Isolation, complete amino acid sequence and characterization of a previously unreported post-synaptic neurotoxin – AlphaN3, from the venom of Bungarus candidus

Abstract

The Malayan krait ( Bungarus candidus) is one of the medically most important snake species in Southeast Asia. The venom from this snake has been shown to posses both presynaptic and post-synaptic neurotoxins. We have isolated a previously uncharacterized post-synaptic neurotoxin – alphaN3 from the venom of B. candidus. Isolation of the toxin was achieved in three successive chromatography steps – gel filtration on a Sephadex G75 column, followed by ion exchange chromatography (Mono-S strong cationic exchanger) and a final reverse-phase chromatography step (PRO-RPC C18 column). Purified toxin alphaN3 was shown to have an apparent molecular weight of ∼7 to 8 kDa on SDS–PAGE. The complete amino acid sequence of toxin alphaN3 was determined by Edman degradation and was found to share a high degree of homology with known post-synaptic neurotoxins (93% with alpha-bungarotoxin from Bungarus multicinctus, 50% with alpha cobratoxin from Naja kaouthia). The intravenous LD 50 of toxin alphaN3 was determined to be 0.16 ± 0.09 μg/g in mice which is comparable to alpha-bungarotoxin from B. multicinctus. Experiments with isolated nerve-muscle preparations suggested that toxin alphaN3 was a post-synaptic neurotoxin that produced complete blockade of neuromuscular transmission by binding to nicotinic acetylcholine receptors.