Singh R, Kats L
ImmunoGen, Inc., Cambridge, Massachusetts 02139, USA.
Anal Biochem. 1995 Nov 20;232(1):86-91. doi: 10.1006/abio.1995.9956.
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(2-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 diselende, is used as the precursor of the catalyst; it is reduced to its selenol in situ.
使用硼氢化钠或二硫苏糖醇作为还原剂将二硫化物还原为硫醇的反应由硒醇催化。使用催化量的硒醇（二硫化物的8摩尔%）时，硼氢化钠还原氧化型谷胱甘肽和双（2-羟乙基）二硫化物的速率分别加快了10倍和12倍。已开发出一种快速简便的二硫化物检测方法，该方法包括在催化量的硒醇存在下用硼氢化钠还原二硫化物，然后进行酸淬灭并用埃尔曼法测量生成的硫醇。使用过量的二硫苏糖醇（5 mM）和硒醇（0.8 mM）可快速还原蛋白质中的二硫键，通过凝胶过滤可方便地将其与还原后的蛋白质分离。在硒醇存在下，二硫苏糖醇还原免疫球蛋白和α-胰凝乳蛋白酶原A中二硫键的速率提高了90倍。速率决定步骤是硒醇阴离子与二硫化物的反应。硒代胱胺是一种市售的二硒化物，用作催化剂的前体；它在原位被还原为硒醇。
