Mitochondrial membrane potential estimated with the correction of probe binding

Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and / or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207–215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0?4) and tetraphenylphosphonium (TPP⁺). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50–800 μM) and low (under 20 μM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the ‘state of no binding’, the membrane potential of intact mitochondria was estimated to be ?147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl₂, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25°C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the Langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 μM.