Bromthymol Blue as a pH Indicator in Mitochondrial Suspensions

The distribution of the functional group of the pH indicator bromthymol blue across the osmotic barrier component (or M phase) of the cristae membrane separating the inner from the outer aqueous phase of rat liver mitochondria has been measured by three partially independent methods. In mitochondria in State 5, under the usual conditions, only about one third of the total bromthymol blue reacts in the inner phase. In State 3, in presence of a high concentration of valinomycin in a KCI medium, approximately the same proportion of the bromthymol blue reacts in the inner phase as in State 5; but in State 4, practically all the bromthymol blue is expelled across the M phase and registers the pH of the outer phase. The bromthymol blue technique is incompetent to measure the pH of the inner phase of mitochondria in State 4; but a method is described that will permit the approximate quantitative measurement of the intramitochondrial pH from the light extinction of bromthymol blue in State 5 (or in State 3 in presence of valinomycin) provided that the distribution of the indicator and the internal and external buffering powers of the mitochondrial suspension are known. The expulsion of the bromthymol blue from the mitochondria in State 4 is attributable to the development of an electric potential across the M phase.