CATALYSIS OF REDUCTION OF DISULFIDE BY SELENOL

The reduction of a disulfide to thiols, using sodium borohydride or dithiothreitol as reductant, is catalyzed by selenol. The rates of reduction of oxidized glutathione and bis(a-hydroxyethyl) disulfide by sodium borohydride are accelerated using a catalytic amount of selenol (8 mol% of the disulfide), by factors of 10 and 12, respectively. A rapid and convenient assay for disulfide has been developed, which involves its reduction with sodium borohydride in the presence of a catalytic amount of selenol, followed by acid quench and measurement of the resulting thiol by Ellman's assay. The disulfide bonds in proteins are reduced rapidly using excess dithiothreitol (5 mM) and selenol (0.8 mM), which can be separated conveniently from the reduced protein by gel filtration. The rates of reduction of disulfide bonds in immunoglobulin and alpha-chymotrypsinogen A by dithiothreitol are enhanced by a factor of 90 in the presence of selenol. The rate-determining step is the reaction of selenolate anion with disulfide. Selenocystamine, a commercially available diselenide, is used as the precursor of the catalyst;it is reduced to its selenol in situ. (C) 1995 Academic Press, Inc.