The Effect of Buffers on Weak Acid Uptake by Vesicles

The assessment of weak acid membrane permeability (P_m) frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpH_in, in response to a sudden augmentation of external acid concentration. However, ΔpH_in may be primarily governed by non-instantaneous protonation and deprotonation reactions of (i) the acid itself, (ii) the buffer molecules, and (iii) the fluorescent pH reporter dye. Moreover, buffer concentration and acid gradient also serve as determinants of ΔpH_in, as we show here. The uniexponential time constant (τ) of ΔpH_in(t) is an invalid measure of P_m as Arrhenius plots of P_m and τ reveal different activation energies for acid influx. We calculate P_m by fitting a mathematical model to experimental stopped-flow traces. The model takes into account not only the time course of total internal buffer capacity but also (i) water self-dissociation, (ii) volume changes due to acid induced osmotic water flow, and (iii) the spontaneous membrane proton leak. It allows extracting a P_m of 30.8 ± 3.5 μm/s for formic acid for 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles.