Deamidation in human gamma S-crystallin from cataractous lenses is influenced by surface exposure

A major component of human nuclear cataracts is water-insoluble, high molecular weight protein. A significant component of this protein is disulfide bonded gamma S-crystallin that can be reduced to monomers by dithiothreitol. Analysis of this reduced gamma S-crystallin showed that deamidation of glutamine and asparagine residues is a principal modification. Deamidation is one of the modifications of lens crystallins associated with aging and cataractogenesis. One proposed hypothesis of cataractogenesis is that it develops in response to altered surface charges that cause conformational changes, which, in turn, permit formation of disulfide bonds and crystallin insolubility. This report, showing deamidation among the disulfide bonded gamma S-crystallins from cataractous lenses, supports this hypothesis.     

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

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.     

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.     

Uptake of75Se in cataractous rat lens proteins

The purpose of the present study was to measure the pattern of uptake of⁷⁵Se into proteins in normal rat lenses and into the proteins of lenses with selenite-induced cataract. Ten-day-old suckling rats received a single injection of⁷⁵Se with or without a cataractous dose of cold carrier sodium selenite. Four days after injection, the proteins from excised lenses were counted for⁷⁵Se radioactivity and subjected to gel permeation chromatography, amino acid analyses, and mass spectrometry. All three soluble crystallin lens proteins took up⁷⁵Se in both normal and cataractous lenses. However, cataractous lenses did not take up⁷⁵Se into a soluble protein in which major quantities of⁷⁵Se were taken up in normal rats. Futhermore,⁷⁵Se in the gamma-crystallins was associated with an unusual acidic amino acid. It was concluded that selenium metabolism by lens proteins may be unusual compared to other soft tissues.     

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

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 μmol/g, in the DM CAT group 1187 vs 382 μmol/g and in the non-DM CAT group 967 vs 252 μmol/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.     

Role of the non-enzymatic glycosylation of protein in the formation of human cataracts

We have measured the free epsilon amino groups in soluble and insoluble proteins of clear human lenses and diabetic and non-diabetic senile cataractous lenses. The free epsilon amino groups content of soluble and insoluble proteins was significantly lower in diabetic cataracts than in clear lenses and non diabetic senile cataracts. Our results seem to demonstrate that non-enzymatic glycosylation of lens protein could play a role in the pathogenesis of cataract in diabetes.     

2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nalpha-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nalpha-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties.     

Estimation of structural changes in the cataractous rat lens using Raman spectroscopy.

For quantitative evaluation of cataract-related changes in lens proteins and lens water, the relative contents of water and SH residues and changes in the microenvironments of aromatic amino acid residues were quantitatively examined in cataract of the rat lens which had been induced by sodium selenite. Using Raman spectroscopy, results were compared with those of age-matched control lenses. The relative contents of water and SH residues decreased with increasing age in normal lenses from 3 to 8 weeks of age. In the cataractous lens, the relative water content increased constantly as compared with that of age-matched controls. The relative SH residue content continued to decline in the cataractous lenses of animals at every age. The microenvironments of tyrosine residues in cataractous lenses also changed progressively.     

3-hydroxykynurenine-mediated modification of human lens proteins: structure determination of a major modification using a monoclonal antibody

Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.     

Detection of nonenzymatic browning products in the human lens

The nonenzymatic browning or Maillard reaction is an aging process in stored foods. The initial stage of this reaction, nonenzymatic glycosylation, has been shown to occur in the human lens. The possible occurrence of further steps of the Maillard reaction involving lysine residues and glucose has been investigated. A lipid-free protein extract from a pool of human cataractous lenses was reduced, alkylated, and digested with pronase. The digest was reduced with [³H]borohydride, acid hydrolyzed and fractionated by Sephadex G-15 chromatography. The fractions eluting ahead of ?-1-deoxyglucitolyllysine were pooled and separated with an amino acid analyzer. Four fluorescent, yellow, and radioactive peaks were obtained. One of these, which co-eluted with tyrosine, was isolated, acetylated, and further analyzed by reverse phase chromatography using HPLC. Two new peaks were separated which co-chromatographed with lysine derivatives isolated from the nonenzymatic browning reaction of α-tert-butyloxycarbonyllysine with glucose. Control experiments showed that they were not artifacts due to acid hydrolysis of ?-glucitolyllysine. These results suggest that dehydration and rearrangement of the Amadori product, ?-fructosyllysine, has occured in vivo, thus leading to the formation of at least two nonenzymatic browning products.     

Comparative study of lens proteins of gray squirrel and human

1. The four crystallins of the gray squirrel lens have been characterized using gel filtration chromatography, polyacrylamide gel electrophoresis, and immunoblotting. Alpha, beta-heavy, beta-light, and gamma crystallins of squirrel lenses have been identified immunologically, and they cross-react strongly with rabbit polyclonal antibodies. The gamma-24 crystallin of the squirrel lens also reacts strongly with monoclonal anti-human lens gamma-24, as shown by its inhibition of the ELISA reaction by 85%. 2. The water-insoluble urea soluble proteins represent non-covalently associated species of soluble crystallins and the lens cytoskeletal proteins. The membrane intrinsic protein in the urea insoluble pellet has a mol. wt of 27,000 but other lower and higher mol. wt components are also present, which were removed by washing with 0.1 NaOH. The N-terminal 30 amino acid of squirrel lens gamma crystallin was found to be identical to that of the bovine (and human) lens. 3. Measurements of the distribution and state of SH and SS compounds in the squirrel lens have shown greater similarities to those of primates than those of rodents. The findings show that on the basis of both protein and sulfur chemistry the squirrel lens is a representative model for studies of oxidative lens changes in diurnal animals, including man.     

Alteration in the ubiquitin structure and function in the human lens: a possible mechanism of senile cataractogenesis

High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73-76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins.     

Temporal relationship between lens protein oxidation and cataract development in streptozotocin-induced diabetic rats

We compared the progression of lens opacification with the time course of oxidation of lens proteins under conditions of streptozotocin-induced experimental diabetes in rats. By the end of the 17th week, approx. 50% of the diabetic animals developed mature cataracts. During the following month, 95% of the eyes in the diabetic group became cataractous. In the course of lens opacification we observed a time-dependent increase in the content of protein carbonyls and decrease in the concentration of protein sulfhydryls in the lenses of diabetic animals. Significantly higher protein carbonyl (p<0.01) and lower protein sulfhydryl (p<0.001) content was found in lenses with the advanced stage of cataract when compared with the diabetic lenses still transparent. We showed that the values of protein carbonyls exceeding 1.2 nmol/mg protein and of sulfhydryls falling below 60 nmol/mg protein corresponded to an approximately 50% incidence of mature cataract development. At the end of the 34th week, when all lenses of diabetic rats became cataractous, the corresponding values of protein carbonyls and sulfhydryls were 2.5 nmol/mg protein and 27 nmol/mg protein, respectively. The main finding of this study is the disclosure of quantitative relationship between the degree of protein oxidation and the rate of advanced cataract development in the widely used model of streptozotocin-induced experimental diabetes in rats.     

Conversion of arginine into ornithine by advanced glycation in senescent human collagen and lens crystallins

Long lived proteins undergo age-related postsynthetic modifications that destabilize them by altering their conformation, charge, and helicity, thereby enhancing their resistance toward proteolysis and propensity to aggregate. The unexpected finding of substantial amounts of ornithine, the nonprotein amino acid, and decarbamidation product of arginine in acid hydrolysates of lens crystallins and skin collagen led us to investigate its source and mechanism of formation. In order to exclude ornithine formation as an artifact of acid hydrolysis, proteins were reductively alkylated with formaldehyde to convert ornithine to dimethyl-ornithine. The proteins were assayed for carboxymethyl-ornithine and glycated ornithine ("furornithine") by liquid chromatography coupled to electrospray ionization mass spectrometry. Ornithine in acid hydrolysates of human lens and skin proteins increased from 1 to 15 nmol/mg protein from ages 10 to 90 years, whereas dimethyl-ornithine increased from 0.5 to 15 and from 0 to 5 nmol/mg protein, respectively. Carboxymethyl-ornithine and furornithine increased with age in lens and skin from approximately 0 to 60 and 0 to 180 pmol/mg protein, respectively. In collagen, ornithine was elevated above levels of nondiabetic controls only when both diabetes and end stage renal disease were present. The age-related increase of these modifications provides evidence for substantial in vivo formation of ornithine in aging human tissue proteins. The mechanism of ornithine formation is not known, but data suggest that arginine-derived advanced glycation end products might serve as precursors for the in vivo conversion of ornithine from arginine.     

Free and protein-bound glutathione in normal and cataractous human lenses

Protein-bound glutathione was identified and measured in normal and cataractous human lenses. In a major group of cataracto us lenses the bound glutathione concentration was higher than normal. Study of normal lenses showed that their glutathione content is age-dependent, decreasing steadily from about 3.5mumol/g of lens at age 20 years to about 1.8mumol/g of lens at age 65 years. Cataract brings further decreases.     

LC-MS display of the total modified amino acids in cataract lens proteins and in lens proteins glycated by ascorbic acid in vitro

We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14-26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A330nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 10(4) - 10(6) response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude that the majority of the major modified amino acids present in early stage brunescent Indian cataract lens proteins appear to arise as a result of ascorbic acid modification, and are presumably advanced glycation end-products.     

Conformational changes in human lens proteins in cataract

The reactivity of protein thiol groups in human lens and the susceptibility of the proteins to tryptic digestion were investigated. Both were found to be greater in some cataractous lenses, indicating that lens proteins have unfolded during cataractogenesis. Almost all the tyrosine in the proteins of the normal human lens reacts with tetranitromethane and is therefore probably on the outside of the major lens proteins.     

Determination of dideoxyosone precursors of AGEs in human lens proteins

Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates.     

Modifications of human betaA1/betaA3-crystallins include S-methylation, glutathiolation, and truncation

Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human gamma-crystallins undergo S-methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that betaA1/A3-crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of betaA1-crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post-translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N-terminal truncations were also found.     

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A(330)-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A(330)-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60.Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins.