In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.
Keywords: 2,4-dinitrophenol; 3,3′-dipropylthiadicarbocyanine iodide; BLM; C(12)-FL; C(18)-FL; C(4)-FL; C(8)-FL; C(n)-FL; CCCP; DNP; DPhPC; DiS-C(3)-(5); EggPC; FCCP; FL; Fluorescein; IMM; Membrane potential; Mitochondria; Protonophore; RLM; TMRE; TPP(+); Uncoupler; bilayer lipid membrane; carbonyl cyanide m-chlorophenyl hydrazone; carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone; diphytanoylphosphatidylcholine; egg yolk phosphatidylcholine; fluorescein n-alkyl ester with n hydrocarbon units; fluorescein n-butyl ester; fluorescein n-dodecyl ester; fluorescein n-octadecyl ester; fluorescein n-octyl ester; inner mitochondrial membrane; rat liver mitochondria; tetramethylrhodamine ethyl ester; tetraphenylphosphonium cation; unsubstituted fluorescein.
© 2013.