Free lysine, glycine, alanine, glutamic acid and aspartic acid reduce the glycation of human lens proteins by galactose.

The amino acids lysine, glycine, alanine, glutamate and aspartate formed adducts with galactose at physiological pH and temperature as shown by incorporation of U[14C] galactose. The percentage of galactose reacting with lysine, glycine, alanine, glutamate and aspartate was 4.5 to 7.8, 7.9 to 10.8, 3.2 to 4.6, 2.8 to 4.8 and 3 to 5.2, respectively. Studies with lysine showed that the extent of glycation of the free amino acid increased with time. Incubation of lens homogenate with galactose, effected glycation of proteins. Addition of lysine in concentrations of 5 and 10 mM to equimolar concentrations of galactose decreased the glycation of lens proteins by 64% to 71%; glycine, alanine, glutamate and aspartate decreased glycation by 23 to 68%, 32 to 61%, 35 to 56% and 26 to 61% respectively. Under similar conditions, glycine reacts to a greater extent than lysine, alanine, glutamic and aspartic acids. However, lysine was more effective than glycine, alanine, aspartic and glutamic acids in decreasing glycation of lens proteins by galactose. The decrease of glycation with added lysine increased with time. In general increase of amino acid concentration rather than that of sugar augmented the decrease of glycation of lens proteins.

Chemical modifications and dissociation characteristics of tyrosine and tryptophan residues in alpha-crystallin.

A quantitative estimation of surface accessibility of aromatic residues in alpha-crystallin from goat lens has been accomplished by chemical modifications using different specific reagents having varying sizes. Results of modification of tyrosine residues with N-acetylimidazole and tetranitromethane when combined with those of ionization studies carried out with hydroxyl ions having the smallest size reveal different classes of tyrosine residues in the native protein: 78 +/- 2 residues have been found to be easily available for modification; among the rest, 94 +/- 2 residues appear to be comparatively less exposed to the reagents while 28 +/- 2 residues are found to be completely unavailable for modification in the native protein and are modified only when the protein is denatured. Modification of tryptophan residues with H2O2 also indicates different classes of these residues available for oxidation at different concentrations of the oxidant. 34 +/- 2 residues of tryptophan are found to be easily oxidized at a lower concentration of H2O2 during the first phase of the reaction. The remaining tryptophan residues appear to be less exposed to the reagent. This is also corroborated from the studies of reactivities of these residues towards another specific but bulkier reagent, 2-hydroxy-5-nitrobenzyl bromide. These surface exposed aromatic residues in alpha-crystallin may be considered to be vulnerable to in vivo oxidative modifications forming insoluble aggregates which may finally contribute to the formation of cataract.

Differential domain folding/unfolding of gamma-crystallins: existence of two distinct groups.

High resolution calorimetry and spectral measurements have been employed to examine folding/unfolding behaviour of gamma-crystallins which are known to contain two homologous domains. Results have been analyzed in terms of selective unfolding of domains, interdomain interactions, conformational stability and the existence of intermediates in the order-disorder transition equilibrium. Both spectral and thermotropic data indicate that, in terms of structural hierarchy, these proteins can be divided into two distinct groups, gamma II and gamma IIIB belonging to one and gamma IIIA and gamma IVA to the other. The unfolding/folding characteristics of these two groups are distinctly different. Equilibrium unfolding of gamma II and gamma IIIB is biphasic indicating the existence of an intermediate in which one domain unfolds and the other remains in the native form. The absence of a cooperative transition in gamma IIIA and gamma IVA in acidic urea has also been attributed to a structured intermediate, most likely a molten globule, which may not be thermodynamically as distinct as of the former group. The difference in the equilibrium folding/unfolding transition of these two groups has been explained by subtle differences in the packing arrangement of their two domains and interactions between them. Since the intermediates, under certain circumstances, are known to aggregate, they are likely to play a critical role in the etiology of human cataract formation.

Physicochemical characterization and phylogenetic comparison of fish lens proteins.

The composition of soluble eye lens proteins from four chondropterygiian and fourteen teleostean fishes were analyzed for heterogeneity in MW and pI. Lens proteins from all the fish species studies are distributed in the pI range 4.3-9.0 with polypeptides in the range 17,500-31,000 Da. Phylogenetic trees are constructed based on the observations.