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ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS
Author(s) -
Ortwerth B. J.,
Linetsky Mikhail,
Olesen Paul R.
Publication year - 1995
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1995.tb02368.x
Subject(s) - chemistry , glycation , ascorbic acid , tryptophan , histidine , dehydroascorbic acid , hydrogen peroxide , lysine , biochemistry , absorbance , incubation , crystallin , chromatography , amino acid , food science , receptor
— ‐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/cm 2 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. The incubation of lens proteins with dehydroascorbic acid or l ‐threose, but not fructose, produced equivalent glycation, protein crosslinking and sensitizer activity. The relative sensitizer activity of the 4 week glycated sample was quantitatively very similar to that of a water‐insoluble fraction from aged human lenses. These data are consistent with the hypothesis that the protein‐bound brunescence in the lens may be advanced glycation endproducts, which are formed in large part by the oxidation products of ascorbic acid, and that these compounds may contribute significantly to the UVA sensitizer activity present in aged human lenses.