Refolding of reduced short neurotoxins: circular dichroism analysis

Abstract

The 4 disulfide bonds of 9 homologous short curare-like polypeptides [Toxin .alpha.1 from Naja nigricollis, cobrotoxin from N. naja atra, toxin d from N. melanoleuca, toxin .alpha. from N. naja philippinensis, erubatoxins a and b Laticauda semifasciata, toxin IV from Hemachatus haemachatus, toxin a from Astrotia stokesii and toxin b from Aipysurus laevis] are cleaved by reduced dithiothreitol. Air oxidation renaturations of the reduced compounds are followed by far-UV circular dichroism analysis, and the kinetics of refolding thus determined are compared. They indicate that 3 toxins refold 4-10 times more slowly than the 6 others. It is shown that a significant difference between the refolding kinetics still subsists when renaturations are made in the presence of various concentrations of thiol-disulfide exchange reagents or at various pH values. From an examination of the toxin sequences, it is proposed that a single additional amino acid insertion is responsible for the difference in the observed kinetics. This proposal is supported by temperature studies of renaturation kinetics.