Received: October 17, 2006.

Accepted: January 15, 2007.

Abstract published online: January 23, 2007.

Full paper published online: August 31, 2007.

PURIFICATION AND FUNCTIONAL CHARACTERIZATION OF TWO FIBRINOGENOLYTIC ENZYMES FROM Bothrops alternatus VENOM

COSTA J. O. (1), PETRI C. B. (1), HAMAGUCHI A. (1), HOMSI-BRANDEBURGO M. I. (1), OLIVEIRA C. Z. (2), SOARES A. M. (2), OLIVEIRA F. (3)

(1) Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, Minas Gerais State, Brazil; (2) Department of Clinical, Toxicological and Bromatological Analyses, School of Pharmaceutical Sciences, University of São Paulo, USP, Ribeirão Preto, São Paulo State, Brazil; (3) Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Minas Gerais State, Brazil.

ABSTRACT: Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aa-chain and more slowly the Bb-chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the g-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

KEY WORDS: snake venom, Bothrops alternatus, metalloproteases, functional characterization, fibrinogenolytic activity, defibrinogenation in vivo.

CONFLICTS OF INTEREST: There is no conflict.

CORRESPONDENCE TO:

FÁBIO OLIVEIRA, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, 38400-902, Uberlândia-MG, Brasil. Phone: 55 34 3218 2200. Fax: 55 34 3218 2247. Email: foliveira@umuarama.ufu.br.