Hydrogen ion concentration changes in mitochondrial membranes.

1. Bromthymol blue added to rat liver mitochondria at concentrations in the range 0.4 to 4.0 mµmoles per mg of protein is tightly bound as indicated by centrifugation and washing of the mitochondria. 2. The absorbance of the dye is increased on adding Ca++ or Mn++ to the mitochondria in the absence of a permeant anion. The extent of increase is 0.03 cm-1 for 167 µ m Ca++ at 618 mµ for 0.5 mµmole of bromthymol blue per mg of protein. 3. The spectrum of the indicator bound to the mitochondria has shifted only slightly to longer wave lengths than in the solution. The indicator shows very nearly the same absorbance changes on alkalinization of the mitochondrial suspension as it does upon addition of small amounts of calcium to the mitochondria. 4. The absorbance increase is independent of the pH of the suspension medium (6.5 to 7.4) and of its buffer capacity from 2.0 m m to 0.3 m sodium succinate. 5. At low protein concentrations (1 to 2 mg per ml), the kinetics of the absorbance increase is compared with the glass electrode recordings and indicates an equal and opposite pH change in the suspension medium of a similar time course. 6. At higher protein concentrations (5 to 10 mg per ml), a similar phenomenon is observed, but only after several prior additions of Ca++; endogenous material (termed Hi+) present, in terms of Ca++, at levels of approximately 40 mµmoles per mg of protein counteracts the initial absorbance increase. A small amount of added phosphate readily serves as Hi+, and endogenous phosphate at 8 mµmoles per mg of protein would afford an appropriate source of Hi+. However, the endogenous material may be phospholipid. 7. The absorbance increase on calcium additions with bromthymol blue as an indicator can be counteracted and even reversed with a 30-fold excess of bovine serum albumin, suggesting that the indicator can be removed from the mitochondrial membranes. 8. The Ca++-induced absorbance increase is nullified rapidly by additions of permeant anions such as phosphate or acetate. Ammonium ions at a 3 m m concentration do not prevent the acidification caused by acetate, and 0.1 m concentrations only slightly facilitate the alkalinization on addition of Ca++. 9. It is concluded that rapid accumulation of a cation such as Ca++ in the absence of a permeant anion can result in an intramitochondrial alkalinization and a pH gradient of about 1 pH unit across the mitochondrial membrane. This gradient develops only as a consequence of cation accumulation in the absence of a permeant anion. In the presence of permeant anions, particularly phosphate under the conditions of oxidative phosphorylation, there is no evidence of a measurable pH gradient across the mitochondrial membrane. 10. The intramitochondrial location of the indicator and of the site of calcium accumulation is tentatively assigned to the intracristal space because of the insensitivity of the bromthymol blue response to changes of osmolarity from 0.1 to 0.8 m . 11. The reaction mechanism for cation accumulation based upon the reaction with the X ∼ I intermediate of energy transfer meets the needs of the available experimental data.