Effect of the UV modification of alpha-crystallin on its ability to suppress nonspecific aggregation

Recent studies have shown that structural modifications of alpha-crystallin during lens aging decrease it's effectiveness as a molecular chaperone. Some of these posttranslational modifications have been linked to UV radiation, and this study was undertaken to investigate the effect of UV irradiation on the ability of alpha-crystallin to suppress nonspecific aggregation. The effect of 3-hydroxykynurenine (3-HK) was also investigated as a model for its glucoside (3-HKG), a main lens chromophore that has been linked to photochemical changes in the human lens. Alpha- and gamma-crystallin solutions (1 mg/mL, 1:0.125 wt/wt) were photolyzed (transmission above 295 nm) for various time intervals. Thermal denaturation of gamma-crystallin with or without alpha-crystallin was carried out at 70 degrees C and increases in light scattering were measured at 360b nm. We found that (1) irradiation of gamma-crystallin increased its susceptibility to heat-induced scattering. The addition of alpha-crystallin protects it against thermal denaturation, although its ability to do so decreases the longer gamma-crystallin is irradiated and (2) irradiation of alpha-crystallin decreases its ability to suppress nonspecific aggregating and the presence of of 3-HK during irradiation decreases it further. Our results indicate that posttranslational modifications of alpha-crystallin due to UV irradiation affect the sites and mechanisms by which it interacts with gamma-crystallin. The kinetics of gamma-crystallin unfolding during thermal denaturation were also analyzed. We found that a simple two state model applies for nonirradiated gamma-crystallin. This model does not hold when gamma-crystallin is irradiated in the presence or absence of alpha-crystallin. In these cases, two step or multistep mechanisms are more likely.