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In an earlier report we showed that incubation of ␣-crystallin with oxidized glutathione results in significant loss of its chaperone-like activity. In the present study, we determined the effect of protein-glutathione mixed disulfides (PSSG), formed at Cys-131 in bovine ␣A-crystallin, and Cys-131 and Cys-142 in human ␣A-crystallin, on the function of ␣-crystallin as a molecular chaperone. After incubation of calf and young human ␣ L -crystallin fractions with oxidized glutathione, levels of PSSG were determined by performic acid oxidation of the mixed disulfides followed by reversed-phase high pressure liquid chromatography separation of phenylisothiocyanate-derivatized glutathione sulfonic acid. Levels of PSSG increased from 0.01 to 0.14 nmol/nmol (20 kDa) in bovine ␣ L -crystallin and from 0.022 to 0.25 nmol/ nmol in human ␣ L -crystallin. The presence of glutathione adducts at Cys-131 and Cys-142 were confirmed by mass spectral analysis. The chaperone-like activity was determined by the heat denaturation assay using ␤ Lcrystallin as the target protein. To examine the reversibility of the effect of mixed disulfides on chaperone activity, studies were done before and after reduction with the glutathione reductase system. Increased levels of PSSG resulted in lower chaperone activities. Treatment with the glutathione reductase system led to 80% reduction in PSSG levels with a concomitant recovery of the chaperone activity. These results suggest that cysteine(s) in the ␣A-crystallin subunit play an important role in the function of ␣-crystallin as a molecular chaperone.

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