4.5. Measurement of Mitochondrial Membrane Potential

The mitochondrial membrane potential was estimated using an ion-selective electrode to measure the distribution of tetraphenylphosphonium (TPP⁺). The voltage response of the TPP⁺ electrode to log (TPP⁺) was linear with a slope of 59 ± 1, in conformity with the Nernst equation. Reactions were carried out at 25 °C, in a temperature-controlled water-jacketed chamber with magnetic stirring. Mitochondria (1 mg) were suspended in 1 mL of standard respiratory medium (130 mM sucrose, 50 mM KCl, 5 mM MgCl₂, 5 mM KH₂PO₄, 50 μM EDTA, 5 mM HEPES (pH 7.4) and 2 μM rotenone supplemented with 3 μM (TPP⁺). A matrix volume of 1.1 μL/mg protein was assumed. The measured parameters were membrane potential (mV), depolarization (mV), lag phase (seconds) and repolarization (mV). Readings were recorded in triplicate.