Synthesis and characterization of μ‐conotoxin IIIa

Abstract

.mu.-Conotoxin IIIa, a voltage-dependent sodium channel neurotoxin, has been synthesised using solid-phase peptide synthesis employing 9-fluorenylmethoxycarbonyl chemistry. After cleavage from resin, the peptide was isolated by reverse-phase HPLC and then the six acetamidomethyl groups were removed by treatment with mercuric acetate. The reduced product so formed was purified by reverse-phase HPLC. Protocols were developed to optimize the oxidation of the cysteine residues to form disulphide bonds. Protocols employed using air oxidation together with 2-mercaptoethanol were the most effective. As complete oxidation was never obtained the oxidised peptide was purified by reverse-phase HPLC. The activity of our products was monitored using [3H]saxitoxin binding to eel membranes. The oxidised product was able to completely block [3H]saxitoxin binding in a competitive manner. Lineweaver-Burke analysis of [3H]saxitoxin binding gave a Ki of 1.5 nM, IC50 was determined as 26.6 nM. It was also shown that the pure synthetic .mu.-conotoxin IIIa had the same retention time on reverse-phase HPLC as the natural conotoxin IIIa. Thus an active toxin has been synthesised that can be used to probe sodium channels.