Refolding of reduced short neurotoxins: circular dichroism analysis.

The four disulfide bonds of nine homologous short curare-like polypeptides are cleaved by reduced dithiothreitol. Air oxidation renaturations of the reduced compounds are followed by far-ultraviolet circular dichroism analysis, and the kinetics of refolding thus determined are compared. They indicate that three toxins refold 4--10 times more slowly than the six 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.