Oxidation of the sulfhydryl forms of insulin A-chain and B-chain

A modified procedure for the preparation of the S-sulfonates of the A- and B-chains of insulin and their conversion to the sulfhydryl forms by tri-n-butylphosphine is described. Air oxidation of the sulfhydryl forms of the A-chain in dilute solution (0.2 mg/ml) either in the presence or absence of urea at pH 9.0 yields primarily monomeric, intrachain disulfides. Similar treatment of the reduced B-chain yield monomeric, intrachain disulfide in 7 M urea but a large number of oligomeric, interchain disulfides in the absence of urea. Electrolytic reduction of insulin in 7 M urea of pH 8.5, followed by oxidation of the sulfhydryls in dilute solution in 7 M urea at pH 9.0 yields primarily a mixture of the monomeric, intrachain disulfides of the A-chain and of the B-chain which can be separated by chromatography on Sp-Sephadex in acidic urea. The rate of the oxidation of the sulfhydryls of the two separate chains was much slower and less complete than that reported for the two chains crosslinked by the carbonylbismethionyl residue.