An optical study of the exchange kinetics of membrane bound molecules.

The kinetics of molecular exchange between lipid bilayers are studied using a special fluorescence technique. Pyrene and pyrene decanoic acid are chosen as typical examples of an apolar and amphiphilic molecule. Their property of forming dimers in the excited state (excimer) is exploited. The time dependencies of monomer and excimer intensities after rapid mixing of vesicles doped with fluorescent probe with undoped ones are studied by stopped-flow technique. The transient curves reveal the information on the exchange kinetics. A theoretical analysis shows that the molecular exchange follows a first order kinetics. Surprisingly short half life-times tex for this exchange process are obtained (for dipalmitoyl phosphatidylcholine tex=3.3 s for T=23 degrees C, tex=0.5 s for T=68 degrees C). Multilamellar systems (onion like structure) show much slower exchange rates. The exchange rates are nearly equal for polar and unpolar molecules. Addition of cholesterol has a strong reducing effect on this rate. Charging of dipalmitoyl phosphatidylcholine vesicle surfaces by the addition of (a) EuCl3 to the aqueous phase and (b) dipalmitoyl phosphatidic acid to the lipid phase reduces the exchange rate by about an order of magnitude above the phase transition. In a separate experiment it is shown that the lipid exchange or fusion for two different lipids is a much slower process compared to the label exchange. In fact vesicles kept below the phase transition temperature Ttr for both lipids, do not fuse even after 70 h. Noticeable fusion occurs after 10 h when the mixture stays above Ttr. Experiment shows that the fusion of pure lipid vesicles is not very much affected by the presence of a charged lipid. Change in concentration of the monovalent ions in the aqueous solution by two orders of magnitude does not have an appreciable effect on the exchange rate of phospholipids.