UVA IRRADIATION OF HUMAN LENS PROTEINS PRODUCES RESIDUAL OXIDATION OFASCORBIC-ACID EVEN IN THE PRESENCE OF HIGH-LEVELS OF GLUTATHIONE

The oxidation products of ascorbic acid (AscH(-)) can rapidly glycate and crosslink lens proteins in vitro, producing fluorophores and brown ing products similar to those present in cataractous lenses, The accum ulation of AscH(-) oxidation products, however, would largely be preve nted by the millimolar levels of glutathione (GSH) present in human le ns, Here we investigate whether protein aggregation could allow the ox idation of AscH(-) by WA-induced reactive oxygen species in the presen ce of physiological levels of GSH. The metal-catalyzed oxidation of 1. 0 mM AscH(-) by 50 mu M Cu(II) was almost complete after 1 h, but no o xidation was seen in the presence of GSH concentrations as low as 0.5 mM. WA irradiation of protein aggregates from human lens, which accumu lated more than 2.0 mM singlet oxygen after 1 h, caused a 50-60% oxida tion 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 oxidi zed 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 i rradiation or when singlet oxygen was chemically generated from 3-(4-m ethyl-1-naphthyl)propionic acid endoperoxide (MNPAE) at 37 degrees C i n the medium. The addition of 50 mu M Cu(II) had no effect on the rate of degradation of dehydroascorbic acid (DHA). Singlet oxygen, either WA-or MNPAE-generated, increased the rate of DHA loss. This secondary oxidation of DHA by singlet oxygen would allow the accumulation of Asc H(-) oxidation products not reducible by GSH. Therefore, the data pres ented here argue that the protein aggregation seen in older human lens es may permit oxidized AscH(-)-induced crosslinking to occur even at p hysiological GSH levels. (C) 1998 Academic Press.