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.

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.

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.

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.

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].

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.

Phototransformations of advanced glycation end products in the human eye lens due to ultraviolet A light irradiation.

Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation.

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.

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.

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).

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.

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.

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 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.

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.

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.

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].

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.

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation.

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion.

Sugar-mediated crosslinking of alpha-biotinylated-Lys to cysteamine-agarose support: a method to isolate Maillard Lys-Lys-like crosslinks.

Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures.

Inhibitors of advanced glycation end product-associated protein cross-linking.

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors.

The non-oxidative degradation of ascorbic acid at physiological conditions.

The degradation of L-ascorbate (AsA) and its primary oxidation products, L-dehydroascorbate (DHA) and 2,3-L-diketogulonate (2, 3-DKG) were studied under physiological conditions. Analysis determined that L-erythrulose (ERU) and oxalate were the primary degradation products of ASA regardless of which compound was used as the starting material. The identification of ERU was determined by proton decoupled (13)C-nuclear magnetic resonance spectroscopy, and was quantified by high performance liquid chromatography, and enzymatic analysis.

Chromatographic comparison of the UVA sensitizers present in brunescent cataracts and in calf lens proteins ascorbylated in vitro.

The water-insoluble (WI) fraction from aged human lenses contains yellow chromophoric sensitizers, which generate reactive oxygen species (ROS) when irradiated with UVA light. The WI proteins from type I to V brunescent cataract lenses were assayed for UVA-dependent superoxide anion synthesis. Rates varied from 8.

Spontaneous generation of superoxide anion by human lens proteins and by calf lens proteins ascorbylated in vitro.

The proteins isolated from aged human lenses and brunescent cataracts exhibit extensive disulfide bond formation. Diabetic rat lenses similarly contain disulfide-bonded protein aggregates. These observations are consistent with the known link between diabetes, glycation and oxidative damage, and suggest a role for reactive oxygen species (ROS) in this process.