PURIFICATION OF NADPH-FREE GLUTATHIONE DISULFIDE REDUCTASE FROM HUMANERYTHROCYTES

Human erythrocyte glutathione disulfide reductase was purified using s erially connected 2',5'-ADP-Sepharose 4B affinity and anion-exchange c olumns. About 11,000-fold purification was achieved with 90% yield. Th e specific activity of the final preparation was 140 units per milligr am of protein. The purified enzyme gave a single band on both native a nd SDS-PAGE with a subunit mass of 58 kDa, Its pH optimum was 7.20. Th e Michaelis constants determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [K-m(NADPH) = 18 mu M at 30-20 0 mu M NADPH; K-m(GSSG) = 72 mu M, at 40-1000 mu M glutathione disulfi de, both at saturating concentrations of the second substrate]. The af finity eluent NADPH and its oxidized form NADP + were successfully rem oved from the enzyme on the ion-exchange column. The purification meth od developed is very useful when the enzyme source material is scarce (e.g, in preparations from human tissues) and may find further applica tion in the purification of other NAD(P)H-dependent enzymes which migh t be inactivated by their affinity eluent(s). (C) 1998 Academic Press.