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

Abstract

The crude venom of Bungarus fasciatus has been fractionated by column chromatography, and the fractionation characteristics of three different resins have been compared. A minimum of 21 fractions can be identified under optimum conditions on Bio-Gel CM-30. Of the major fractions tested for neurotoxic activity, three showed postsynaptic (alpha) and four showed presynaptic (beta) neurotoxic activity. The major protein component (an alpha-neurotoxin) has an isoleucyl N terminus and a calculated molecular weight of 14 200 based on amino acid composition. This main component contains 127 amino acid residues including 16 cysteine residues. A second less abundant alpha-neurotoxin of similar molecular weight has a methionyl N terminus. The isoelectric points of these toxins are 9.1 and 8.8, respectively. A third fraction also has postsynaptic (alpha) activity. Four other, very basic proteins have presynaptic (beta) activity. Their apparent molecular weights are approximately 10 800 (two fractions), 13 100, and 19 100 as determined by sodium dodecyl sulfate gel electrophoresis. All alpha-toxin fractions showed a high tendency to aggregate in aqueous media; however, the presence of L-cysteine in molar excess prevents dimer formation. In the absence of L-cysteine, freeze/thaw cycling of aqueous solutions of alpha-toxins invariably leads to the formation of dimers which can be dissociated only under reducing conditions (beta-mercaptoethanol). Conversely, only one out of four beta-toxins examined tended to form dimers.