Agkistrodon acutus venom. Our results with thrombin agree with the finding of CALDWELL and S>?t:Gmts (196 . TOXlCON 197~ Yd. 12
Thrombin-like principle of Agkistrodon acutus venom
431
Sulfhydryl groups can be oxidized by cystine to form disulfide bonds while phenylmercuric acetate and 4-chloromercuric benzoic acid combine with sulfhydryl groups and form mercaptides, and N-ethylmaleimide or iodoacttamide induce alkylation. 4Chloromercuric benzoic acid and phenylmercuric acetate inhibited the activities of both thrombin and the thrombin-like principle of Agkistrodon acutus venom, while N-ethyl-maleimide and iodoacetamide were inactive . According to Fxf xxa.-CoxxnT (1957), phenylmercuric acetate and 4-chloro-mercuric benzoic acid have relatively strong actions on masked SH groups, while N-ethyl-maleimide and iodoacetamide act only on free SH group . Cystine, N-ethyl-maleimide and iodoacetamide possibly did not inhibit activity due to their weak action on masked SH groups . From our results we may conclude that SH groups are essential in both thrombin and the thrombin-like principle of Agkistrodon acutus venom. M~~rsustna~ and St~ATA (1967) and NARAYA and SHIHATA (1967) state that ß-naphthoquinone-4-sulfonic acid ($eikagaku Co .) and monochlorotrißuoroquinone (Seikagaku Co .) act on free (unburied) amino groups specifically (chiefly lysine). These two enzyme inhibitors inhibited the activity of thrombin markedly, while they showed only a slight inhibition on the thrombin-like activity of Agkistrodvn acutus venom. Hence we know that a free amino group is an essential group in the thrombin molecule, but not important for the thrombin-like activity of Agkistrodon acutus venom. Our result on thrombin agrees with the observations of Cu.nw~-r- and SE6GERS (1%~ . Hydrazine and hydroxylamine react with aldehyde and ketone groups . Both thrombin and the thrombin-like principle of Agkistrodon acutus venom retained full activity following incubation with hydroxylamine, and decreased only slightly with phenylhydrazine. These results indicate that the carbonyl group is not important for the activities of thrombin or the thrombin-like principle of Agkistrodon acutus venom. HIROO and SHIBATA (1964) stated that although diazonium-l-H-tetrazide acted on histidine, the action was not very specific since it also reacts with tyrosine and lysine . Diazonium-l-H-tetrazide markedly inhibited the activities of both thrombin and the throm bin-like principle of Agkistrodon acwtus venom. As lysine and tyrosine are not the essential group in the molecule of the thrombin-like principle of Agkistrodon acutus venom (see Table 1), the effect of diazonium-l-H-tetrazide on the thrombin-like principle of Agkistrodon acutus venom may be due to an action on histidine. Our results on thrombin agrees with the statement of M~cirn~x and $EEGERS (1966) that histidine is an essential group in the thrombin molecule . Stow (1%~ reported that N-ap-tosyl-L-lysine chloromethyl ketone (TLCK) (Sigma Co .) inactivates trypsin stoichiometrically by alkylation of a histidine residue. This action of TLCK is very specific . The molecule of this reagent contains lysine, trypsin can break the peptide linkage. Hence TLCK can inhibit trypsin, while it has no action on chymotrypsin . Tosyl-amide-phénylalanine chloromethyl-ketone (TPCK) (Sigma Co .) contains phenylalanine, and chymotrypsin can break its peptide linkage, hence TPCK can inhibit chymotrypsin, while it has no action on trypsin (SCHOII.LMANN and Stow, 1%3). The steric effects of TLCK and TPCK on the molecular structure are highly specific . Mwttcixtwx and Sa~fiRS (1966) and Swats et al. (196 reported that the activity of thrombin was inhibited by TLCK, but not by TPCK . TLCK could inhibit the activity of thrombin, but not the thrombin-like activity of Agkistrodon acutus venom . As the water solubility of TPCK is very small, organic solvents such as methanol should be used . However, all of the organic solvents we used precipitated the venom, thus we could not study the effects of TPCK . N~tuY~ et al. (1967) reported that glyoxal acted on the guanidyl group of arginine . 7YJXlCON 1971 Yot. 1 2
45 2
C. OUYANG and J.-S. HONG
Glyoxal had no action on thrombin, and only a slight action on the thrombin-like action of
Agkistrodon status venom. This indicated that arginine is not a part of the active centers of thrombin or the thrombin-like principle of Agkistrodon status venom. Jnlrtss et al. (1965) reported that N-acetyl-imidazole could acetylate free (unburied)
tyrosine in a relatively specific manner. N-Acetyl-imidazole showed only a slight inhibitory action on thrombin, and no significant action on the thrombin-like action of Agkistrodon status venom. Hence we know that a free tyrosine residue is not a part of the active centers of thrombin or the thrombin-like principle of Agkistrodon status venom. GLADNER and Lexl (195 and MILLER and VAN VwlvuKts (1956) found that düsopropylfluorophosphate destroyed the activity of thrombin, and the combination of düsopropylfluorophosphate with a single serine amino acid residue was demonstrated (GLADNF-R and LAx1,1958) . This indicates that serine is a part of the active center of the thrombin molecule. In our study the activities of both thrombin and the thrombin-like principle of the venom were inhibited completely by düsopropylfluorophosphate, hence serine is a part of the active centers of both thrombin and the thrombin-like principle of Agkistrodon status venom. FAHRNSY and GOLD (1963) reported that phenyl-methane sulfonyl fluoride (Sigma Co.) inhibits the enzymatic activities of acetylcholinesterase, a-chymotrypsin and trypsin. CALDWm-t- and S$ecExs (196 stated that the clotting activity of thrombin was also in hibited by phenyl-methane sulfonyl fluoride. Phenyhnethane-sulfonyl fluoride inhibited the activities of both thrombin and the thrombin-like principle of Agkistrodon status venom markedly, although the effect on the former was greater than on the latter . Sulfonation can thus affect the activities of both thrombin and the thrombin-like principle of Agkistrodon status venom. From the above results, we may say that the sulfhydryl group, histidine and serine are essential for the activities of both thrombin and the thrombin-like principle of Agkistrodon status venom, while the carbonyl group, disulfide group, arginine and tyrosine are not important for their activities . A free amino group is essential for the activity of thrombin, but not for the thrombin-like activity of Agkistrodon status venom. Acknowledgements-This work was supported by the National Science Council Research Grant of the Republic of China. The authors express their sincere appt+xiation to Prof. C. Y. Lee, Dean of the College of Medicine, National Taiwan University for his invaluable advice. REFERENCES Bt oMaxcs:, H. (1958) Studies on the action of thrombic enzymes on bovine fibrinogen as measured by N-terminal analysis . Ark. Kem. 12, 321. Cwi.nwet,t., M. J. and Seeceas, W. H. (1965) Inhibition of prothrombin, thrombin and autoprothrombin with enzyme inhibitors. 79rromb. Dlath. haemorrh . 132, 373. CLwKe, H. T. (1932) The action of sulfite upon cystine. J. blot. Chem . 97, 235. ESNOUF, M . P . and Tutvwwx, G. W. (1967) The isolation and properties of the thrombin-like activity from Ancistrodon rhodartoma venom. Br. J. Hasmat . 13, 581. FAHRNEY, D. E. and Gow, A. M. (1963) Sulfonyl-fluoride as inhibitor of esterase. J. Am . them . Soc. 85, 997 . FRANREIrCONRAT, H. (1957) Methods for investigating the essential groups for enzyme activity. Meth . Enzymology 4, 256. Giwnnex, J. A. and Lwxt, K. (1956) The inhibition of thrombin by düsopropylphosphomfluoridate . .lrchs Bioohem. Biophys. 62, SOI . Gt .wnxea, J. A. and Lwtcr, K. (1958) The active site of thrombin . J. .Im. chsm. Soc. 80, 1263 . Hntoo, H. and Swtawrw, K. (1964) States of amino acid residues in proteins. III. Histidine residues in insulin, lysozyme, albumin and proteinases determined with a new reagent of diazonium-l-H-tetrazok . Biochlm. biophys. .lcta 86, 475. 7~OXJCON 197~ Yol. J2
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Jas, F. R., WNexe, E. C. W. and B©tT, L. V. (1965) A reagent for determination of `free tyrosyl' residues of protein. Biochemistryy, 4,1758. Mwxcmaex, E. and S~oERS, W. H. (19C~ Characteristics of the prethrombin subunit of prothrombin . ?fuomb. Diatlr . lulemorrb .l5, 633. M~~ravsfmu, A. and Sz~Tly K. (1967) Amino groups with different reactivities toward naphthoquinone sulfonic acid . l. Biochtm. 63, 1328 . Mn.l~e, K. D. and vl r~ Venvms, H. (1956) The effect of diisopropyl-fiuorophosphate on the proteinase and esterase activities of thrombin and on prothrombin and its activators. l, bioi. Clam. 223, 227. NAICAYA, K., HonnvlsHa, H. and Sluenre, K. (1967) Glyoxal as a reagent for discrimination of arginine residues . l. Biochem. 61, 345. NAiCAYA, K, and S1m~Te, K. (1967) Monochlorotrifluoroquinone as a new reagent for discrimination of amino groups . I. Biocbem. 61, 337. t7uverao, C., Hor~ct, J. S. and Tr~a, C, M. (1971) Purification and properties of the thrombin-like principle of Agkistrodon acutru venom and its comparison with bovine thrombin . 7lrromb. Diath. haemorrh. 26, 224. Scxoiz raurrN, G. and Sll~w, E. (1963) Direct evidence for the presence of histidine in the active center of chymotrypain . Biochumistr 2, 252. S~asRS, W. H., Hr~re, D., MNtcavl~tc, E., Iwuvovtc, N, and CALDWELL, M. J. (1965) Sensitivity of thrombin and autoprothrombin C to selected enzyme inhibitors . L{fe Sct. 4, 425. S~omes, W. H, and ~rx, H. P, (1942) Factors which influence the activity of purified thrombin . l. PhysioJ. 137, 348. Sxsw, E. (1965) Evidenz for an active center histidine in trypsin through use of a specific reagent l~hloro3-toaylamido-7-amino-2-heptanone, the cliloromethylketone derived from N-artoayl-l-lysine. Biochemistry 4, 2219.
TOXICON 1971 Yol. 11