The interaction of reactive oxygen species with biomolecules has been implicated in the etiology or manifestation of several pathological processes. To guard against this oxygen toxicity, organisms have developed a battery of antioxidant defenses, including the synthesis of enzymes such as catalase, glutathione peroxidase and superoxide dismutase that catalyze the destruction of some reactive oxygen species. Studies in the Laboratory of Biochemistry at NHLBI have led to the discovery of an antioxidant protein, called thiol specific antioxidant (TSA). This protein specifically protects enzymes from oxidative damage by a nonenzymatic Fe(III)/oxygen/thiol mixed-function oxidase system. TSA does not contain a heme or a flavin prosthetic group, nor tightly bound metal ions. Furthermore, TSA does not possess catalase, glutathione peroxidase, superoxide dismutase or iron chelation activities. In order to understand how TSA protects biomolecules from oxidative damage, we have studied the inactivation of glutamine synthetase by ascorbate/Fe(III)/ oxygen in the presence of thiols. We demonstrated that TSA can protect glutamine synthetase from inactivation by ascorbate/Fe(III)/oxygen, only if a thiol also is present in the reaction mixture. The concentrations of 2-mercaptoethanol required to activate TSA is ten times greater than that of DTT. These results suggest that TSA has a higher specificity for dithiols than monothiols or, alternatively, that a high reducing power is necessary to activate this protein. We also have studied the effect of TSA on DTT oxidation catalyzed by Fe(III). In this system, TSA inhibits oxygen and sulfhydryl consumption.
