THE AGGREGATION IN HUMAN LENS PROTEINS BLOCKS THE SCAVENGING OF UVA-GENERATED SINGLET OXYGEN BY ASCORBIC-ACID AND GLUTATHIONE

One hour of UVA irradiation of air-saturated solutions of 2 mg/mL solu bilized lens protein aggregates from aged human lens is able to produc e an accumulated concentration of more than 2 mM O-1(2), along with ox idation of 120 nmol/mL of both Trp and His amino acid residues. Increa sing concentrations of either sodium azide or ascorbic acid (up 60 10 mM) during the irradiation decreased the His destruction by no more th an 50-60% with the intact aggregates, but completely prevented the His loss with proteolyzed aggregates. Glutathione (up to 10 mM) was able to protect less than 10% of the aggregate His residues from oxidative damage, whereas His loss was almost completely prevented in the proteo lyzed aggregates. Similar data were obtained for the WA photolysis of the Trp residues. This finding led us to study the role a protein conf ormation of these aggregates plays in the diminishing of antioxidant a bility to prevent WA-mediated photolysis of O-1(2)-sensitive amino aci d residues. We found that Trp, His, and Cys are buried in the aggregat es and cannot be oxidized by a relatively high concentration of O-1(2) generated externally to the protein. Increasing urea denaturation of the aggregates caused exposure of the buried Trp residues as determine d by the red shift of the fluorescence maximum and by a marked increas e in the acrylamide and iodide fluorescence quenching. The ability of glutathione to prevent Trp oxidation by WA Light correlated directly w ith the extent of Trp exposure. These data suggest that the aggregatio n of the lens crystallins during aging produces a barrier, which preve nts the access of water-soluble antioxidants to the sites of WA-depend ent singlet oxygen generation. In this case WA photolysis of the lens proteins can proceed even in the presence of physiological levels of a ntioxidants. (C) 1998 Academic Press.