Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

Barros LC (1, 2), Soares AM (3), Costa FL (4), Rodrigues VM (4), Fuly AL (5), Giglio JR (6), Gallacci M (7), Thomazini-Santos IA (1), Barraviera SRCS (2, 8), Barraviera B (1, 2), Ferreira Junior RS (1, 2)

(1) Department of Tropical Diseases and Imaging Diagnosis, Botucatu Medical School, São Paulo State University (UNESP - Univ Estadual Paulista), Botucatu, São Paulo State, Brazil; (2) Center for the Study of Venoms and Venomous Animals, São Paulo State University (UNESP - Univ Estadual Paulista), Botucatu, São Paulo State, Brazil; (3) Department of Clinical, Toxicological and Bromatological Analysis, School of Pharmaceutical Sciences, University of São Paulo, USP, Ribeirão Preto, São Paulo State, Brazil; (4) Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, Minas Gerais State, Brazil; (5) Department of Cellular and Molecular Biology, Institute of Biology, Fluminense Federal University, UFF, Niterói, Rio de Janeiro State, Brazil; (6) Department of Biochemistry and Immunology, Medical School of Ribeirão Preto, University of São Paulo, USP, Ribeirão Preto, São Paulo State, Brazil; (7) Department of Pharmacology, Institute of Biology, São Paulo State University (UNESP - Univ Estadual Paulista), Botucatu, São Paulo State, Brazil; (8) Department of Dermatology, Botucatu Medical School, São Paulo State University (UNESP - Univ Estadual Paulista), Botucatu, São Paulo State, Brazil

Abstract: Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn2+, Cu2+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.

Key words: gyroxin, neurotoxicity, coagulant activity, Crotalus durissus terrificus, serine proteinase.

COPYRIGHT

© CEVAP 2011

SUBMISSION STATUS

Received: June 24, 2010.

Accepted: October 26, 2010.

Abstract published online: November 5, 2010.

Full paper published online: February 28, 2011.

CONFLICTS OF INTEREST

There is no conflict.

FINANCIAL SOURCE

The State of São Paulo Research Foundation (FAPESP) provided the financial grants (process number 2007/05159-7).

ETHICS COMMITTEE APPROVAL

The present study was approved by the Ethics Committee on Animal Experimentation of the Botucatu Medical School, UNESP.

CORRESPONDENCE TO

RUI SEABRA FERREIRA JÚNIOR, Centro de Estudos de Venenos e Animais Peçonhentos, CEVAP-UNESP, Caixa Postal 577, Botucatu, 18618-000, SP, Brasil. Phone: +55 14 3814 54446. Email: rseabra@cevap.org.br.