Oxidative damage of the lens causes disulfide bonds between cysteinyl residues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H 2 O 2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H 2 O 2 . The H 2 O 2 treatment decreased the glutathione and increased the protein-glutathione and proteincysteine disulfides in the lens. The molecular mass of the ␥B-crystallin isolated from lenses, not treated with H 2 O 2 , agreed with the published sequence (M r 20,966). Some lenses also had a less abundant ␥B-crystallin component 305 Da higher (M r 21,270), suggesting the presence of a glutathione adduct. The ␥B-crystallins from H 2 O 2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified ␥B-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of ␥B-crystallins from different lenses indicated that the ؉305 Da modifications were not at a specific cysteine. For the lenses incubated without H 2 O 2 , there was evidence of adducts at Cys-41 and in peptide 10 -31, which includes 3 cysteines. Analysis of modified peptide 10 -31 by tandem mass spectrometry showed GSH adducts at Cys-15, Cys-18, and Cys-22. In addition, ␥B-crystallins from H 2 O 2 -treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.Age-related cataract is one of the major causes of blindness in humans. The pathology of such a condition involves opacification and decreased transparency of the lens, which can lead to loss of vision. Although the mechanisms for age-related cataractogenesis are not understood, oxidation of the lens proteins, known as crystallins, is associated with cataract formation in humans (1, 2). Protein thiolation, which involves the formation of disulfide bonds between the cysteinyl residues of lens proteins and other low molecular weight thiols in the lens, is one of the modifications caused by oxidative stress of the lens (3). Thiols in the lens that participate in this reaction are GSH and cysteine, which form protein-S-S-glutathione (PSSG) 1 and protein-S-S-cysteine (PSSC), respectively (4). These proteinthiol products are referred to as mixed disulfides. The conformational changes caused by protein thiolation (5, 6) may allow some of the buried functional groups to be exposed and modified. This may cause proteins to aggregate and disrupt the close packing of the crystallins, decreasing their solubility (7) and leading to cataract formation. In the H 2 O 2 -induced cataract model, the progression of cataract is associated with sequential events involving first the formation of PSSG, followed by protein-protein disulfide cross-links, decreased protein solubility, and finally an increase in the formation of high molecular weight aggregates (8). Protein-thiol mixed disulfides accumulate in older human lenses (9) and in all types of human cataractous lenses ...
