Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

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Abstract
Oxygen radical generating systems, namely, Cu(II)/ H2O2, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H2O2/catecholamine and Cu(II)/H2O2/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H2O2 enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H2O2 depended on Cu(II) and H2O2 concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO˙ scavengers prevented GR inactivation by Cu(II)/H2O2 and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H2O2 in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H2O2 effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H2O2 inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*+. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.