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

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±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.