Diamphotoxin. The arrow poison of the !Kung Bushmen

We have purified the arrow poison extracted from Diamphidia nigro-ornata pupae by the !Kung Bushmen of Southern Africa, and named it diamphotoxin. The toxin is a single chain polypeptide of Mr = 60,000 with an isoelectric point of pH 9.5. It blocks neuromuscular function and is cardiotoxic and hemolytic, and the minimum lethal dose for mice is 25 pg (less than 0.5 fmol). The toxin binds tightly to cells, permitting the resolution of two distinct phases. Erythrocytes exposed to toxin in the absence of divalent cations show no apparent lesion (phase I). After washing and addition of 1 mM Ca2+, there occurs a rapid efflux of K+ followed by the loss of hemoglobin (phase II). The pH optimum for phase I is pH 6.7 and for phase II pH 8.6. The action of the toxin is noncatalytic, requiring a solution concentration of approximately 65 toxin molecules/cell for hemolysis of sheep erythrocytes under standard conditions. Ca2+ ions induce a conformational change in the free, purified toxin molecule. We propose that this change also occurs in membrane-bound toxin. Hemolysis would result from the formation of channels permitting the diffusion of small cations.