RESPONSE OF THE ELECTROCHROMIC DYE, MEROCYANINE 540, TO MEMBRANE-POTENTIAL IN RAT-LIVER MITOCHONDRIA

Merocyanine binds extensively to rat liver mitochondria in spite of the presence of a sulfonic acid group which would suggest only limited penetration through the membrane. Passive binding shows both tight and weak binding components and is dependent on salt concentration and ionic strength in accord with the Gouy-Chapman theory. The binding of merocyanine to mitochondria is accompanied by both a fluorescence enhancement and a spectral shift. Induction of an electrical field by either respiration or K+ diffusion potential results in a partial reversal of the spectral shift seen on dye binding. At low temperature, the merocyanine spectral response to an electrical field is biphasic, consisting of a fast phase with a t1/2 of less than 1 sec at 15-degrees-C and a slower phase which may vary considerably in rate and extent with conditions. The spectral shift during the two phases appears similar, but differ in sensitivity to ionic strength and temperature. The spectral shift during the fast phase at 15-degrees-C indicates that the major component is a decrease in bound monomer and an increase in the aqueous dimer, indicating an "on-off" mechanism. It is suggested that the fast and slow phases of the merocyanine response may be due to two different populations of dye, possibly located at the outer and inner surfaces, respectively, of the mitochondrial membrane. The electrophoretic movement of the dye located in the membrane interior would result in the temperature-sensitive slow phase response. Demonstration of the proportionality of the fast phase response to the magnitude of the membrane potential suggests the usefulness of merocyanine in studies with mitochondrial systems.