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

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