Charge and pH dependent drug binding to model membranes. A 2H-NMR and light absorption study

The binding of the local anesthetics tetracaine and procaine to model membranes of egg phosphatidylcholine and bovine phosphatidylserine has been studied by 2H-NMR and light absorption. Dispersions of drug-lipid mixtures in 0.1 M NaCl were centrifuged and the concentration of drug in the supernatant was measured by ultraviolet light absorption. Several freeze-thaw cycles of the sample were used before centrifugation to facilitate equilibration of the drug between the bilayers. Binding curves for the drug were obtained as a function of pH. The results were simulated by a theoretical model based on the Gouy-Chapman theory, in which both the charged and the uncharged forms of the drug, and the equilibrium between them, were included. Two deuterated forms of the drugs, [2H6] tetracaine and [2H4] procaine, were used for the 2H-NMR experiments. In most cases the 2H-NMR spectrum contained a broad central resonance and an underlying quadrupolar pattern. However, after five freeze-thaw cycles only a single broad resonance was observed under most conditions. Particle size measurements showed that freeze-thawing resulted in a more uniform population of liposomes of smaller average diameter than those obtained by simple vortex mixing. The single broad resonance observed in both cases is interpreted as due to rapid exchange of the anesthetic between lipid and bulk solution. In the absence of freeze-thawing, the quadrupolar pattern is attributed to anesthetic species in exchange with only a limited amount of water. The data suggest that a true equilibrium between lipid, water and anesthetic is only attained after freeze-thawing.