Finley E L, Dillon J, Crouch R K, Schey K L
Department of Cellular and Molecular Pharmacology, Medical University of South Carolina, Charleston 29425, USA.
Photochem Photobiol. 1998 Jul;68(1):9-15.
Radiolysis of water by ionizing radiation results in the production of pure hydroxyl radicals. This technique, combined with analysis by tandem mass spectrometry (MS/MS), has been used to study the effect of hydroxyl radicals on the intact bovine alpha-crystallin protein. After exposure to gamma-irradiation, the oxidized alpha-crystallin was digested with trypsin and the resulting peptides were fractionated by reverse-phase HPLC. The isolated fractions were analyzed by matrix-assisted laser desorption ionization and by MS/MS to determine the locations and identities of the modifications. Structural analysis revealed that methionine 1 of alpha A- and alpha B-crystallin and methionine 68 of alpha B-crystallin were oxidized to methionine sulfoxide. Hydroxytryptophan was formed from each tryptophan residue in alpha-crystallin, although only tryptophan 9 of alpha A-crystallin was converted into N-formylkynurenine. This study has, for the first time, identified the sites of modification and the structures produced in the intact alpha-crystallin protein by exposure to hydroxyl radicals. By determining the consequences of in vitro exposure of alpha-crystallin to pure hydroxyl radicals, the in vivo contribution of this reactive oxygen species to the overall oxidative stress of the lens will be achieved from the identification of the modifications to alpha-crystallin purified from intact human lenses.
电离辐射对水的辐射分解会产生纯羟基自由基。这项技术与串联质谱分析（MS/MS）相结合，已被用于研究羟基自由基对完整的牛α-晶体蛋白的影响。在接受γ辐射后，将氧化的α-晶体蛋白用胰蛋白酶消化，所得肽段通过反相高效液相色谱进行分离。对分离出的组分进行基质辅助激光解吸电离和MS/MS分析，以确定修饰的位置和性质。结构分析表明，αA-晶体蛋白和αB-晶体蛋白的甲硫氨酸1以及αB-晶体蛋白的甲硫氨酸68被氧化为甲硫氨酸亚砜。α-晶体蛋白中的每个色氨酸残基都形成了羟基色氨酸，不过只有αA-晶体蛋白的色氨酸9被转化为N-甲酰犬尿氨酸。本研究首次确定了完整的α-晶体蛋白暴露于羟基自由基后发生修饰的位点和产生的结构。通过确定α-晶体蛋白在体外暴露于纯羟基自由基的后果，从对从完整人晶状体中纯化的α-晶体蛋白修饰的鉴定中，将能够了解这种活性氧物种对晶状体整体氧化应激的体内贡献。
