Determination of membrane potential with lipophilic cations. Comparison of estimated values with various phosphonium ions

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–5) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the binding of probes to the membrane was measured. For the probes of n = 0 and n = 1, and for TPP⁺, binding followed the Langmuir adsorption isotherm. For other probes, analysis revealed the presence of two, high- and low-affinity, binding sites. Upon illumination, which generated the membrane potential, the probe molecules were accumulated into the vesicles. If we ignore the membrane-potential-dependent binding of the probe molecules, the estimated values are larger when the probe used is more hydrophobic. We have tested some models describing the amount of probe bound on membranes in terms of concentration of free probe inside and outside the vesicles. No model has fulfilled the criterion of valid estimation that the membrane potentials estimated are independent of probes used. An experimental method for the estimation of true membrane potential is proposed. Effects of tetraphenylboron on the estimation of membrane potential and on the transport rate of phosphonium cations were examined.