Neurotoxins from Bungarus fasciatus venom: a simple fractionation and separation of .alpha.- and .beta.-type neurotoxins and their partial characterization

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Abstract
The crude venom of B. fasciatus was fractionated by column chromatography, and the fractionation characteristics of 3 different resins was compared. A minimum of 21 fractions were identified under optimum conditions on Bio-Gel CM-30. Of the major fractions tested for neurotoxic activity, 3 showed postsynaptic (.alpha.) and 4 showed presynaptic (.beta.) neurotoxic activity. The major protein component (an .alpha.-neurotoxin) had an isoleucyl N terminus and a calculated MW of 14,200 based on amino acid composition. This main component contained 127 amino acid residues including 16 Cys [cysteine] residues. A 2nd less abundant .alpha.-neurotoxin of similar MW had a methionyl N terminus. The isoelectric points of these toxins were 9.1 and 8.8, respectively. A 3rd fraction also had postsynaptic (.alpha.) activity. Four other basic proteins had presynaptic (.beta.) activity. Their apparent MW were .apprx. 10,800 (2 fractions), 13,100 and 19,100 as determined by sodium dodecylsulfate gel electrophoresis. All .alpha.-toxin fractions showed a high tendency to aggregate in aqueous media; the presence of L-Cys in molar excess prevented dimer formation. In the absence of L-Cys, freeze/thaw cycling of aqueous solutions of .alpha.-toxin invariably led to the formation of dimers which could be dissociated only under reducing conditions (.beta.-mercaptoethanol). Only 1 of 4 .beta.-toxins formed dimers.

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