UVA Irradiation of Human Lens Proteins Produces Residual Oxidation of Ascorbic Acid Even in the Presence of High Levels of Glutathione

The oxidation products of ascorbic acid (AscH−) can rapidly glycate and crosslink lens proteinsin vitro,producing fluorophores and browning products similar to those present in cataractous lenses. The accumulation of AscH−oxidation products, however, would largely be prevented by the millimolar levels of glutathione (GSH) present in human lens. Here we investigate whether protein aggregation could allow the oxidation of AscH−by UVA-induced reactive oxygen species in the presence of physiological levels of GSH. The metal-catalyzed oxidation of 1.0 mM AscH−by 50 μM Cu(II) was almost complete after 1 h, but no oxidation was seen in the presence of GSH concentrations as low as 0.5 mM. UVA irradiation of protein aggregates from human lens, which accumulated more than 2.0 mM singlet oxygen after 1 h, caused a 50–60% oxidation of 1.0 mM AscH−. The addition of 2–4 mM GSH, however, decreased AscH−oxidation by less than half, and 30% of the AscH−was oxidized even in the presence of 15 mM GSH. This diminished protection may be due, in part, to the ability of AscH−, but not GSH, to penetrate to the sites of singlet oxygen generation located within the protein. Consistent with this hypothesis, greater GSH protection was seen when a proteolytic digest of the human proteins was subjected to the same irradiation or when singlet oxygen was chemically generated from 3-(4-methyl-1-naphthyl)propionic acid endoperoxide (MNPAE) at 37°C in the medium. The addition of 50 μM Cu(II) had no effect on the rate of degradation of dehydroascorbic acid (DHA). Singlet oxygen, either UVA- or MNPAE-generated, increased the rate of DHA loss. This secondary oxidation of DHA by singlet oxygen would allow the accumulation of AscH−oxidation products not reducible by GSH. Therefore, the data presented here argue that the protein aggregation seen in older human lenses may permit oxidized AscH−-induced crosslinking to occur even at physiological GSH levels.