Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

Abstract

We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the "on-off" model (E.B. George et al., J. Membrane Biol., 103:245-253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a "sigmoidally shaped" increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.

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

“…The hemispherical bilayer lipid membrane system was originally developed by Dragsten and Webb (1978) and the optical detection system is similar to that described by Waggoner, Wang and Tolles (1977). The structures of the three membrane potential probes used in this work are given in Nyirjesy et al (1988). In order to identify systematic variations in the signal as a function of time (Materials and Methods section, George et al, 1988), membrane voltage settings were regularly returned to a reference setting.…”

“…If the potential drops linearly across the membrane,/3 would be equal to the "depth" of the chromophore in population I divided by the membrane thickness (see Fig. 4 in Nyirjesy et al, 1988). The larger the value of/3 the greater the driving force of the membrane potential to displace the chromophore to a new location.…”

“…The model, which is depicted in Fig. 4 of Nyirjesy et al (1988), describes the voltage-dependent movement of dye between population I, which is in the membrane hydrocarbon, and population II, which is in the unstirred aqueous region of thickness 0 just off the surface of the membrane. The dye in population II is in equilibrium with the dye in the bathing solution and has the same average concentration.…”

“…The dye in population II is in equilibrium with the dye in the bathing solution and has the same average concentration. In Part II of this series, Nyirjesy et al (1988) developed an equilibrium thermodynamic equation based on the "on-off" model in order to analyze the dependence of the signal on voltage and on the membrane-binding properties of the dye. According to the theory, the absorbance signal is proportional to the amount of probe An moving between populations I and II and is given by their Eq.…”

“…Potential-dependent light absorption changes have been determined with oxidized cholesterol bilayer lipid membranes bathed in an aqueous solution containing the dye. The previous papers were concerned with the dependence of the absorption signal on the wavelength and polarization of the illumination and on the length of alkyl groups attached to the chromophore structure Nyirjesy et al, 1988). The present paper reports experimental mea- surements of the dependence of the absorption signal on the size of the voltage steps and on the offset voltage of the pulse train used to generate the signal.…”