ASCORBIC-ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

Soluble calf lens proteins were extensively glycated during a 4 week i ncubation with ascorbic acid in the presence of oxygen. Amino acid ana lysis of the dialyzed proteins removed at weekly intervals showed an i ncreasing loss of lysine, arginine and histidine, consistent with the extensive protein crosslinking observed. Irradiation of the dialyzed s amples with UVA light (1.0 kJ/cm(2) total illumination through a 338 n m cutoff filter) caused an increasing loss of tryptophan, an additiona l loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photoly tic effects were seen in proteins incubated without ascorbic acid or i n proteins incubated with glucose for 4 weeks. The rate of hydrogen pe roxide formation was linear with each glycated sample with a maximum p roduction of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of trypt ophan was eliminated by the presence of a heavy metal ion chelator dur ing the incubation and by showing equivalent effects with ascorbate-in cubated ribonuclease A, which is devoid of tryptophan. The ascorbate-i ncubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. T he spectra of the 4 week glycated proteins were identical to those obt ained with a solubilized water-insoluble fraction from human lens, whi ch is known to have UVA sensitizer activity. The incubation of lens pr oteins with dehydroascorbic acid or L-threose, but not fructose, produ ced equivalent glycation, protein crosslinking and sensitizer activity . The relative sensitizer activity of the 4 week glycated sample was q uantitatively very similar to that of a water-insoluble fraction from aged human lenses. These data are consistent with the hypothesis that the protein-bound brunescence in the lens may be advanced glycation en dproducts, which are formed in large part by the oxidation products of ascorbic acid, and that these compounds may contribute significantly to the UVA sensitizer activity present in aged human lenses.