Impermeant potential-sensitive oxonol dyes: III. The dependence of the absorption signal on membrane potential

We have measured potential-dependent changes in the absorption of light by oxidized cholesterol bilayer lipid membranes in the presence of impermeant oxonol dyes. The magnitude of the absorption signal increased linearly with the size of potential steps over a range of 500 mV. The signal also increased when the offset voltage of the pulse train was increased from -150 to +150 mV. The data are consistent with the "on-off" mechanism proposed by E. B. George et al. (J. Membrane Biol. 103:245-253, 1988) in which the probe undergoes potential-dependent movement between a binding site in the membrane and an aqueous region just off the surface of the membrane. An equilibrium thermodynamic analysis of the experimental data indicates that the negatively charged oxonol chromophore senses only 5-10% of the total membrane potential difference across the membrane when it is driven into a nonpolar binding site on the membrane.