Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase
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Singlet oxygen ((1)O(2)) is a reactive oxygen species generated during photo-oxidation, inflammation, and via peroxidase-catalyzed reactions (e.g., myeloperoxidase and eosinophil peroxidase). (1)O(2) oxidizes the free amino acids Trp, Tyr, His, Cys, and Met, and those species present on peptides/proteins, with this resulting in modulation of protein structure and function. Impairment of the activity of antioxidant enzymes may be of relevance to the oxidative stress observed in a number of pathologies involving either light exposure or inflammation. In this study, the effects of (1)O(2) on glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activity, including the mechanisms of their inactivation, were investigated. Exposure of GPx or TrxR, either as purified proteins or in cell lysates, to Rose Bengal and visible light (an established source of (1)O(2)) resulted in significant, photolysis time-dependent reductions in enzyme activity (10-40%, P<0.05). More extensive inhibition (ca. 2-fold) was detected when the reactions were carried out in D(2)O, consistent with the intermediacy of (1)O(2). No additional inhibition was detected after the cessation of photolysis, eliminating a role for photo-products. Methionine, which reacts rapidly with (1)O(2) (k~10(7)M(-1) s(-1))(,) significantly reduced photo-inactivation at large molar excesses, presumably by acting as an alternative target. Reductants (NaBH(4), DTT, GSH, or NADPH) added after the cessation of (1)O(2) formation were unable to reverse enzyme inactivation, consistent with irreversible enzyme oxidation. Formation of nonreducible protein aggregates and/or fragments was detected for both photo-oxidized GPx and TrxR by SDS-PAGE. An oxidant concentration-dependent increase in protein carbonyls was detected with TrxR but not GPx. These studies thus demonstrate that the antioxidant enzymes GPx and TrxR can be irreversibly inactivated by (1)O(2).
|Journal||Free Radical Biology & Medicine|
|Number of pages||9|
|Publication status||Published - 15 Sep 2012|
- Animals, Borohydrides, Cattle, Cell Extracts, Cell Line, Dithiothreitol, Enzyme Stability, Free Radical Scavengers, Glutathione, Glutathione Peroxidase, Hydrogen Peroxide, Methionine, Mice, Oxidants, Photochemical, Oxidation-Reduction, Photolysis, Protein Carbonylation, Rats, Reducing Agents, Rose Bengal, Singlet Oxygen, Thioredoxin-Disulfide Reductase