BLEACHING OF MEMBRANE-BOUND MEROCYANINE-540 IN CONJUNCTION WITH FREE RADICAL-MEDIATED LIPID-PEROXIDATION

The lipophilic dye merocyanine 540 (MC540) can photosensitize potentia lly lethal cell membrane damage as well as its own degradation (bleach ing). Photobleaching in a test membrane, the human erythrocyte ghost h as been examined. White light irradiation of MC540-sensitized ghosts r esulted in lipid hydroperoxide (LOOH) formation, low-level thiobarbitu ric acid (TBA) reactivity, and dye bleaching (A(568) decay). When the reaction was carried out in the presence of ascorbate (AH(-)), and add ed Fe3+, there was a large enhancement of TBA reactivity (indicative o f free radical-mediated lipid peroxidation) and concomitant increase i n 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 ext ent of bleaching in this system was found to be proportional to the st arting level of LOOH. Like the wave of free radical lipid peroxidation that accompanied it, dye bleaching in AH(-)-treated, preperoxidized g hosts was stimulated by supplemental Fe3+, inhibited by desferrioxamin e or butylated hydroxytoluene (BHT), but unaffected by catalase or sup eroxide dismutase. From this and related evidence, we deduce that: (1) in the absence of Fe3+/AH(-), photoperoxidation and photobleaching oc cur independently and are nonradical, singlet oxygen-mediated processe s; and (2) in the presence of Fe3+/AH(-), 1-electron reduction of phot ogenerated LOOHs results in a surge of lipid peroxidation that amplifi es dye loss via free radical processes. MC540 bleaching might be explo ited as a relatively simple and sensitive indicator of lipid autoxidat ion in isolated membranes and cells.