Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to ∼40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (/o) and the liquid disordered (/d) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the /d phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (EA) in the range of 28-40 kJ/mol. The /o phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The EA for this phase was significantly larger (55-65 kJ/mol) than for the /d phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the /d and /o regions in the bilayer on the timescale of the NMR experiment (100ms). Thus, strong evidence has been obtained that fluid domains (with size of μm or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained EA:s indicate that these systems are in the /d phase at all CHOL contents for temperatures above 25°C.
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