Generation of oxidants in the near-UV photooxidation of human lens α-crystallin

In this study we report on the generation of superoxide anion (O₂^-) and hydrogen peroxide in the near-UV irradiation of human lens α-crystallin by monochromatic light at 300 nm. Photolysis of human lens α-crystallin at 300 nm, at irradiances similar to those encountered in sunlight causes an alteration of protein tertiary structure, a loss of tryptophan fluorescence and increase of nontryptophan fluorescence. The nontryptophan fluorescence is likely to be due to the photooxidation of tryptophan to N-formylkynurenine (N-FK or related species), which is a good photodynamic sensitizer, has significant absorption at 300 nm, and can thus react via its triplet state with O₂ to generate ¹O₂ or with reducing substrates (amino acids of the protein) to generate free radicals. The latter, in the presence of O₂ can lead to the generation of O₂^- and H₂O₂. These species have been directly assayed in this study in photolyzed solutions of fetal, young and old human lens α-crystallin. The addition of superoxide dismutase (SOD) to the protein solution prior to photolysis increased the amount of H₂O₂ generated by 3- to 4-fold. This observation not only provides definitive evidence for the photogeneration of O₂^-, but also indicates that only a fraction of this species is transformed into H₂O₂ in the absence of SOD. Significant amounts of O₂^- and H₂O₂ were formed by 340 nm irradiation of old human lens α-crystallin, in which the basal level of N-FK is high. The role of ¹O₂ in these photoreactions has been studied by investigating the quenching effect of azide and the enhancing effects of D₂O on the rate of loss of tryptophan fluorescence yield and the effect of azide on the rate of H₂O₂ generation.