Membrane potentials in mitochondrial preparations as measured by means of a cyanine dye

Changes in the fluorescent intensity of the dye 3,3′-dipropylthiodicarbocyanine iodide were measured in suspensions of hamster liver mitochondria upon the development of a K⁺ diffusion potential by the addition of valinomycin and upon the development of the energized state by the addition of succinate or ATP. The changes (large decreases) seen with the addition of succinate or ATP (inhibitable by NaCN and oligomycin respectively) were comparable to those recorded upon the addition of valinomycin to mitochondria suspended in media containing low concentrations of K⁺. The change observed with succinate was partially reversed by the addition of either 2,4-dinitrophenol or ADP. Oligomycin prevented the reversal seen with ADP. Decreases in fluorescent intensity were also recorded when succinate was added to suspensions of inner membranes (prepared from rat liver mitochondria) containing the dye. With submitochondrial particles (also from rat liver mitochondria), however, increases in fluorescent intensity were seen upon the addition of succinate or ATP. These observations are consistent with the idea that a large negative (internal) potential develops across the inner membrane of the mitochondrion during energization and with other aspects of the chemiosmotic hypothesis.