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N-Cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4) and N-cyclohexyl-N'-(1-pyrenyl)carbodiimide (NCP) are two novel fluorescent analogues of the mitochondrial inhibitor dicyclohexylcarbodiimide (DCCD). Although nonfluorescent in aqueous media, both compounds form fluorescent conjugates with mitochondrial electron transport particles (ETPH) or purified H+-ATPase (F1-F0) vesicles. DCCD prevents the reaction of ETPH with both NCD-4 and NCP. The fluorescent probes are effective inhibitors of ATPase activity and ATP-driven membrane potential, although their reaction rates are considerably slower than that of DCCD. The fluorescence of NCD-4- or NCP-treated H+-ATPase is quenched by hydrophobic spin-label nitroxide derivatives of stearic acid (chi-NS) in the order 16-NS greater than 12-NS greater than 7-NS approximately equal to 5-NS, whereas membrane-impermeant iodide ions have negligible effect. The quenching behavior of 16-NS (the most effective quencher) suggests that a small fraction of labels remain inaccessible to the quencher. It is concluded that the DCCD-binding sites are oriented toward the membrane lipids and are located in the lipid bilayer ca. 18 A from the membrane surface.

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