Thiolation of the gammaB-crystallins in intact bovine lens exposed to hydrogen peroxide.
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 H2O2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H2O2. The H2O2 treatment decreased the glutathione and increased the protein-glutathione and protein-cysteine disulfides in the lens. The molecular mass of the gammaB-crystallin isolated from lenses, not treated with H2O2, agreed with the published sequence (Mr 20,966). Some lenses also had a less abundant gammaB-crystallin component 305 Da higher (Mr 21,270), suggesting the presence of a glutathione adduct. The gammaB-crystallins from H2O2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified gammaB-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of gammaB-crystallins from different lenses indicated that the +305 Da modifications were not at a specific cysteine. For the lenses incubated without H2O2, 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, gammaB-crystallins from H2O2-treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.
Hanson
S R
SR
Department of Chemistry, Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0905, USA.
Chen
A A
AA
Smith
J B
JB
Lou
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism*