The transfer of a fatty acid, 9-(3-pyrenyl)nonanoic acid (PNA), between synthetic phospholipid single-bilayer vesicles is a first-order process, with no dependence on either the concentrations of donor and acceptor vesicles or the chemical composition of acceptor vesicles. The invariance in transfer rates over 50-fold change in concentration of vesicles and an identical rate constant for transfer of the fluorescent fatty acid from vesicles to solution is compelling evidence that the transfer of PNA between phospholipid vesicles proceeds through the aqueous phase. High ionic strength, 4 M NaCl, reduces the reaction rate 25-60-fold, depending on the pH and temperature. The rate of transfer of ionized PNA is faster than that of the protonated form. At 28 °C and pH 7.4, the rate constant for transfer of PNA between dimyristoylphosphatidylcholine is 5.4 s-1; at pH 2.8, it is 0.08 s-1. Between pH 2.8 and 7.4, observed rates are the arithmetic sum of the rates of protonated and ionized PNA; transbilayer “flip-flop” appears to be faster than transfer between vesicles. The activation energy for this process is determined by donor lipid phase and by the ionization state of the fatty acid. The activation energy for PNA transfer is lower below the transition temperature of the donor lipid vesicle than it is above it. The difference in the activation energy above and below the transition temperature is the same at pH 2.8 and 7.4. Arrhenius plots for transfer for protonated and ionized PNA show that the activation energy includes energies to offset phasedependent and phase-independent free energies of association for the aryl-alkyl side chain and excess free energy of association of the protonated carboxyl group and the vesicle. Thermodynamics of the transition state and distribution parameters indicate that the rate-limiting step appears to be the solvation of PNA in the interfacial water at the phospholipid surface. Furthermore, the calculated pKa of the activated state molecule is that of an aqueous carboxyl group, about 4.5. From kinetic considerations, the pKa of the fatty acid in the phospholipid vesicle is 7.1. An excess free energy of association between the protonated carboxyl and the phospholipid membrane accounts for a 100-fold suppression of PNA ionization. The fatty acid anion is the chemical entity that transfers at physiological pH. These results imply that the properties of interfacial water limit the transfer rates of hydrophobic compounds between membrane surfaces.
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