Determination of free Mg2+ in isolated heart mitochondria using fluorescent probes.

The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored using the fluorescent probe furaptra. The techniques used for loading, calibrating and using furaptra fluorescence to monitor matrix free Mg2+ are described. Furaptra is loaded as the membrane-permeable acetoxymethyl ester which is hydrolyzed to the impermeant acid form by nonspecific esterases. Loading is sufficient in a 20-min period at 25 degrees C to give signals approximately 5-fold greater than nonloaded mitochondria. Uncertainties concerning the apparent dissociation constant value for Mg2+ for entrapped furaptra and the ability of ionophores to equilibrate Mg2+ across the mitochondrial membrane are discussed. We conclude that our best estimate for matrix Mg2+ in isolated mitochondria is 0.5 mM and that it can change significantly with changing Mg2+ ligand availability in the matrix.