Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations

Abstract

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.