Kinetics of Proton-Hydroxyl Transport across Lecithin Vesicle Membranes as Measured with a Lipoid pH-Indicator

Abstract

When unilamellar vesicles are prepared in the presence of 3-Palmitoyl-7-oxy-coumarin (abbreviation 3P-UBF) this lipoid pH-Indicator is anchored by its fatty acid chain to the membrane and can be used to measure pH-changes at the outer and inner membrane surface (ranging from pH 5 to pH 9.5). By rapidly-changing the pH of the outer aqueous phase a pH- gradient is set up across the vesicle membrane. The rate of the subsequent H+ or OH- influx into the vesicles can be measured as a change of the 3P-UBF absorbance at 424 nm. This was done with a stopped-flow-spectrophotometer at temperatures between 10 °C and 50 °C. Suspensions of vesicles prepared from egg-lecithin or ʟ-dipalmitoyl-lecithin were investigated in buffered salt solutions. The influence of Na+, K+, Cl-, SO4 2- and valinomycin on the rate of absorbance changes was studied at different temperatures. It was found that the rate of the pH-equilibration between the aqueous phase outside and inside the vesicles depends on the direction of the pH-gradient. This new result together with a high H+/OH- permeability of vesicle membranes found in recent studies from other laboratories and confirmed by this investigation is interpreted to indicate a higher permeability of the vesicle membrane to OH--ions compared to H+-ions. (Calculated values are: POH = 1.4 × 10-4cm/s at pH 9 and PH = 8.3 × 10-7 cm/s at pH 5 and 20 °C.) All data described in the literature in detail agree with this suggestion but a pH-dependence of POH and PH cannot be excluded.