Protein-Bound UV Filters in Normal Human Lenses: The Concentration of Bound UV Filters Equals That of Free UV Filters in the Center of Older Lenses

Abstract

PURPOSE. To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters. METHODS. Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins. RESULTS. The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total proteinbound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts. CONCLUSIONS. The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people. (Invest Ophthalmol Vis Sci. 2007;48:1718 -1723 DOI: 10.1167DOI: 10. /iovs.06-1134 T he primate lens is unique among both other primate tissues and the lenses of other species, in that it synthesizes 3-hydroxykynurenine glucoside (3OHKG) as a UV filter from the amino acid tryptophan (Trp). 1 The immediate precursors kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) are metabolites found in all organs of the body. All three of these Trp metabolites are unstable at physiological pH and undergo side chain deamination to yield ␣,␤-unsaturated ketones that are prone to nucleophilic attack. 2 In tissues that contain sufficient glutathione (GSH), it is likely that GSH will react with the deamination products before they bind to proteins 3 and that the adducts thus formed will diffuse out of the lens. In the interior of the lens, once the barrier to diffusion forms at middle age, 4 -6 the nucleus becomes a partially uncoupled region in which metabolites spend a longer time than in the young lens. This factor leads to an environment in the older lens nucleus that favors greater decomposition of intrinsically unstable molecules. Coupled with a diminished flux of GSH from the cortex, this results in increased covalent binding of UV filters to nuclear proteins after middle age. In this study, we set out to examine the levels of all three bound UV filters in normal lenses using a novel assay system in which lens proteins were incubated with excess GSH at pH 9.5. 9 Under these conditions, the UV filters that are attached to proteins are released, and the GSH adducts thus formed can be quantified by HPLC. UV filters were found to be covalently bound to proteins from all lenses older than 50 years. Such posttranslational modifications may have important consequences in terms of the susceptibility of the proteins to photooxidation and, in the case of 3OHKyn, the sensitivity to an oxidative environment such as that in the nuclei of lenses with age-related nuclear cataract. METHODS Purified water (purified to 18.2 M⍀/cm 2 ; Milli-Q; Millipore, Bedford, MA) was used in the preparation of all solutions. All organic solvents were HPLC grade (Ajax, Auburn, NSW, Australia). 3OHKyn, reduced GSH, trifluoroacetic acid (TFA), and guanidine HCl were obtained from Sigma-Aldrich (St. Louis, MO). Reversed-Phase HPLC Reversed-phase (RP)-HPLC was performed on a Shimadzu system (Kyoto, Japan). For analytical scale separations, a column (Jupiter, 5 m, C18, 300 Å, 250 ϫ 4.6 mm; Phenomenex, Torrance, CA) was used with the following mobile phase conditions: solvent A (aqueous 0.1% vol/vol TFA) for 5 minutes followed by a linear gradient of 0% to 50% solvent B (80% vol/vol acetonitrile/H 2 O, 0.1% vol/vol TFA) over 20 minutes, followed by a linear gradient of 50% to 100% B over 15 minutes and re-equilibration in the aqueous phase for 15 minutes. The flow rate was 0.5 mL/min and with detection at 360 nm. From th