THE ISOLATION AND STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF SERINE PROTEINASES IN THE VENOM OF Bothrops jararaca

THESIS: S. M. de T. Serrano developed her thesis partly in the Department of Clinical Chemistry and Clinical Biochemistry at the University of Munich in Germany and partly in the Escola Paulista School of Medicine, UNIFESP, São Paulo, Brazil. This thesis was submitted for her Doctor of Philosophy degree in Molecular Biology publicly examined in the Escola Paulista School of Medicine, UNIFESP, São Paulo, SP, Brazil in 1994.

Advisors: Prof. Dr. Claudio A. M. Sampaio and Prof. Dr. Edwin Fink

ABSTRACT. Three functionally distinct serine proteinases -TL-BJ, KN-BJ and PA-BJ were isolated from the venom of Bothrops jararaca. They were isolated by ammonium sulfate precipitation followed by ion-exchange chromatography on DEAE-Sephacel, SP-Sephadex C-50, Mono Q and Mono S (FPLC), and reversed-phase HPLC. TL-BJ exhibits thrombin-like coagulant activity. It is an acidic glycoprotein and at least three electrophoretically different forms occur: TL-BJ 1, TL-BJ 2 and TL-BJ 3- with molecular masses ranging from 30,000 to 32,000. The pI values of these forms ranged from 3.9 a 4.1. The N-terminal amino acid sequences of the TL-BJ forms are also quite similar. However, the incubation with N-glycosidase F revealed that they differ in their carbohydrate moieties. These forms also vary in their specific activity on synthetic substrates and in their coagulant activity on plasma and fibrinogen. Specific coagulant activities of TL-BJ forms 1, 2, and 3 on fibrinogen were 16.8 NIH U/mg, 16.7 NIH U /mg and 20.8 NIH U/mg, respectively. KN-BJ, a kinin-releasing and coagulant enzyme, occurs in two forms -KN-BJ 1 and KN-BJ 2 with molecular masses 8,000 and 39,000 respectively, and pIs values ranging from 4.3 to 4.7. Both KN-BJ 1 and KN-BJ 2 have similar amino acid sequences, specific activities on synthetic substrates and both also release bradykinin from bovine L-kininogen. Their coagulant activities on fibrinogen are also similar (245 NIH U/mg and 219 NIH U/mg, respectively) but nearly 10 times higher than that of TL-BJ. PA-BJ caused 70% platelet aggregation on platelet rich plasma (PRP) at the concentration of 4x10 M, and 40% aggregation at the concentration of 10 M on washed platelet suspensions. PA-BJ is a basic glycoprotein and its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation. The protein has 232 amino acid residues which bear both an N- and an O-linked glycosilation sites. The determined amino acid sequence of PA-BJ shows a higher degree of similarity to that of snake venom thrombin-like enzymes, trypsin and tissue kallikrein, than to that of thrombin itself. Kinetic parameters of TL-BJ, KN-BJ and PA-BJ were determined for several chromogenic substrates. The most sensitive of these for measuring the amidolytic activity of these snake venom proteinases were the thrombin substrate D-Phe-Pip-Arg-pNA and the glandular kallikrein substrate D-Val-Leu-Arg-pNA. KN-BJ also presents high activity on the plasma kallikrein substrate Bz-Pro-Phe-Arg-pNA. Amidolytic and biological activities of TL-BJ, KN-BJ and PA-BJ were affected by phenylmethylsulfonyl fluoride (PMSF). Benzamidine derivatives which are competitive, reversible inhibitors of trypsin-like serine proteinases, also inhibited the enzymatic activity of these B. jararaca venom enzymes. Among the various compounds tested Na-(2-naphthylsulfonyl-glycil)-4-amidinophenylalanine piperidide) (NAPAP) exerted a more pronounced inhibitory activity.

CORRESPONDENCE TO:

S. M. de T. SERRANO - Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brasil, 1500, CEP: 05503-900, São Paulo, SP, Brasil.

Fone/Fax: [phone], E-mail: [e-mail]

Publication Dates

• Publication in this collection
  17 Sept 1999
• Date of issue
  1999