Tryptophan metabolites from young human lenses and the photooxidation of ascorbic acid by UVA light.

PURPOSE: To determine whether there are UVA light-responsive sensitizers in young human lenses capable of initiating the oxidation of ascorbic acid in the absence of oxygen. METHODS: Lens homogenates were fractionated, and low-molecular-weight (LMW) components were separated from the proteins by filtration through a 3000-MWt cutoff filter. Aliquots of each fraction were assayed for sensitizer activity by UVA irradiation (337-nm cutoff filter) with 0.1 mM ascorbic acid, measuring ascorbate oxidation by loss of absorbance at 265 nm. Two major peaks were isolated from a human lens water-soluble (WS)-LMW fraction on a reversed-phase column and were identified by mass spectrometry. RESULTS: All human lens fractions oxidized ascorbate when irradiated by UVA light. Most of the sensitizer activity in young human lenses was in the LMW fractions. An action spectrum for the WS-LMW fraction from human lens showed activity throughout the UVA region. Assays with and without oxygen showed little or no difference in ascorbate oxidized, arguing for a direct transfer of an electron in a so-called type 1 reaction. A human lens WS-LMW fraction contained two major peaks of activity. The greater peak was identified as 3-hydroxykynurenine glucoside (3OHKG) by mass spectrometry and its absorption spectrum, whereas the lesser peak was identified as 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG). The activities were 1.1 and 2.8 nmol of ascorbate oxidized in 30 minutes/nmol 3OHKG and AHBG, respectively. CONCLUSIONS: The filter compounds present in human lenses can absorb UVA light and cause the oxidation of ascorbic acid in the presence and absence of oxygen, possibly initiating the glycation of lens proteins.

Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins.

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.

Effect of a single AGE modification on the structure and chaperone activity of human alphaB-crystallin.

During aging, human lens proteins undergo several post-translational modifications, one of which is glycation. This process leads to the formation of advanced glycation end products (AGEs) which accumulate with time possibly leading to the formation of cataract. alphaB-Crystallin, a predominant protein in the lens, is a member of the small heat shock proteins (sHSPs) which are a ubiquitous class of molecular chaperones that interact with partially denatured proteins to prevent aggregation. This chaperone function is considered to be vital for the maintenance of lens transparency and in the prevention of cataract. In the present study, we introduced an analog of the advanced glycation end product, OP-lysine, at the 90th position of a mutated human alphaB-crystallin (K90C) by covalent modification of the cysteine residue with N-(2-bromoethyl)-3-oxidopyridinium hydrobromide. The AGE-modified K90C-alphaB-crystallin is termed as K90C-OP. We compared the structural and functional properties of K90C-OP with the original K90C mutant, with K90C chemically modified back to a lysine analog (K90C-AE), and with wild-type human alphaB-crystallin. Modified K90C-OP showed decreased intrinsic tryptophan fluorescence and bis-ANS binding without significant alterations in either the secondary, tertiary, or quaternary structure. K90C-OP, however, exhibited a reduced efficiency in the chaperoning ability with alcohol dehydrogenase, insulin, and citrate synthase as substrates compared to the other alpha-crystallin proteins. Therefore, introduction of a single AGE near the chaperone site of human alphaB-crystallin can alter the chaperoning ability of the protein with only minor changes in the local environment of the protein.

Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model.

Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5- to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products.

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.

K2P--a novel cross-link from human lens protein.

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC-MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure as being 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)-3-hydroxy-2, 3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues and a five-carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of water-soluble [WS] protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross-link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.

Structure elucidation of a novel yellow chromophore from human lens protein.

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and (1)H, (13)C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract development in vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.

Regulation of tissue oxygen levels in the mammalian lens.

Opacification of the lens nucleus is a major cause of blindness and is thought to result from oxidation of key cellular components. Thus, long-term preservation of lens clarity may depend on the maintenance of hypoxia in the lens nucleus. We mapped the distribution of dissolved oxygen within isolated bovine lenses and also measured the rate of oxygen consumption (QO2) by lenses, or parts thereof. To assess the contribution of mitochondrial metabolism to the lens oxygen budget, we tested the effect of mitochondrial inhibitors on (QO2) and partial pressure of oxygen (PO2). The distribution of mitochondria was mapped in living lenses by 2-photon microscopy. We found that a steep gradient of PO2 was maintained within the tissue, leading to PO2 < 2 mmHg in the core. Mitochondrial respiration accounted for approximately 90% of the oxygen consumed by the lens; however, PO2 gradients extended beyond the boundaries of the mitochondria-containing cell layer, indicating the presence of non-mitochondrial oxygen consumers. Time constants for oxygen consumption in various regions of the lens and an effective oxygen diffusion coefficient were calculated from a diffusion-consumption model. Typical values were 3 x 10(-5) cm(2) s(-1) for the effective diffusion coefficient and a 5 min time constant for oxygen consumption. Surprisingly, the calculated time constants did not differ between differentiating fibres (DF) that contained mitochondria and mature fibres (MF) that did not. Based on these parameters, DF cells were responsible for approximately 88% of lens oxygen consumption. A modest reduction in tissue temperature resulted in a marked decrease in (QO2) and the subsequent flooding of the lens core with oxygen. This phenomenon may be of clinical relevance because cold, oxygen-rich solutions are often infused into the eye during intraocular surgery. Such procedures are associated with a strikingly high incidence of postsurgical nuclear cataract.

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.

Activation of protein-bound copper ions during early glycation: study on two proteins.

This study proposes several possible pathways by which hyperglycemia could make protein-bound metal ions more redox active. These mechanisms were tested on bovine serum albumin and calf lens protein. Proteins rich in early glycation products were less capable of competing for copper ions in the presence of other ligands (e.g., glycine and calcein), suggesting that glycated proteins might have diminished stability constants of their copper chelates compared to control counterparts. When protein-copper complexes were tested for their ability to cause the oxidation of ascorbic acid, as well as the reduction of molecular oxygen to hydrogen peroxide, glycated and control proteins differed considerably in their redox abilities. Oxidative damage on proteins documented by protein carbonyl content and amino acid analysis indicates the involvement of Fenton chemistry upon metal chelation. The possible biological consequences of the observed activation of metal ions bound to early glycated proteins are discussed.

Studies on the mechanism of the UVA light-dependent loss of glutathione reductase activity in human lenses.

PURPOSE: To determine the mechanism that leads to the UVA light-dependent loss of glutathione reductase (GR) activity in human lens (HL). METHODS: Both the HL water-soluble (WS) fraction and yeast GR were irradiated with UVA light (200 mW/(cm(2). h) for 1 hour at +20 degrees C, and the specific activity (SA) was observed. GR apoenzyme (apo-GR) was prepared from either HL-WS fractions or yeast GR by treatment with a cold solution of acidic ammonium sulfate. Reconstitution of apo-GR was conducted by mixing enzyme with an excess of flavine adenine dinucleotide (FAD) and purification of GR on a size-exclusion separation column. RESULTS: One hour of UVA photolysis of an HL-WS fraction resulted in a 96% decrease in the SA of GR (6.32 +/- 0.22 vs. 0.39 +/- 0.01 mU/mg lens protein). Action spectra of GR SA in the WS fraction from HL within the range 320 to 500 nm showed that the enzyme was most vulnerable to the wavelengths in the UVA region with the highest decrease in the SA at 320 to 350 nm ( approximately 23%-28% activity loss within 1 hour of irradiation), and lowest with the wavelengths beyond 400 nm (7%-8% SA loss). UVA irradiation of apo-GR in the crude HL-WS fraction, followed by reconstitution with FAD, showed that 90% of the original SA was recovered. The original GR activity either in HL or yeast GR, however, was not recovered by (NH(4))(2)SO(4) (pH 2.25) treatment followed by reconstitution with FAD after UVA photolysis. Experiments with UVA-photolyzed yeast GR revealed that UVA photolysis caused the formation of additional SH groups within the enzyme, as shown by the incorporation of an SH-specific fluorescent probe, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Similar results were obtained on the photolyzed iodoacetamide-alkylated yeast GR, which was evaluated by matrix-assisted desorption ionization-time of flight (MALDI-TOF) mass spectrometry. CONCLUSIONS: The results show that the reduction of HL GR activity by UVA light was directly linked to the presence of FAD within the enzyme. That the irradiated GR showed de novo formed SH groups argues that UVA photolysis of GR leads to the reduction of the redox-active disulfide within the reaction center of the enzyme, making it inactive.

Separation of the yellow chromophores in individual brunescent cataracts.

Quantitative changes in the 330 nm absorbing chromophores and 350/450 nm fluorophores of water-soluble (WS) and water-insoluble (WI) proteins of individual human cataract lenses were characterized and compared with aged normal human lens. Twenty-five brunescent cataract lenses from India were selected from five different stages (types I-V) based upon the color of the lens. The WS and WI proteins from each lens were collected and subjected to an extensive enzymatic digestion procedure under argon. The lens protein digests were separated by Bio-Gel P-2 size-exclusion chromatography and individual peaks were analyzed further by reversed-phase HPLC. The total WI proteins increased and the total WS protein decreased with the development of cataract, especially in the late stages of cataract (III-V). The total 330 nm absorbance and 350/450 nm fluorescence of the WI fraction also increased, however, the A(330) and fluorescence per mg lens protein were constant except for type V (black) lenses. Bio-Gel P-2 chromatography separated the chromophores and fluorophores into four fractions. The main fraction (designated as peak 2+3) from the cataract WI proteins was several times higher than that present in aged normal human lens WI proteins. A significant increase of this fraction was observed in WI proteins, but not in WS proteins with cataract development. Similarly, fractions 1 and 4 in the WI proteins also increased gradually but fraction 5 did not. Reversed-phase HPLC resolved fraction (2+3) of the water-insoluble sonicate supernatant proteins into four 330 nm absorbing peaks and eight fluorescent peaks. Among these peaks, a late-eluting peak (peak 8) increased 10 to 15-fold with the progress of cataract, and accounted for 80% of the total chromophores in type V lenses. This peak may represent limit digests of advanced glycation end-products (AGEs) derived protein cross-links. HPLC profiles of fraction 5 from both WS and WI proteins showed numerous new peaks which were not observed in either WS protein from cataract or WI proteins from aged normal human. The severe coloration and the higher levels of numerous novel chromophores and fluorophores in brunescent cataractous lenses reveal the possibility that a different chemistry occurs during cataract development.

The effect of UVA light on the anaerobic oxidation of ascorbic acid and the glycation of lens proteins.

PURPOSE: To determine whether UVA-excited human lens chromophores can cause the oxidation of ascorbic acid in the absence of oxygen, and whether these oxidation products are capable of glycating lens proteins. METHODS: The oxidation of ascorbic acid, mediated by UVA irradiation in the presence of aged human lens proteins, was measured in the absence of oxygen by the decrease in absorbance at 265 nm in vitro. An action spectrum from 320 to 400 nm was determined for both ascorbate oxidation and the photobleaching of the lens yellow pigments at lambda = 350 nm. The UVA-mediated oxidation products of [U-(14)C]ascorbate were quantified by HPLC. Glycation was assayed by the UVA-dependent incorporation of [U-(14)C]ascorbate into lens proteins with a water-insoluble (WI) fraction in vitro, with incubated whole human lenses, and with a WI fraction after a 5- to 7-day exposure to ambient sunlight. An enzymatic digest of [U-(14)C]ascorbate-labeled proteins was fractionated over HPLC columns and compared with the 330-nm absorbance profile of a proteolytic digest of aged human lens proteins. RESULTS: Aged human lens WI proteins absorbed UVA light (86 J/h per square centimeter) and oxidized 33 to 45 nanomoles of ascorbate over 1 hour in the absence of oxygen. No ascorbate oxidation was detected, however, in the dark control. An action spectrum showed that ascorbate oxidation occurred throughout the UVA region, with lambda(max) at 350 nm, which was similar to the action spectrum obtained for the photobleaching of the lens chromophores. Anaerobic UVA irradiation of aged human lens proteins for 2 hours with [U-(14)C]ascorbate resulted in a 40% loss of ascorbate with the accumulation of dehydroascorbic acid, diketogulonic acid, and oxalate. After subsequent incubation for 24 hours, the ascorbate oxidation products disappeared, with a corresponding incorporation of radioactivity into lens proteins. Chromatography of enzymatic digests of the labeled proteins produced peaks that coeluted with several of the 330-nm absorbing peaks in an aged human lens protein digest. Irradiation of whole human lenses for 2 hours caused a 33% loss of total lens ascorbate. UVA irradiation of aged human lenses for 2 hours resulted in the incorporation of ascorbate into lens proteins during the ensuing 24 hours in the dark. Exposure of aged human lens WI proteins to reflected ambient sunlight (1.1 J/h per square centimeter) for 5 to 7 days in the absence of oxygen also produced an increased incorporation of [(14)C]ascorbate into protein when compared with dark control samples. CONCLUSIONS: These data argue that UVA light can cause an oxidation of ascorbic acid in the absence of oxygen, due to the activation of the sensitizers present in aged human lens WI proteins. The oxidation products formed were the same as those seen in the presence of oxygen, and were rapidly incorporated into protein, apparently by Maillard-type chemistry. These data argue that ascorbate glycation can occur under the low oxygen levels thought to exist in the human lens nucleus in vivo.

Isolation and characterization of a new advanced glycation endproduct of dehydroascorbic acid and lysine.

Proteins are subject of posttranslational modification by sugars and their degradation products in vivo. The process is often referred as glycation. L-Dehydroascorbic acid (DHA), an oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. A new product of modification of lysine epsilon -amino group by DHA was discovered as a result of the interaction between Boc-Lys and dehydroascorbic acid. The chromatographic and spectral analyses revealed that the structure of the product was 1-(5-ammonio-5-carboxypentyl)-3-oxido-4-(hydroxymethyl)pyridinium. The same compound was isolated from DHA modified calf lens protein after hydrolysis and chromatographic separation. The study confirmed that L-erythrulose is an important intermediate of modification of proteins by DHA. The structure of the reported product and in vitro experiments suggested that L-erythrulose could further transform to L-threose, L-erythrose and glycolaldehyde under conditions similar to physiological. The present study revealed that the modification of epsilon -amino groups of lysine residues by DHA is a complex process and could involve a number of reactive carbonyl species.

Effect of UVA light on the activity of several aged human lens enzymes.

PURPOSE: To determine the effect of UVA irradiation on the specific activities of several protective and metabolic enzymes in aged human lenses. METHODS: Intact human lenses (ages 55-75) in artificial aqueous humor were irradiated in a quartz cuvette with UVA light (925 J/cm(2) per hour) at +20 degrees C. The lenses were homogenized and the activities of enzymes in the water-soluble (WS) fraction were measured in irradiated and nonirradiated lenses. RESULTS: One hour of UVA photolysis of human lens resulted in a 70% loss in glutathione reductase (GR)-specific activity and a 24% loss in glyceraldehyde-3-phosphate dehydrogenase (G3PD)-specific activity. At the same time, glutathione peroxidase (GPx) and superoxide dismutase (SOD) showed little or no loss in specific activity. GR and G3PD showed similar losses when human lenses were photolyzed with the same dose of UVA light delivered to the lens over 8 hours, using a 12.5% neutral-density filter (ndf), or over 24 hours with a 4.25% ndf. One hour photolysis of the human lens WS fraction under anaerobic conditions yielded an almost complete inactivation of GR, but only an 18% loss of G3PD activity. Under aerobic conditions, however, both enzyme activities were almost completely lost. Clear fetal bovine lenses, photolyzed under the identical conditions, displayed essentially the same loss of GR activity. CONCLUSIONS: UVA light causes inactivation of GR in human and fetal calf lenses under both anaerobic and aerobic conditions. This suggests that flavine adenine dinucleotide (FAD), the prosthetic group of GR, may be responsible for the enzyme's self-sensitizing properties. WS proteins from aged human lens generate reactive oxygen species (ROS) during UVA irradiation, which may be responsible for the inactivation of G3PD.

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.

Studies on singlet oxygen formation and UVA light-mediated photobleaching of the yellow chromophores in human lenses.

The protein-bound chromophores, which increase with aging in the human lens, act as UVA sensitizers, producing almost exclusively singlet oxygen in vitro. Direct irradiation of whole, aged human lenses with high intensity UVA light (200 mW cm(-2) for 24 hr), however, failed to produce singlet oxygen damage, as evidenced by the lack of either His or Trp photodestruction. Total homogenates of human lenses prepared in a cuvette under air did show destruction of His and Trp residues by UVA light, but no destruction was seen when equivalent homogenates were prepared under argon. These data are consistent with the idea that the low oxygen levels in the lens prevent singlet oxygen damage in vivo.UVA irradiation of aged human lenses in culture caused an extensive photobleaching of the yellow chromophores. A time course indicated that the photobleaching increased with time, with significant color loss apparent after 6 hr. Homogenization of the irradiated and dark control lenses in 6 M guanidine-HCl, followed by determination of the difference spectrum, showed approximately 50% bleaching of compounds with a lambda(max) at 355 nm. Similarly, fluorophores with a lambda(max) for excitation of 355 nm and for emission of 420 nm were 50% destroyed by the UVA light. Similar results were obtained in vitro by the anaerobic irradiation of a sonication-solubilized WI fraction from type II brunescent cataracts and from aged human lenses. In this system, there was an initial bleaching of 15% after 30 min of irradiation, followed by a slow increase over the next 6 hr to a final bleaching of 30%. The addition of 1.0 m M ascorbic acid, but not 1.0 m M glutathione (GSH), increased the photobleaching to 60% under argon, and the loss of ascorbate could be detected under these anaerobic conditions. In the presence of air, UVA light produced no photobleaching, but rather caused a three-fold increase in absorbance at 345 nm, which was prevented by the inclusion of 1.0 m M ascorbic acid and almost 50% inhibited by 1.0 m M GSH. The data are consistent with the conversion of the triplet state of the sensitizers to anion and cation radicals in the absence of oxygen. Photobleaching may occur either by dismutation of the anion radical or by reduction of the anion radical by ascorbate via type I chemistry. UVA irradiation of an enriched fraction of sensitizers from a proteolytic digest from type II cataract lenses produced a 63% bleaching at 330 nm in the absence of oxygen, and the almost complete loss of the A(330) absorbing and 350/450 nm fluorescent peaks upon HPLC separation. This loss correlated with the loss of the ability of the irradiated fraction to produce singlet oxygen in vitro upon subsequent UVA irradiation.