Titration of mitochondrial buffer by accumulated anions

Acidimetric titration of intact rat liver mitochondria discloses a buffer power of about 45 mEquiv per g between pH 7 and 8, the value rises to 60 mEquiv per g per pH unit after lysis using Triton X-100. The existence and properties of this buffer system have been related to mitochondrial anion accumulation. The uptake of permeant anions by mitochondria occurs to a charge-dependent extent and they are in electrochemical equilibrium with each other and the protons as in a Donnan system. Adding permeant anion causes the intramitochondrial anion content to rise towards a saturation level, the inside to outside concentration ratio falls and concomitantly the transmembrane proton gradient diminishes, making the interior less alkaline. The falling internal pH is associated with protonation of the internal buffer, thus providing a second method for measuring the buffer power, a method which also tests the arguments used in the calculations. The titration curve is constructed by relating the internal pH (deduced from the permeant anion ratio) to the total internal anion equivalents which in turn determines the ionization state of the buffer because the sum of the internal anion equivalents, including the buffer anion, equals the equivalents of internal cation. The buffer power so measured agrees with the acidimetric method applied to lysed mitochondria. The disparity between the acidimetric data from lysed and unlysed mitochondria follows theoretical predictions.