The purpose of this study was to quantitatively study the changes that occur upon irradiation of 3-hydroxykynurenine (3-HK) in the presence of α-crystallin under conditions similar to those in the lens. The samples were prepared in 10 mM phosphate buffer at pH 7.4, bubbled with O2 or Ar and irradiated with 300–400 nm light. The amount of light absorbed by the samples (Iabs) was measured using azobenzene as an actinometer. Modifications to α-crystallin were monitored by ultraviolet–visible and fluorescence spectroscopy. Aerobic samples had increased absorption around 320 nm and above 400 nm while the 3-HK maximum at 368 nm decreased. The isolated modified protein showed that there was increased absorption throughout the spectrum. Changes in the anaerobic samples were similar to those of the aerobic but occurred more slowly. As irradiation time increased fluorescence emission of the isolated protein red shifted and quantum yields of fluorescence (Φf) were calculated at different irradiation time intervals by comparison to 3-HK. By comparing OD320/OD365 for the model system to values from primate lenses, Iabs can be correlated with age and transmission of the sample in the blue region of the spectrum and thus allows lenticular aging to be quantitated.
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Vol. 73 • No. 6