Purification and N-terminal sequencing of two presynaptic neurotoxic PLA(2), neuwieditoxin-I and neuwieditoxin-II,from Bothrops neuwiedi pauloensis (jararaca pintada) venom

Abstract

Two presynaptic phospholipases A(2) (PLA(2)), 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 (mu Bondapak 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 10 mu g/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.