Identification of tryptophan oxidation products in bovine α-crystallin

被引:171
作者
Finley, EL
Dillon, J
Crouch, RK
Schey, KL
机构
[1] Med Univ S Carolina, Dept Cell & Mol Pharmacol & Expt Therapeut, Charleston, SC 29425 USA
[2] Columbia Univ, Dept Ophthalmol, New York, NY 10032 USA
[3] Med Univ S Carolina, Dept Ophthalmol, Charleston, SC 29425 USA
关键词
alpha-crystallin; Fenton reaction; mass spectrometry; N-formylkynurenine; oxidation; tryptophan;
D O I
10.1002/pro.5560071116
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal-catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of alpha-crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine alpha-crystallin sequence. After oxidative exposure, alpha-crystaIlin was digested with trypsin, and the resulting peptides were fractionated by reverse-phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of alpha A- and tryptophan 60 of alpha beta-crystallin were each converted into hydroxytryptophans (HTRP), N-formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of alpha beta-crystallin. Oxidation of methionine 1 of alpha A- and methionine 1 and 68 of alpha beta-crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact alpha-crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.
引用
收藏
页码:2391 / 2397
页数:7
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