The effect of fatty acids on the surface potential of phospholipid vesicles measured by condensed phase radioluminescence.

(1) The carboxyl group of fatty acids has a very low pK value, which is shifted into the physiological pH range when they are incorporated in a phospholipid membrane. As a result of a pH increase the surface charge and surface potential of the membrane increase. (2) The titration of the carboxyl group was observed with condensed phase radioluminescence. This technique uses the electron emitted by the tritiated membrane probe (oleic acid or cholesterol) to excite a fluorophore also incorporated in the bilayer. (3) The phase transition of dimyristoyl phosphatidylcholine vesicles labelled within 12-(9-anthroyloxy)stearic acid was measured by condensed phase radioluminescence at different pH values. (4) We related the condensed phase radioluminescence signal to the asymmetrical distribution of the fluorophore between the inner and outer layer of the lipid membrane which is induced by the repulsion of the negatively charged fatty acids. (5) We showed that the condensed phase radioluminescence signal is proportional to the protonation of the carboxyl group. On this basis, the broadening of the titration curve can be explained as an effect of the self-induced membrane potential calculated using the Gouy-Chapman theory. (6) Ca2+ drastically reduces the flip-flop rate of fatty acids across the membrane and also caused a decrease in the asymmetric distribution. (7) We concluded that a fatty acid can act as a membrane surface buffer. The pK value of 12-(9-anthroyloxy)stearic acid in a dimyristoyl phosphatidylcholine membrane is 7.0 +/- 0.3. (8) We discuss the results with respect to aggregation, fusion and clustering.