Thiolation of the gamma B-crystallins in intact bovine lens exposed to hydrogen peroxide

Oxidative damage of the lens causes disulfide bonds between cysteinyl resid ues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H2O2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H2O2. The H2O2 treatment de creased the glutathione and increased the protein-glutathione and proteincy steine disulfides in the lens. The molecular mass of the gamma B-crystallin isolated from lenses, not treated with H2O2, agreed with the published seq uence (M-r 20,966). Some lenses also had a less abundant gamma B-crystallin component 305 Da higher (M-r 21,270), suggesting the presence of a glutath ione adduct. The gamma B-crystallins from H2O2 treated lenses had three com ponents, the major one with one GSH adduct, another one with the mass of un modified gamma B-crystallin, and a third with a mass consistent with additi on of two GSH adducts. Mass spectrometric analysis of tryptic peptides of g amma B-crystallins from different lenses indicated that the +305 Da modific ations were not at a specific cysteine, For the lenses incubated without H2 O2, there was evidence of adducts at Cys-41 and in peptide 10-31, which inc ludes 3 cysteines. Analysis of modified peptide 10-31 by tandem mass spectr ometry showed GSH adducts at Cys-15, Cys-18, and Cys-22, In addition, gamma B-crystallins from H2O2-treated lenses had an adduct at Cys-109, partial o xidation at all 7 Met residues, and evidence for two disulfide bonds.