Photooxidized products of recombinant alpha A-crystallin and W9F mutant

Human lenses contain many photosensitizers that absorb light at wavelengths above 300 nm, most notably UVA light (320-400 nm). Kynurenine (Kyn) and 3- hydroxykynurenine (HK), two of the best-known photosensitizers in the human lens, may play a significant role in photooxidation-related changes in len s proteins, such as conformational change and aggregation, In vitro irradia tion experiments with proteins indicate that the Trp residue (with maximal absorption at 295 nm) is more susceptible to photooxidation by UVB light (2 80-320 nm) than by UVA light, but most UVB light below 300 nm is screened b y the cornea and little reaches the lens, especially the nuclear region whe re nuclear color develops. Therefore, if photooxidation is an important con tributor to nuclear color or nuclear cataract, it must arise from a photose nsitized reaction, In the present study, we use recombinant alpha A- and it s Trp-deficient mutant W9F as models to study the effects of UVA irradiatio n in the presence of HK or Kyn and of UVB (300 nm) irradiation on alpha-cry stallins. (alpha A- crystallin showed a large decrease in Trp fluorescence and a large increase in non-Trp (blue) fluorescence after the HK-sensitized or 300 nm photooxidation. For the W9F mutant, a smaller decrease in protei n fluorescence (lambda(ex) at 280 nm) and a smaller increase in blue fluore scence than for the wild-type alpha A-crystallin were observed. A decrease in the near-UV CD was also observed for both photooxidized (YA and the W9F mutant. The effect of Kyn sensitization is smaller than that of HK sensitiz ation, A study of chaperone-like activity indicated that only 300 nm photoo xidized alpha A and the W9F mutant increased the ability to protect insulin from dithiothreitol-induced aggregation. Thus, sensitized photooxidation c an occur in amino acids other than Trp by UVA in the presence of HK or Kyn with effects similar to, albeit smaller than, those of direct UVB (300 nm) photooxidation.