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Abstract:
μ‐Conotoxin IIIa, a voltage‐dependent sodium channel neurotoxin, has been synthesised using solid‐phase peptide synthesis employing 9‐fluorenylmethoxycarbonyl chemistry. After cleavage from the 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 acitivity 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 μ‐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.
