Estimation of the pH gradient and donnan potential in de-energized heart mitochondria

The transmembrane pH gradient maintained by nonrespiring, uncoupled heart mitochondria has been estimated using the distribution of methylamine and of 5,5-dimethyl-2,4-oxazolidinedione (DMO) and compared with the delta pH reported by the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under these conditions the protonmotive force approaches zero and the membrane potential (delta psi) should equal 59 delta pH (P. Mitchell and J. Moyle (1969) Eur. J. Biochem. 7, 471-484). The delta pH reported by DMO corresponds closely to that estimated by BCECF and is consistent with a Donnan potential of no greater than about -30 mV (interior negative) for nonenergized mitochondria in a sucrose medium. This potential appears to result from the presence of immobile negative charges in the matrix and is eliminated by addition of 10 to 25 mM KCl. Measurements of delta pH using the methylamine and of delta tsi using the distribution of 42K+ in the presence of valinomycin result in an apparent overestimation of these parameters due to binding of these components to negative sites on the membrane. Increasing ionic strength decreases this contribution of surface potential, but significant binding can still be detected in 100 mM KCl. These studies suggest that 42K+ (or 86Rb+) is far from an ideal probe for measuring delta tsi in respiring mitochondria and may significantly overestimate this parameter, especially in sucrose media.