Safranine as a probe of the mitochondrial membrane potential

One of the most central postulates of the chemiosmotic hypothesis of oxidative phosphorylation is the existence of a large electrochemical proton gradient (protonmotive force) across the mitochondrial membrane [ 1,2] . This gradient, which is composed of a membrane potential and a pH differential, is postulated to be the obligatory link between respiration and phosphorylation in the process of oxidative phosphorylation. The membrane potential has been suggested to be the major component of the electrochemical proton gradient under most conditions, and measurements of this potential are therefore essential for the understanding of the mechanism of oxidative phosphorylation and mitochondrial ion transport. The mitochondrial membrane potential has mainly been estimated from the distribution of K’ or Rb’ across the mitochondrial membrane in the presence of the ionophore valinomycin [3-g]. This method has given fairly consistent results and membrane potentials in the range 130190 mV (positive polarity extramitochondrially) have generally been reported in coupled mitochondria supplemented with either substrate or ATP. The differences in the values reported by different workers may for the most part be attributed to the use of different values for the matrix volume. The use of a method of this kind, which requires partially artificial experimental conditions, makes confirmation by other independent methods most desirable. This has prompted several workers to search for suitable spectroscopic membrane potential probes. The fluorescent probe MC V has been used by Chance and collaborators [9,10] as a membrane potential indicator in mitochondria, and Laris et al. [ 111 have employed a cyanine dye