Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Abstract

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.

Roles of the tyrosine isomers meta- tyrosine and ortho- tyrosine in oxidative stress

“…Indeed, o -tyrosine concentration within protein often is found to be higher than that of m -tyrosine, particularly in tissues in which there is minimal protein synthesis (e.g. lens) (Fu et al, 1998b; Molnar et al, 2005a). What accounts for this variability is currently unknown, but it could be that in addition to the preference for the non-enzymatic hydroxylation of free phenylalanine to generate o -tyrosine (see section 2.1), o -tyrosine formation may be favored when phenylalanine in intact protein is hydroxylated by nature of phenylalanine’s orientation within peptides.…”

Endogenous 3,4-Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine

“…Recent studies have suggested that protein-bound DOPA is involved in triggering antioxidant defenses (30) and mediating oxidative damage to DNA (31). Moreover, elevated levels of protein-bound DOPA have been reported in several diseases, including atherosclerosis, cataracts, and myocardial disease, and in PD patients undergoing levodopa therapy (26,(32)(33)(34)(35)(36). However, the specific DOPAmodified proteins, which could provide mechanistic knowledge of the progression of these diseases, have not been identified (27,28).…”

Conformational and vibrational analyses of meta-tyrosine: An experimental and theoretical study

“…The m-tyr and o-tyr isomers are non-proteinogenic amino acids and rarely found in nature. However, they are available synthetically and can also arise through non-enzymatic free-radical hydroxylation of phenylalanine under conditions of oxidative stress like hydroxyl radicals or peroxynitrite [1][2][3][4]. Beside, tyrosine positional isomers can be generated from radiation induced chemical reactions of free phenylalanine as well as phenylalanine containing proteins, and so they can be used as markers in irradiated foods http://dx.…”