Abstract
The assessment of weak acid membrane permeability (Pm) frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpHin, in response to a sudden augmentation of external acid concentration. However, ΔpHin 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 ΔpHin, as we show here. The uniexponential time constant (τ) of ΔpHin(t) is an invalid measure of Pm as Arrhenius plots of Pm and reveal different activation energies for acid influx. We calculate Pm 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 Pm of 30.8 ± 3.5 μm/s for formic acid for 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles.
Highlights
Many pharmacologically important substances belong to the class of weak acids and bases [1,2].Such drugs are appealing because they may permeate membranes in their neutral form and yet attain high water solubility in their charged form at physiological pH values [3]
In the most widely used assay, large unilamellar lipid vesicles (LUV) are exposed to a jump in external [ AH ] concentration and Pm is derived from the time constant (τ ) of exponential intravesicular pH change [6,7]
The volume distribution returned by the Dynamic Light Scattering (DLS) measurements
Summary
Many pharmacologically important substances belong to the class of weak acids and bases [1,2] Such drugs are appealing because they may permeate membranes in their neutral form and yet attain high water solubility in their charged form at physiological pH values [3]. Cellular uptake of these substances is intricately linked to protonation and deprotonation reactions [4]. Where k+ and k− denote the reaction rates for the deprotonation and protonation reaction, respectively These pH changes are used to assess weak acid membrane permeability, Pm [5]. The assumed proportionality between Pm and τ is only valid in a very limited set of experimental conditions as demonstrated by the following derivation: The flux density ( J ) of a weak acid across the membrane of area (S) into a spherical vesicle of radius (r ) is defined as:
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