Mikhail Linetsky

The relative UV sensitizer activity of purified advanced glycation endproducts.

Abstract— The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L‐Threose, the most active of these oxidation products, was reacted with N‐acetyl lysine and six AGE peaks were isolated by RP‐HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 μg/mL) generated5–10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100‐fold less than the superoxide anion generated by kynurenic acid and 400‐fold less than riboflavin.

THE GENERATION OF HYDROGEN PEROXIDE BY THE UVA IRRADIATION OF HUMAN LENS PROTEINS

Abstract—

Quantitation of the singlet oxygen produced by UVA irradiation of human lens proteins.

Ultraviolet irradiation of aged human lens proteins in vitro causes extensive photolytic damage of His and Trp residues. Protection by sodium azide argues for a process mediated by singlet oxygen (1O2). In the work described here, the synthesis of 1O2 was measured by the bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), the oxidation of added histidine and the oxidation of furfuryl alcohol. To obtain a more accurate value for 1O2 generation, a known quantity of 1O2 was generated by the thermal dissociation of 3-(4-methyl-naphthyl)propionic acid endoperoxide, and the efficiency of each assay method to report on the 1O2 generated was determined. The values obtained were 0.003 mol of RNO bleached/mol of 1O2 generated, 0.55 mol of furfuryl alcohol oxidized/mol 1O2 and 0.5 mol of His oxidized/mol 1O2 generated. Irradiation of the human lens proteins with UVA light produced from 2.1 to 2.4 mM of 1O2 by RNO bleaching, 2.6-2.8 mM 1O2 by furfuryl alcohol oxidation and up to 1.9 mM of 1O2 by histidine oxidation during a 1 h irradiation period. The average value (2.2 mM of 1O2) corresponds to the theoretical production of 30 nmol of singlet oxygen at UVA light intensities equivalent to a 1 h exposure to sunlight at noon in the northern hemisphere.

ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

‐Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acid analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross‐linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm2 total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid or in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate‐induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate‐incubated ribonuclease A, which is devoid of tryptophan. The ascorbate‐incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water‐insoluble fraction from human lens, which is known to have UVA sensitizer activity.

Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid‐Glycated Lens Proteins

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein‐bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol. 62, 454–463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/ cm2 of absorbed UVA light (λ > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 μM as determined by the superoxide dismutase (SOD)‐depen‐dent increase in hydrogen peroxide formation and of 52 μM by the SOD‐inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 μM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system.

Glutathionylation of lens proteins through the formation of thioether bond

Formation of lanthionine, a dehydroalanine crosslink, is associated with aging of the human lens and cataractogenesis. In this study we investigated whether modification of lens proteins by glutathione could proceed through an alternative pathway: that is, by the formation of a nonreducible thioether bond between protein and glutathione. Direct ELISA of the reduced water-soluble and water-insoluble lens proteins from human cataractous, aged and bovine lenses showed a concentration-dependent immunoreactivity toward human nonreducible glutathionyl-lens proteins only. The reduced water-insoluble cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. These data were confirmed by dot-blot analysis. The level of this modification ranged from 0.7 to 1.6 nmol/mg protein in water-insoluble proteins from aged and cataractous lenses. N-terminal amino acid determination in the reduced and alkylated lens proteins, performed by derivatization of these preparations with dansyl chloride followed by an exhaustive dialysis, acid hydrolysis and fluorescence detection of dansylated amino acids by RP-HPLC, showed that N-terminal glutamic acid was present in concentration of approximately 0.2 nmol/mg of lens protein. This evidence points out that at least some of the N-terminal amino groups of nonreducible glutathione in the reduced human lens proteins are not involved in a covalent bond formation. Since disulfides were not detected in the reduced and alkylated human lens proteins, GSH is most likely attached to lens proteins through thioether bonds. These results provide, for the first time, evidence that glutathiolation of human lens proteins can occur through the formation of nonreducible thioether bonds.

Sugar-mediated crosslinking of α-biotinylated-lys to cysteamine-agarose support

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. The method is based on a sugar-dependent incorporation of Nα-biotinyl-l-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of Nα-biotinyl-l-Lys equal to 8% of the total support amino groups with methylglyoxal after 7d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.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. The method is based on a sugar-dependent incorporation of N(alpha)-biotinyl-L-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N(alpha)-biotinyl-L-Lys equal to 8% of the total support amino groups with methylglyoxal after 7 d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.