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ISSN 1672-9145                                                Acta Biochim Biophys Sin 2004, 36(12): 798–802                                                  CN 31-1940/Q

Purification and Characterization of Jararassin-I, A Thrombin-like Enzyme from Bothrops jararaca Snake Venom

Débora F. VIEIRA1, Leandra WATANABE1, Carolina D. SANT´ANA2,3, Silvana MARCUSSI2,4, Suely V. SAMPAIO3, Andreimar M. SOARES2*, and Raghuvir K. ARNI1*

 

1Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista,

UNESP, 15054-000, São José do Rio Preto, SP, Brazil;

2Unidade de Biotecnologia, Universidade de Ribeirão Preto, UNAERP, Ribeirão Preto, SP, Brazil;

3Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, Universidade de São Paulo, USP, Ribeirão Preto, SP, Brazil;

4Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, USP, Ribeirão Preto, SP, Brazil

 

Abstract        A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bb chain of fibrinogen while the Aa chain and g chain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bb chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mm´0.2 mm´0.2 mm) and used for X-ray diffraction experiments.

 

Key words        snake venom; Bothrops jararaca; serine protease thrombin-like; fibrinogenolytic activity; crystallization

 

 

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Received: September 22, 2004        Accepted: October 29, 2004

Abbreviations: SVTLE, snake venom thrombin-like enzyme; SDS, sodium dodecyl sulphate; EDTA, ethylene diaminetetraacetic acid

*Corresponding authors:

Andreimar M. SOARES: Tel, 55-16-603-6892; Fax, 55-16-603-7030; E-mail, [email protected] 

Raghuvir K. ARNI: E-mail, [email protected]