Purification and n-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom

• Published in: The journal of venomous animals and toxins including tropical diseases Vol. 13; no. 1; pp. 103 - 121
• Main Authors: Borja-Oliveira, C. R.(State University of Campinas School of Medical Sciences Department of Pharmacology), Kassab, B. H.(State University of Campinas Institute of Biology Department of Biochemistry), Soares, A. M.(University of São Paulo FCFRP School of Pharmaceutical Sciences), Toyama, M. H.(State University of Campinas Institute of Biology Department of Biochemistry), Giglio, J. R.(University of São Paulo School of Medicine Department of Biochemistry and Immunology), Marangoni, S.(State University of Campinas Institute of Biology Department of Biochemistry), Re, L.(University of Ancona Laboratory of Pharmacology Institute of Experimental and Clinical Sciences), Rodrigues-Simioni, L.(University of São Paulo FCFRP School of Pharmaceutical Sciences)
• Format: Journal Article
• Language: English
• Published: Centro de Estudos de Venenos e Animais Peçonhentos - CEVAP, Universidade Estadual Paulista - UNESP 2007; Centro de Estudos de Venenos e Animais Peçonhentos; SciELO
• Subjects: Bothrops neuwiedi pauloensis; chick biventer cervicis; loose patch clamp; nerve-muscle preparation; neuromuscular junction; neurotoxicity; Neuwieditoxin-I; Neuwieditoxin-II; PLA2 neurotoxin; presynaptic action; TOXICOLOGY; TROPICAL MEDICINE; Bothrops neuwiedi pauloensis; neurotoxicity; Neuwieditoxin-I; loose patch clamp; presynaptic action; neuromuscular junction; Neuwieditoxin-II; chick biventer cervicis; nerve-muscle preparation; PLA2 neurotoxin
• ISSN: 1678-9199; 1678-9199
• DOI: 10.1590/S1678-91992007000100008

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 (µBondapak 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 10µg/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&plusmn;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.