E L S E V I E R lmmunopharmacology 32 (1996) 163-165

Immunopharmacology

Kininase activities of some snake venoms

Abdusalam M.H. A1-Joufi a, Sandra Andrews b, Graham S. Bailey Department of Science, Sanaa Unit:ersitv, Sanaa. Yemen

b Department ~ Biological and Chemical Sciences, Unil'ersity ~f Essex. Colehester. Essex. UK

b. *

Keywords: Snake: Venom; Kininase

1. Introduction

Certain crotalid venoms are known to possess both kinin-releasing (kininogenase, kallikrein-like) and kinin-degrading (kininase) activities (Deutsch and Diniz, 1955). Other snake venoms have been assayed for the presence of kinin-releasing enzymes but not directly for kinin-degrading enzymes (Oshima et al., 1969; Mebs, 1970). Chymotrypsin is a potent kininase (Schachter, 1969) but chymotrypsin-like en- zymes have been reported to be absent from snake venoms (Tu, 1977). This paper describes a compara- tive study of kininase and chymotrypsin-like activi- ties in some crotalid and viperid venoms.

2. Materials and methods

Lyophilized samples of the venoms of Bitis ari- etans (Puff Adder), Vipera ammodytes ammodytes (Southern European Sand Viper), Vipera russelli (Russell's Viper), Crotalus atrox (Western Dia- mondback Rattlesnake), Crotalus adamanteus (East- ern Diamondback Rattlesnake), Crotalus horridus

atricaudatus (Canebrake Rattlesnake), Agkistrodon

pisci~'orus pisci~,orus (Eastern Cottonmouth Moc- casin), Agkistrodon piscit,orus leukostoma (Western Cottonmouth Moccasin), and Bothrops atrox (Fer- de-lance) were purchased from Sigma Chemical Company. Incubation of bradykinin with bovine chy- motrypsin or crude venom was carried out at 20°C in 0.01 M sodium phosphate buffer (pH 8.0). Analysis by TLC was performed using silica gel plates (Sigma T-6145) developed with n-butanol/glacial acetic acid/water (2:1 : 1, v /v) . After drying, the plates were immersed in 20% (w/v ) trichloracetic acid solution for 10 rain and then stained with 0.3% (w/v ) Serva Blue W solution (in distilled water) for 5 rain (EI-Thaher and Bailey, 1994). Bradykinin fragments were identified by comparison with stan- dard partial bradykinin peptides run and detected under identical conditions. Chymotrypsin-like activ- ity was measured at 25°C by a titrimetric assay using 10 mM N-acetyl-L-tyrosine ethyl ester (ATEE) as substrate (Wilcox, 1970).

3. Results and discussion

* Corresponding author.

Incubation of bradykinin (50 /xg) with chy- motrypsin (1 /.tg) at pH 8.0 and 20°C produced a number of fragments which were identified by TLC.

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164 A.M.H. AI-Joufi et al. / lmmunopharmacology 32 (1996) 163-165

Table 1 Cleavage of bradykinin by 30 minute incubation with crude venoms analyzed by thin layer chromatography

Venom R F Identify of Fragment

B. arietans 0.46 Bradykinin 1-5 V.a. ammodytes 0.31 des-Arg9-bradykinin

0.48 Arginine C. atrox 0.31 des-Arg'Lbradykinin

0.48 Arginine C. adamanteus 0.31 des-Argg-bradykinin

0.48 Arginine C.h. atricaudatus 0.31 des-Argg-bradykinin

0.48 Arginine A.p. piscir, orus 0.31 des-Argg-bradykinin

0.48 Arginine A.p. leucostoma 0.29 Bradykinin V. russelli 0.29 Bradykinin B. atrox 0.29 Bradykinin

The fragmentation pattern of bradykinin produced by the action of chymotrypsin as seen on TLC was consistent with the known behaviour of the enzyme towards the peptide. Chymotrypsin rapidly cleaves the PheS-Arg 9 peptide bond and that hydrolysis is followed by cleavage of the PheKSer 6 peptide bond to give the bradykinin 1-5 fragment (Arg-Pro-Pro- Gly-Phe) (Sampaio et al., 1976). In our hands the bradykinin 6 - 8 fragment (Ser-Pro-Phe) could not be detected by the Serva blue stain.

Incubation of bradykinin (50 /zg) at pH 8.0 and 20°C with a number of crude venoms (400 /xg) for

Table 3 Chymotrypsin-like activity of crude venoms

Venom Specific activity ( #mol /min per mg venom)

B. arietans 0.59 _+ 0.01 V.a. ammodvtes 0.39 _+ 0.02 C. atrox 0.28 _+ 0.03 C. adamanteus 0.20 _+ 0.0 I C.h. atricaudatus 0.19 _+ 0.02 A.p. pisci~,orus 0.32 _+ 0.01 A.p. leucostoma O. 13 +_ 0.01 14 russelli 0.05 _+ 0.01 B. atrox 0.02 _+ 0.01

Results are expressed as mean_+ SEM (n = 9).

30 min and 2 h produced various fragments which were identified by TLC and the results are shown in Tables 1 and 2, respectively.

It can be seen that all of the venoms tested showed kininase activity. The most active venom was that of B. arietans and the venoms of A.p. leucostoma, V. russelli and B. atrox were the least active. The cleavage patterns were indicative of chy- motryptic activity although the kininase activity of the three least active venoms could be due to the presence of carboxypeptidase B-like enzymes.

So the chymotrypsin-like activity of each venom was quantitatively measured by a titrimetric assay at pH 8.0 using ATEE as substrate. The results are presented in Table 3.

It can be seen that chymotryptic activities of the

Table 2 Cleavage of bradykinin by 2-h incubation with crude venoms analyzed by thin-layer chromatography

Venom R F Identify of fragment

B. arietans 0.46 Bradykinin 1-5 V.a. ammodytes 0.46 Bradykinin 1-5 C. atrox 0.46 Bradykinin 1-5 C. adamanteus 0.46 Bradykinin 1-5 C.h. atricaudatus 0.46 Bradykinin 1-5 A.p. pisci~,orus 0.46 Bradykinin 1-5 A.p. leucostoma 0.31 des-Argg-bradykinin

0.48 Arginine V. russelli 0.3 l des-Argg-bradykinin

0.48 Arginine B. atrox 0 . 3 1 des-Argg-bradykinin

0.48 Arginine

Table 4 pH optima and pH stabilities of the crude venoms

chymotryptic activities of the

Venom pH % activity remaining optimum after 72 h

at pH 7.4 at pH 5.0

B. arietans 8.5 78 88 V.a. ammodytes 8.3 69 87 C. atrox 8.2 57 63 C. adamanteus 8.2 40 71 C.h. atricaudatus 8.0 76 80 A.p. piscicorus 8.0 63 91 A.p. leucostoma 8.2 58 77 V. russelli 8.1 86 91 B. atrox 8. I 88 95

A.M.H. Al-Joufi et al. / Immunopharmacology 32 ( ! 996) 163-165 165

crude venoms showed the same order of magnitude as the kininase activities. Thus it is likely that chy- motrypsin-like enzymes are responsible for much of the kininase activity exhibited by the venoms.

The pH optima of the chymotryptic activities of the crude venoms and an indication of their stabili- ties at pH 7.4 and pH 5.0, both at 20°C, are recorded in Table 4.

It can be seen that the chymotryptic activities were more stable when the venoms were kept at pH 5.0 than at pH 7.4. Thus purification of the kininase enzymes should be carried out at the lower pH.

It has previously been reported that snake venoms do not hydrolyse tyrosine esters and thus do not contain chymotrypsin-like enzymes (Tu, 1977). However, those earlier reports were based on the results from spectrophotometric assays where much lower concentrations of ester were used than in the titrimetric assay (Walsh and Wilcox, 1970; Schwert and Takenaka, 1955). Much lower levels of es- terolytic activity were seen when the titrimetric assay was performed at 1 mM substrate concentration (AI- Joufi and Bailey, unpublished data). Elucidation of the reason(s) for those differences (substrate activa- tion, high values of K m, presence of competitive inhibitors) will form part of the characterization of the enzymes when they have been isolated from the crude venoms.

References

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