Purification and biochemical characterization of an 11 000-dalton β-bungarotoxin

Abstract The chromatographic separation and biochemical characterization of a β-bungarotoxin is described. This toxin is isolated as the most basic eluting protein of Bungarus multicinctus venom when separated by column chromatography on CM-Sephadex C-25. The protein migrated as a single band on pH 4.3 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of this toxin was estimated to be 10 000 ± 1000 by analytical sedimentation analysis. This value was consistent with the electrophoretic mobility of the toxin in SDS-polyacrylamide gels. The amino acid composition of this 11 000-dalton β-bungarotoxin was similar to that of the 22 000-dalton β-bungarotoxin previously reported (Lee et al. (1972) J. Chromatogr. 72, 71–82; Kelly, R.B. and Brown, III, F.R. (1974) J. Neurobiol. 5, 135–150; Kondo et al. (1978) J. Biochem. Tokyo 83, 91–99), suggesting that the 11 000-dalton toxin may be one of the polypeptide chains of the large toxin. The 11 000-dalton β-bungarotoxin was toxic to mice when injected intravenously. Animals that received lethal doses exhibited hyperexcitability followed by ataxia, convulsions, and death. The minimum lethal dose was 0.12 μg/g body weight. This β-bungarotoxin exhibited Ca 2+ -dependent phospholipase A activity comparable to that of the 22 000-dalton β-bungarotoxin. The enzyme exhibited phospholipid substrate specificity in the rank order of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. The enzyme activity was destroyed by boiling for 3 min at pH 8.6. In addition, an enzymatically inactive quantity of the 11 000-dalton toxin, equivalent to five times the minimum lethal dose of enzymatically active toxin, was not lethal when injected into mice. To test whether phospholipase A activity is responsible for lethality, bee venom phospholipase A 2 was injected into mice at similar and greater concentrations with no toxic effect. Thus, while phospholipase A activity may be required for the lethal effect of the 11 000-dalton β-bungarotoxin, the specificity of action of the toxin is not determined by its enzyme activity.