Thiol-disulfide interchange reactions between serum albumin and disulfides

Abstract

Thiol-disulfide interchange between serum albumins and l-cystine or lysine vasopressin was studied by p- chloromercuribenzoate (PCMB) titration and by incubation with radioactive disulfides followed by separation of reaction products using Sephadex G-15 gel filtration. Good agreement between these two techniques was obtained. Serum albumins were purified by Sephadex G-150 gel filtration, and lysine vasopressin was purified by CM-cellulose ion-exchange chromatography and Sephadex G-25 gel filtration. [ 3H]Lysine vasopressin was prepared by reduction of the iodinated hormone with 3H and purified by ion-exchange chromatography and gel filtration. The PCMB technique was primarily used to confirm the gel filtration technique. The gel filtration technique was used to measure the kinetic and equilibrium parameters of reactions between l-[ 14C]cystine and human serum albumin and between [ 3H]lysine vasopressin and bovine mercaptalbumin. The pH profiles for these reactions were also determined. Kinetic experiments indicated that these thiol-disulfide interchange reactions proceeded at two distinct rates, and equilibrium experiments were measured only with respect to the fast-reacting component. Mg 2+ was demonstrated to inhibit the rate and extent of reaction between [ 3H]lysine vasopressin and bovine serum albumin, while Ca 2+ had no significant effect at similar concentrations.