title: Purification of NADPH-free glutathione disulfide reductase from human erythrocytes.
abstract: Human erythrocyte glutathione disulfide reductase was purified using serially connected 2',5'-ADP-Sepharose 4B affinity and anion-exchange columns. About 11,000-fold purification was achieved with 90% yield. The specific activity of the final preparation was 140 units per milligram of protein. The purified enzyme gave a single band on both native and SDS-PAGE with a subunit mass of 58 kDa. Its pH optimum was 7.20. The Michaelis constants determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [K(m)(NADPH) = 18 microM, at 30-200 microM NADPH; K(m)(GSSG) = 72 microM, at 40-1000 microM glutathione disulfide, both at saturating concentrations of the second substrate]. The affinity eluent NADPH and its oxidized form NADP+ were successfully removed from the enzyme on the ion-exchange column. The purification method developed is very useful when the enzyme source material is scarce (e.g., in preparations from human tissues) and may find further application in the purification of other NAD(P)H-dependent enzymes which might be inactivated by their affinity eluent(s).
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
authors.forename: I H
authors.initials: IH
authors.lastname: Ogüs
authors.forename: N
authors.initials: N
authors.lastname: Ozer
mesh_terms: D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione) / Q000097:blood / Q000302:isolation & purification*
chemical_list: D009249:NADP; D011490:Protein Disulfide Reductase (Glutathione)