Kappa-neurotoxins: heterodimer formation between different neuronal nicotinic receptor antagonists

The kappa-neurotoxins are a family of snake venom polypeptides that are competitive antagonists of acetylcholine at a variety of neuronal nicotinic receptors. We have previously determined that kappa-bungarotoxin, purified from the venom of Bungarus multicinctus, exists in solution entirely as a dimer of identical subunits. We now report that the three other known kappa-neurotoxins, namely, kappa 2-bungarotoxin and kappa 3-bungarotoxin from Bungarus multicinctus and kappa-flavitoxin from Bungarus flaviceps, also self-aggregate in solution. Furthermore, when two different kappa-neurotoxins are mixed, a heterodimer species spontaneously forms and reaches an equilibrium with the two homodimers after which 40-50% of the protein exists as the heterodimer. A cation-exchange high-pressure liquid chromatography procedure is described which readily separates kappa-neurotoxin heterodimers from the homodimers. Sedimentation equilibria experiments give an Mr = 15,500 +/- 1000 for kappa-flavitoxin and an Mr = 14,500 +/- 700 for a mixture of kappa-bungarotoxin and kappa-flavitoxin. Since the subunit molecular weights of kappa-bungarotoxin and kappa-flavitoxin are respectively 7313 and 7242, self-aggregation of these toxins in solution results in a preponderance of kappa-neurotoxin dimers. The stoichiometry of the heterodimer formed by kappa-bungarotoxin and kappa-flavitoxin is 1:1, as determined by amino acid sequence analysis. After isolation, the kappa-neurotoxin heterodimer partially dissociates and again reaches equilibrium with the homodimers, a process which requires 2-4 h at 23 degrees C. The ability to self-aggregate to form heterodimers and homodimers thus appears to be a common property of the kappa-neurotoxins.