Autosensitized oxidation of glycated bovine lens proteins irradiated with UVA-visible light at low oxygen concentration

Abstract

Advanced glycation endproducts (AGEs) are generated in lens proteins following ascorbic acid glycation. Autosensitized photo-processes induced by these AGEs exposed to UVA-visible light at low oxygen concentrations, such as those found in the eye lens, were studied in order to establish their photo-damaging potential. Ascorbic acid glycated bovine lens proteins, irradiated with UVA and/or visible light at 5% oxygen concentration showed an increase in the amount of carbonyl groups and an increase in the emission of chemiluminescence. Both are indicators of the occurrence of amino acid oxidation within the proteins. A good correlation was found between the chemiluminescence intensity and the extent of the glycation process. No modifications in the SDS-PAGE patterns were observed when 6-day glycated proteins were irradiated with UVA-visible light at low oxygen pressure, excluding the occurrence of photochemical crosslinking. When the same samples were derivatized with 2,4-dinitrophenylhydrazine and analyzed by Western blot, using polyclonal antibodies to the DNP moiety, an increase in the carbonyl content restricted to the AGE-crosslinked proteins fractions was observed. These results indicate that photo-oxidations are limited to the nearby environment of the sensitizers. On the contrary, when native lens proteins were irradiated in the presence of small molecular weight models of AGEs, prepared by incubating ascorbate with Nalpha-acetylated lysine and arginine, the occurrence of both photochemically induced crosslinking and oxidative processes were observed, emphasizing the importance of the localization of the sensitizer. Taken together, these results provide information with respect to the actual photosensitizing ability of endogenous AGEs to induce photo-oxidative processes in lens proteins.