Long-chain free fatty acids (FFAs) play an important role in several physiological and pathological processes such as lipid fusion, adjustments of membrane permeability and fluidity, and the regulation of enzyme and protein activities. FFA-facilitated membrane proton transport (flip-flop) and FFA-dependent proton transport by membrane proteins (e.g., mitochondrial uncoupling proteins) are governed by the difference between FFA’s intrinsic pK(a) value and the pH in the immediate membrane vicinity. Thus far, a quantitative understanding of the process has been hampered, because the pK(a) value shifts upon moving the FFA from the aqueous solution into the membrane. For the same FFA, pK(a) values between 5 and 10.5 were reported. Here, we systematically evaluated the dependence of pK(a) values on chain length and number of double bonds by measuring the ζ-potential of liposomes reconstituted with FFA at different pH values. The experimentally obtained intrinsic pK(a) values (6.25, 6.93, and 7.28 for DOPC membranes) increased with FFA chain length (C16, C18, and C20), indicating that the hydrophobic energy of transfer into the bilayer is an important pK(a) determinant. The observed pK(a) decrease in DOPC with increasing number of FFA double bonds (7.28, 6.49, 6.16, and 6.13 for C20:0, C20:1, C20:2, and C20:4, respectively) is in line with a decrease in transfer energy. Molecular dynamic simulations revealed that the ionized carboxylic group of the FFAs occupied a fixed position in the bilayer independent of chain length, underlining the importance of Born energy. We conclude that pK(a) is determined by the interplay between the energetic costs for 1) burying the charged moiety into the lipid bilayer and 2) transferring the hydrophobic protonated FFA into the bilayer.
