Received: April 12, 2006.

Accepted: June 7, 2006.

Abstract published online: August 31, 2006.

Full paper published online: February 28, 2007.

PURIFICATION AND N-TERMINAL SEQUENCING OF TWO PRESYNAPTIC NEUROTOXIC PLA2, NEUWIEDITOXIN-I AND NEUWIEDITOXIN-II, FROM Bothrops neuwiedi pauloensis (JARARACA PINTADA) VENOM

BORJA-OLIVEIRA C. R. (1), KASSAB B. H. (2), SOARES A. M. (3), TOYAMA M. H. (2), GIGLIO J. R. (4), MARANGONI S. (2), RE L. (5), RODRIGUES-SIMIONI L. (1)

(1) Department of Pharmacology, School of Medical Sciences, State University of Campinas, UNICAMP, Campinas, São Paulo State, Brazil; (2) Department of Biochemistry, Institute of Biology, State University of Campinas, UNICAMP, Campinas, São Paulo State, Brazil; (3) Department of Clinical, Toxicological and Bromatological Analyses, School of Pharmaceutical Sciences, FCFRP, University of São Paulo, USP, Ribeirão Preto, São Paulo State, Brazil; (4) Department of Biochemistry and Immunology, School of Medicine, University of São Paulo, USP, Ribeirão Preto, São Paulo State, Brazil; (5) Institute of Experimental and Clinical Sciences, Laboratory of Pharmacology, University of Ancona, Ancona, Italy.

ABSTRACT: Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (mBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10mg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0% (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71% homology with bothropstoxin-II and 54% homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92% homology with Basp-III and 62% homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.

KEY WORDS: chick biventer cervicis, loose patch clamp, nerve-muscle preparation, neuromuscular junction, neurotoxicity, PLA2 neurotoxin, presynaptic action, Bothrops neuwiedi pauloensis, Neuwieditoxin-I, Neuwieditoxin-II.

CONFLICTS OF INTEREST: There is no conflict.

CORRESPONDENCE TO:

LÉA RODRIGUES-SIMIONI, Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6111, 13083-970, Campinas, SP, Brasil. Phone: 55-19-3788-9534; Fax: 55-19-3289-2968, Email: simioni@unicamp.br.