PHOTOOXIDATION OF CELL MEMBRANES USING EOSIN DERIVATIVES THAT LOCATE IN LIPID OR PROTEIN TO STUDY THE ROLE OF DIFFUSIBLE INTERMEDIATES

Abstract

Eosin derivatives that bind primarily to lipid or protein sites in erythrocyte membranes were studied in solution and as sensitizers of erythrocyte membranes. In 50% ethanol-water mixtures eosin maleimide (EYMA) and 5-N-hexadecanoyl amino eosin (E16) had nearly identical absorption spectra. Higher ethanol concentrations did not change peak absorbances. In the presence of neutral detergent both sensitizers had equivalent absorbance at all ethanol concentrations. In water, EYMA was more effective than E16 at bleaching RNO, probably because of E16 aggregation into micelles, while in ethanol-water mixtures E16 was slightly more effective at bleaching DPBF, indicating equivalent singlet oxygen generation when the sensitizers are in monomeric form. In water with neutral detergent, azide in the 20 microM range inhibited the majority of RNO bleaching with both sensitizers; in 50% ethanol-water mixtures azide at 1 mM showed a 50% inhibition of DPBF bleaching with both sensitizers. Iodide in the 30 mM range reduced DPBF bleaching by 50% in 50% ethanol-water mixtures. When matched for amount loaded in erythrocyte membranes these sensitizers were about equally effective at sensitizing induction of cation permeability, assayed as rate of delayed photohemolysis, while E16 was slightly more effective at sensitizing loss of cholinesterase (AchE) activity. The relation of lysis rate to load was somewhat steeper for E16 than EYMA. For both sensitizers lysis rate increased at about the 1.5 power of light dose. Deoxygenation of the reaction media with argon totally blocked detectable photomodification. Ghost membranes made from sensitizer-treated cells were effective generators of singlet oxygen, assayed by RNO bleaching. However, when mixtures of EYMA-treated and untreated cells were illuminated together, only the EYMA-treated cells showed evidence of photomodification. Azide at 5 mM slowed the initial rate of AchE loss by about 75% with E16 and EYMA. Azide partially slowed photohemolysis. Azide decreased RNO bleaching by sensitizer-treated ghosts as it did in water with detergent micelles. A deuterium oxide solvent increased photohemolysis rate with E16 by 41%, but did not increase photohemolysis rate with EYMA. Deuterium oxide had a positive, but statistically insignificant effect on loss of AchE with both sensitizers. Deuterium oxide following illumination slowed lysis sensitized by both sensitizers more than 50%. Iodide exerted a modest inhibition of photohemolysis and loss of AchE sensitized by E16, but had virtually no influence on sensitization by EYMA. The results in solution indicate that EYMA and E16 have nearly identical photochemical properties when in monome