Pharmacological and partial biochemical characterization of Bmaj-9 isolated from Bothrops marajoensis snake venom

Galbiatti C (1), Rocha T (2), Randazzo-Moura P (1), Ponce-Soto LA (3), Marangoni S (3), Cruz-Höfling MA (2), Rodrigues-Simioni L (1)

(1) Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo State, Brazil; (2) Department of Histology and Embryology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, São Paulo State, Brazil; (3) Department of Biochemistry, Institute of Biology, State University of Campinas (UNICAMP), Campinas, São Paulo State, Brazil.

Abstract: Bmaj-9, a basic PLA2 (13679.33 Da), was isolated from Bothrops marajoensis snake venom through only one chromatographic step in reversed phase HPLC on μ-Bondapak C-18 column. The amino acid composition showed that Bmaj-9 had a high content of Lys, His, and Arg, typical of a basic PLA2. The sequence of Bmaj-9 contains 124 amino acid residues with a pI value of 8.55, such as DLWQWGQMIL KETGKLPFSY YTAYGCYCGW GGRGGKPKAD TDRCCFVHDC, revealing a high homology with Asp49 PLA2 from other snake venoms. It also exhibited a pronounced phospholipase A2 activity when compared with crude venom. In chick biventer cervicis preparations, the time for 50% and 100% neuromuscular paralysis was respectively (in minutes): 110 ± 10 (1 µg/mL); 40 ± 6 and 90 ± 2 (5 µg/mL); 30 ± 3 and 70 ± 5 (10 µg/mL); 42 ± 1 and 60 ± 2 (20 µg/mL), with no effect on the contractures elicited by either exogenous ACh (110 µM) or KCl (20 mM). Bmaj-9 (10 µg/mL) neither interfered with the muscular response to direct electrical stimulation in curarized preparations nor significantly altered the release of CK at 0, 15, 30 and 60 minutes incubations (27.4 ± 5, 74.2 ± 8, 161.0 ± 21 and 353.0 ± 47, respectively). The histological analysis showed that, even causing blockade at the maximum dosage (5 µg/mL), the toxin does not induce significant morphological alterations such as necrosis or infiltration of inflammatory cells. These results identified Bmaj-9 as a new member of the basic Asp49 PLA2 family able to interact with the motor nerve terminal membrane, thereby inducing a presynaptic neuromuscular blockade.

Key words: Bothrops marajoensis, snake venom, Asp49 PLA2, neuromuscular blockade.

ACKNOWLEDGMENTS

The authors are grateful to Mr. Paulo A. Baldasso and Mr. Gildo B. Leite for technical assistance. This work was supported by the National Council for Scientific and Technological Development (CNPq) and was part of a master’s thesis developed by Charlene Galbiatti.

COPYRIGHT

© CEVAP 2012

SUBMISSION STATUS

Received: June 22, 2011.

Accepted: November 4, 2011.

Abstract published online: November 28, 2011.

Full paper published online: February 28, 2012.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

FINANCIAL SOURCE

The National Council for Scientific and Technological Development (CNPq) provided the financial grants.

ETHICS COMMITTEE APPROVAL

The present study was approved by the Institutional Committee for Ethics in Animal Use (CEUA/IB/Unicamp, protocol number 1027-1). Moreover, all tests were in accordance with the guidelines of the Brazilian Society of Laboratory Animal Science (SBCAL/COBEA).

CORRESPONDENCE TO

Léa Rodrigues-Simioni, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Rua Tessália Vieira de Camargo, 126, Cidade Universitária "Zeferino Vaz", Barão Geraldo, Campinas, SP, 13083-887, Brasil. Phone: +55 19 3521 9533. Fax: +55 19 3289 2968. Email: simioni@unicamp.br.