IDENTIFICATION OF PHOTOOXIDATION SITES IN BOVINE ALPHA-CRYSTALLIN

Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine alpha-crystallin, a major lens prot ein with molecular chaperone activity, were identified using tandem ma ss spectrometry (MS/MS), Bovine alpha-crystallin was irradiated with U V light (>293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser desorption ionization, time-of-flight mass s pectrometry (MALDI) to identify the oxidized sequences, Tryptic peptid es were purified by reverse-phase HPLC and oxidized peptides were sequ enced by MS/MS to determine the sites of oxidation, Tryptophan fluores cence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of alpha A-crystallin and 1-11, 12-2 2 and 57-69 of alpha B-crystallin were determined to be oxidized by sh ifts of 16 D or multiples of 16 Da above the mass of the unmodified pe ptide, The MALDI analysis revealed single oxidation of all four sequen ces, which increased with increasing time of UV exposure and possible double oxidation of alpha B 12-22, The specific sites of photooxidatio n indicate that the N-terminal regions of alpha A- and alpha B-crystal lin are exposed to an aqueous environment and are in the vicinity of t ryptophan residues from neighboring subunits.