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JP Dillon, L Ervin, ER Gaillard; Detection of a Beta-glucosidase in the Primate Lens: a Possible Mechanism of Age-related Yellowing . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2390.
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Purpose: To test the hypothesis that yellowing of lens proteins is due to the covalent attachment of 3-hydroxykynurenine (3-HK) via its aromatic ring. In order for this to occur, 3-HKG must be converted to 3-HK. One mechanism would involve the presence of a beta-glucosidase in the primate lens. Methods: Two methods were used to detect the beta-glucosidase. The first relies on the fact that the glucoside of 3-HK has a fluorescent quantum yield of .04, whereas 3-HK is less than .001. Cleavage of the glycosidic bond will result a decreased fluorescence. Additionally, an assay was developed to confirm the presence of a beta-glycosidase activity. The assay consists of adding glucose oxidase to the 3-HKG/glucosidase solution and then allowing the hydrogen peroxide, generated from the interaction of glucose with glucose oxidase, to oxidize 3-hydroxykynurenine to xanthomattin (XAN). This fluoresces in the visible region and is more accurate than the traditional salicin assay. Results:The investigation of beta-glucosidase activity in the primate lens led to the following results; 1) Incubation of the total supernatant from either monkey or human lenses led to the loss of 25-50 % of 3-HKG fluorescence (ex/em=365/440 nm) over a period of ca 30 minutes, at which point the reaction stopped. 2) The addition of authentic 3-HKG to the supernatant devoid of small molecular weight components gave the same results. 3) The addition of 3-HKG to almond beta-glucosidase resulted in the total loss of 3-HKG fluorescence over 2 hours. 4) The second assay (above) produced XAN fluorescence. Conclusion: The primate lens contains a relatively unstable beta-glucosidase which converts 3-HKG to 3-HK. This may play an important role in the age-related yellowing of the primate lens.
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