Determination of Intramitochondrial pH and Intramitochondrial-Extramitochondrial pH Gradient of Isolated Heart Mitochondria by the Use of 5,5-Dimethyl-2,4-oxazolidinedione

A newly developed method is described for calculating the intramitochondrial pH of isolated bovine heart mitochondria based on the distribution of 5,5 -dimethyl -2,4-oxazolidinedione between mitochondrial compartments, pK, of 5,5-dimethyl-2,4-oxazolidinedione, and pH of the extramitochondrial medium.The measurement of intramitochondrial pH also permitted calculation of the intra-and extramitochondrial pH gradient.2. Intramitochondrial pH and pH gradient values were calculated for respiration and in ATP-dependent Ca++-, Mgf+-, and Z&+-activated bovine heart mitochondria.3. The intramitochondrial pH of succinate-supported State 4 mitochondria ranged from 7.40 to 7.51 while the extramitochondrial pH ranged from 6.96 to 7.00.The pH gradient ranged from 0.40 to 0.51, depending upon the pH of the extramitochondrial medium.The addition of either 0.565 mM or 6.237 mM Ca++ gave rise to an intramitochondrial pH ranging from 8.31 to 8.58 while the extramitochondrial pH was between 6.70 and 7.00.The pH gradient ranged from 1.51 to 1.75 pH units.Under these conditions the stoichiometry of H+ ejected from and OH-accumulated within the mitochondria was 1.00.