Isolation of “ceruleotoxin” from Bungarus fasciatus venoms

Abstract

Ceruleotoxin is a potent neurotoxin which irreversibly blocks the neuromuscular transmission at a postsynaptic level, without preventing the binding of acetylcholine to its receptor. We have originally purified this toxin from a batch of venom obtained from the Pasteur Institute, which was thought to be Bungarus caeruleus venom. Recently Noble and co-workers have observed that the protein composition of Bungarus caeruleus provided by Miami Serpentarium significantly differed from that of the Pasteur Institute batch, which they concluded therefore to be of a different origin. In order to clarify this point, the venom composition of various Bungarus species from several origins have been analysed by electrophoresis and by electrofocusing on polyacrylamide gels. Although individual variations exist between samples of the same snake species, the venom from Bungarus caeruleus, Bungarus fasciatus and Bungarus multicinctus possess distinct and characteristic protein compositions. The results of this study allowed us to identify unambiguously the batch used to purify the ceruleotoxin, as a Bungarus fasciatus venom. We identified a neurotoxin similar to ceruleotoxin in each of the five samples of Bungarus fasciatus venoms that we tested. On the contrary we did not detect such a toxin either in Bungarus caeruleus or in Bungarus multicinctus venoms. All purified ceruleotoxins are acidic proteins with a high toxicity (their LD 50 by intravenous injection in mice are from 0.04 to 0.06 mg per kg), which irreversibly block the postsynaptic response of Electrophorus electricus electroplaque to cholinergic agonists. They also possess a phospholipase A 2 activity (200 nmoles of egg lecithins hydrolysed per min per mg of protein). In this respect, ceruleotoxin is analogous to crotoxin and β-bungarotoxin.