Figure 1. Reaction of photooxidized bisretinoid with glutathione and glyceraldehyde-3-phosphate dehydrogenase. Data are mean±standard error of mean (SEM) of 3–5 experiments. A: Incubation of glutathione (GSH) with photooxidized-A2E (photooxo-A2E) results in a concentration-dependent decrease in GSH-mediated reduction of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) to 2-nitro-5-thiobenzoic acid (TNB). Background absorbance (DPBS) and absorbance values for photooxo-A2E samples alone were subtracted. B: Total glutathione (GSH/GSSG) in ARPE-19 cells that had accumulated A2E and were exposed to 430 nm light. Total glutathione was measured in a DTNB-based assay in the presence of glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH), and values were normalized to untreated controls. C: Incubation of GSH (650 µM) with photooxo-A2E and peroxy-A2E (100 µM) diminishes the ability of GSH to subsequently reduce DTNB to TNB. DPBS and absorbance values for photooxo-A2E and peroxy-A2E alone were subtracted and data were normalized to GSH control. D: A2E and all-trans-retinal dimer were irradiated in the presence of GSH, after which DTNB was added. The background was subtracted and values normalized to the GSH control. E: Prior incubation of GSH with photooxo-A2E decreases the ability of GSH to reduce DTNB even in the presence of GSH reductase (GR) and NADPH. DPBS and absorbance values for photooxo-A2E samples alone were subtracted and data were normalized to GSH only.