FORMATION OF DISULFIDE BONDS BETWEEN GLUTATHIONE AND MEMBRANE SH-GROUPS IN HUMAN-ERYTHROCYTES

In erythrocytes treated with the SH-oxidizing agent diamide, mixed disulfide bonds between membrane proteins and GSH [glutathione] were formed involving 20% of the membrane SH groups. To study the distribution of these mixed disulfides over the membrane protein fractions, intracellular GSH was labeled biosynthetically with [2-3H]glycine prior to diamide treatment of the cells and the radioactivity of defined membrane peptide fractions determined. Mixed disulfides preferentially occurred in the extrinsic protein, spectrin (6 SH groups), in addition to the formation of peptide disulfides. Intrinsic proteins were much less reactive, only 1 SH group of the major intrinsic protein (band 3) reacted with GSH, which accounted for the previously observed impossibility to dimerize band 3 via disulfide bonds in intact cells. The labeling method described offered a promising strategy to label and map exposed endofacial SH groups of membrane proteins with a physiological, impermeable marker, GSH. In ghosts treated with diamide and GSH, the number of mixed disulfides formed was greater than in erythrocytes. Polymerization of spectrin via intermolecular disulfide bridges was suppressed, while intramolecular disulfides were still formed, providing a means for the analysis of spectrin structure. Diamide-induced mixed membrane-GSH disulfides were readily reduced by GSH. GSH may also be able to reduce mixed disulfides formed in the erythrocyte membrane under oxidative stress in vivo. The reversible formation of mixed disulfides may serve to protect sensitive membrane structures against irreversible oxidative damage.