Estimation of matrix pH in isolated heart mitochondria using a fluorescent probe

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the pH-sensitive 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The resulting charged forms of the probes are retained in the mitochondrial matrix and appear well-suited for the estimation of pCa and pH in this compartment. The mitochondria esterase activity is stimulated by Ca2+, inhibited by butacaine and quinine, and shows an alkaline pH optimum. The esterase has a similar affinity for the two probes (Km about 1.5 microM) and a somewhat higher Vmax for BCECF. Intramitochondrial pH can be determined by recording the ratio of the fluorescence of matrix BCECF at its excitation maximum of 509 nm to that at 450 nm, an excitation wavelength that is unresponsive to pH. A calibration plot relating the fluorescence ratio to pH is constructed using detergent-lysed mitochondria and the excitation maximum of 500 nm for BCECF in aqueous solution. Estimates of matrix pH by BCECF fluorescence in its useful range (pH 6 to 8) agree well with values obtained using the distribution of 5,5-dimethyl-2,4-oxazolidenedione. In protocols in which the fluorescence with excitation at 450 nm does not vary, a direct recording of BCECF fluorescence with excitation at 509 nm can be used to follow the kinetics of matrix pH changes.