Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid‐Glycated Lens Proteins

Abstract

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol. 62, 454-463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/cm2 of absorbed UVA light (gamma > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 microM as determined by the superoxide dismutase (SOD)-dependent increase in hydrogen peroxide formation and of 52 microM by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 microM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system. Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N-dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H2O2 were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.