QUANTITATION OF THE REACTIVE OXYGEN SPECIES GENERATED BY THE UVA IRRADIATION OF ASCORBIC-ACID GLYCATED LENS PROTEINS

The oxidation products of ascorbic acid rapidly glycate proteins and p roduce protein-bound, advanced glycation endproducts. These endproduct s can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol. 62, 454-463, 199 5), which is mediated by the formation of reactive oxygen species. A d ialyzed preparation of calf lens proteins, which had been incubated fo r 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/cm(2) of absorbed UVA light (lambda > 338 nm), and the concent ration of individual oxygen free radicals was measured. Superoxide ani on attained a level of 76 mu M as determined by the superoxide dismuta se (SOD)-dependent increase in hydrogen peroxide formation and of 52 m u M by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxid e formation increased linearly to 81 mu M after 1 h. Neither superoxid e anion nor hydrogen peroxide, however, could account for the UVA phot olysis of Trp and His seen in this system. Singlet oxygen levels appro ached 1.0 mM as measured by the oxidation of histidine, which was cons istent with singlet oxygen measurements by the bleaching of N,N-dimeth yl-4-nitrosoaniline. High concentrations of sodium azide, a known sing let oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl rad ical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H2O2 were generated by the UVA irradiation of asc orbate advanced glycation endproducts, however, the major reactive oxy gen species formed was singlet oxygen.