Chromatographic comparison of the UVA sensitizers present in brunescent cataracts and in calf lens proteins ascorbylated in vitro.
The water-insoluble (WI) fraction from aged human lenses contains yellow chromophoric sensitizers, which generate reactive oxygen species (ROS) when irradiated with UVA light. The WI proteins from type I to V brunescent cataract lenses were assayed for UVA-dependent superoxide anion synthesis. Rates varied from 8.4-15 nMol h(-1)mg protein(-1), but there was no significant difference in specific activity between cataract types. When calf lens soluble proteins were incubated with ascorbic acid for 4 weeks and dialyzed, they were capable of generating 30-40 nMol h(-1)mg protein(-1)superoxide anion when irradiated with UVA light. Two preparations each of brunescent cataract WI proteins and bovine lens proteins ascorbylated in vitro were extensively digested with proteolytic enzymes and the released amino acids separated by normal phase HPLC. The elution profiles of the digests were very similar based upon the absorbance at 330 nm and fluorescence at 350 nm excitation/450 nm emission. Each peak was pooled and analyzed for the UVA-dependent generation of both superoxide anion and singlet oxygen. Every peak exhibited sensitizer activity, and the UVA-dependent ROS generation was roughly proportional to the absorbance at 330 nm. In addition, the ratio of superoxide anion to singlet oxygen generated was similar with both preparations. These data argue that it is the brown, fluorescent compounds which accumulate during aging and cataract formation that are responsible for the UVA-dependent ROS formation, and that these browning products may be similar to the advanced glycation endproducts produced by ascorbylation of lens proteins under oxidative conditions. This work also presents an initial report of a chromatographic method to separate the UVA-sensitizers present in each of these protein preparations without the use of acid or base hydrolysis.
Lee
K W
KW
Mason Eye Institute, University of Missouri, Columbia, MO 65212, USA.
Meyer
N
N
Ortwerth
B J
BJ
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002386:Cataract / Q000378:metabolism*; D002417:Cattle; D003459:Crystallins / Q000378:metabolism* / Q000528:radiation effects; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D011838:Radiation-Sensitizing Agents / Q000378:metabolism*; D012995:Solubility; D013481:Superoxides / Q000378:metabolism; D014466:Ultraviolet Rays
D003459:Crystallins; D011838:Radiation-Sensitizing Agents; D013481:Superoxides; D001205:Ascorbic Acid