Characterization of the thiol/disulfide chemistry of neurohypophyseal peptide hormones by high-performance liquid chromatography

Article abstract of DOI:10.1021/ac00069a019

Methodology is described for characterization of the kinetics and equilibria of thiol/disulfide interchange reactions of the disulfide bonds in the neurohypophyseal peptide hormones arginine vasopressin and oxytocin and the related peptides pressinoic acid and tocinoic acid. Thiol/disulfide interchange reaction mixtures are analyzed by reversed-phase high-performance liquid chromatography. The effect of mobile-phase composition and pH on the HPLC capacity factors for the native disulfide and reduced dithiol forms of each peptide was examined. In each case, the capacity factor decreases as the acetonitrile content of the mobile phase increases. For each disulfide/dithiol peptide pair, the capacity factor is larger for the dithiol form of the peptide, indicating that the hydrophobic side chains of the linear peptide are more accessible for interaction with the hydrophobic stationary phase. To illustrate application of the methodology, rate and equilibrium constants are reported for the thiol/disulfide interchange reactions of cysteine with arginine vasopressin at pH 7.0. Cysteine reacts with arginine vasopressin to form two mixed disulfides, which in turn react with another molecule of cysteine to give the dithiol form of arginine vasopressin and cystine. Rate and equilibrium constants were determined for each step by analysis of reaction mixtures by HPLC. The results are compared to rate and equilibrium constants for reaction of cysteine with oxidized glutathione.