Bleaching of membrane-bound merocyanine 540 in conjunction with free radical-mediated lipid peroxidation

The lipophilic dye merocyanine 540 (MC540) can photosensitize potentially lethal cell membrane damage as well as its own degradation (bleaching). Photobleaching in a test membrane, the human erythrocyte ghost has been examined. White light irradiation of MC540-sensitized ghosts resulted in lipid hydroperoxide (LOOH) formation, low-level thiobarbituric acid (TBA) reactivity, and dye bleaching (A568 decay). When the reaction was carried out in the presence of ascorbate (AH-), and added Fe3+, there was a large enhancement of TBA reactivity (indicative of free radical-mediated lipid peroxidation) and concomitant increase in the rate of photobleaching. Rapid bleaching also occurred when MC540 was incubated in the dark with ghosts that had been photoperoxidized with another dye (a phthalocyanine) and then exposed to AH-. The extent of bleaching in this system was found to be proportional to the starting level of LOOH. Like the wave of free radical lipid peroxidation that accompanied it, dye bleaching in AH(-)-treated, preperoxidized ghosts was stimulated by supplemental Fe3+, inhibited by desferrioxamine or butylated hydroxytoluene (BHT), but unaffected by catalase or superoxide dismutase. From this and related evidence, we deduce that: (1) in the absence of Fe3+/AH-, photoperoxidation and photobleaching occur independently and are nonradical, singlet oxygen-mediated processes; and (2) in the presence of Fe3+/AH-, 1-electron reduction of photogenerated LOOHs results in a surge of lipid peroxidation that amplifies dye loss via free radical processes. MC540 bleaching might be exploited as a relatively simple and sensitive indicator of lipid autoxidation in isolated membranes and cells.