The aggregation in human lens proteins blocks the scavenging of UVA-generated singlet oxygen by ascorbic acid and glutathione.
One hour of UVA irradiation of air-saturated solutions of 2 mg/mL solubilized lens protein aggregates from aged human lens is able to produce on accumulated concentration of more than 2mM 1O2, along with oxidation of 120 nmol/mL of both Trp and His amino acid residues. Increasing concentrations of either sodium azide or ascorbic acid (up to 10 mM) during the irradiation decreased th His destruction by no more than 50-60% with the intact aggregates, but completely prevented the His loss with proteolyzed aggregates. Glutathione (up to 10 mM) was able to protect less than 10% of the aggregate His residues from oxidative damage, whereas His loss was almost completely prevented in the proteolyzed aggregates. Similar data were obtained for teh UVA photolysis of the Trp residues. This finding led us to study the role a protein conformation of these aggregates plays in the diminishing of antioxidant ability to prevent UVA-mediated photolysis of 1O2-sensitive amino acid residues. We found that Trp, His, and Cys are buried in the aggregates and cannot be oxidized by a relatively high concentration of 1O2 generated externally to the protein. Increasing urea denaturation of the aggregates caused exposure of the buried Trp residues as determined by the red shift of the fluorescence maximum and by a marked increase in the acrylamide and iodide fluorescence quenching. The ability of glutathione to prevent Trp oxidation by UVA light correlated directly with the extent of Trp exposure. These data suggest that the aggregation of the lens crystallins during aging produces a barrier, which prevents the access of water-soluble antioxidants to the sites of UVA-dependent singlet oxygen generation. In this case UVA proteolysis of the lens proteins can proceed even in the presence of physiological levels of antioxidants.
Linetsky
M
M
Mason Eye Institute, University of Missouri, Columbia, Missouri 65212, USA.
Ranson
N
N
Ortwerth
B J
BJ
D000375:Aging / Q000502:physiology; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology*; D003459:Crystallins / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005453:Fluorescence; D016166:Free Radical Scavengers / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D010545:Peroxides / Q000494:pharmacology; D010782:Photolysis; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D019810:Sodium Azide / Q000494:pharmacology; D014364:Tryptophan / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects; D014508:Urea / Q000494:pharmacology
D000975:Antioxidants; D003459:Crystallins; D016166:Free Radical Scavengers; D010545:Peroxides; D017382:Reactive Oxygen Species; D006639:Histidine; D014364:Tryptophan; D014508:Urea; D019810:Sodium Azide; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid