THE GENERATION OF HYDROGEN-PEROXIDE BY THE UVA IRRADIATION OF HUMAN LENS PROTEINS

The water-insoluble proteins from aged human lens are known to contain protein-bound chromophores that act as UVA sensitizers. The irradiati on of a sonication-solubilized, water-insoluble fraction from human le nses (55-75 years) with UVA light (1.5 kJ/cm(2), lambda > 338 nm) caus ed an oxygen-dependent photolysis of tryptophan, not seen when either alpha-crystallin or lysozyme were irradiated. The suggested requiremen t for active oxygen species was consistent with a linear increase in h ydrogen peroxide formation, which was also observed. A final concentra tion of 55 mu M H2O2 was attained, with no H2O2 being detected in eith er dark-incubated controls or in irradiated samples of native proteins . The UVA-dependent H2O2 formation was increased 50% by superoxide dis mutase (SOD) and abolished by catalase, arguing for the initial genera tion of superoxide anion. A linear photolysis of histidine and tryptop han was also seen; however, the addition of SOD or SOD and catalase ha d no effect on the photolytic destruction of either amino acid. Supero xide dismutase increased the oxidation of protein SH groups implicatin g H2O2, but SOD and catalase caused a decrease in SH oxidation only at later time periods. The direct addition of H2O2 to a water-insoluble sonicate supernatant fraction caused only a slight oxidation of SH gro ups, but this was increased four- to eight-fold when the protein was d enatured in 4.0 M guanidine hydrochloride. Overall, the data suggest a UVA-dependent oxidation of protein SH groups via H2O2 generated withi n the large protein aggregates of the water-insoluble fraction. These data also provide a mechanism for oxidation of the sulfur-containing a mino acids in vivo-a process that is known to accompany the formation of age-onset cataracts.