OXIDATIVE-STRESS INDUCED PROTEIN GLUTATHIONE MIXED-DISULFIDE FORMATION IN THE OCULAR LENS

The biochemistry of protein-glutathione mixed disulfide formation in t he ocular lens was examined by C-13-NMR spectroscopic measurements of glutathione oxidative metabolism in intact rabbit lenses maintained in organ culture. Lenticular amino acid uptake and glutathione biosynthe tic mechanisms were employed to facilitate the incorporation of L-[3-C -13]cysteine from the incubation medium into the cysteinyl residue of glutathione. Subsequent exposure to increasing levels of oxidative str ess induced by tert-butylhydroperoxide resulted in decreased levels of ([3-C-13]cysteinyl)-glutathione and a loss of C-13 NMR resonance inte nsity, a reflection of protein-glutathione mixed disulfide formation. The rate of ([3- C-13]cysteinyl)-glutathione loss depended on the conc entration of tert-butylhydroperoxide; C-13-labeled oxidized glutathion e was observed only at the highest concentration (2 mM) of oxidant tes ted. Removal of the oxidative stress led to a partial recovery of ([3- C-13]cysteinyl)-glutathione levels and C-13 resonance intensity. Reco very was significantly enhanced by the addition of 2-mercaptoethanol. The mechanism of protein-glutathione adduct formation was further char acterized by the in vitro monitoring of the reaction of oxidized gluta thione with bovine lens gamma-II crystallin protein using proton NMR s pectroscopy, These experiments provided insight into the role of the c ellular glutathione redox-couple, [GSH]/[GSSG], in maintaining reduced protein thiol groups, and suggested that protein-glutathione adduct f ormation may function as a mechanism for modulating the glutathione re dox buffer under conditions of oxidative stress in ocular tissue. In a ddition, the results demonstrate the feasibility of direct chemical re duction of protein-glutathione disulfide bonds in vivo which may refle ct a mechanism for the inhibition of disulfide-linked light scattering protein aggregate formation.