NMR studies on phospholipid bilayers. Some factors affecting lipid distribution.

1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phospholipid mixture contains negative lipids. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4-5 Hz and the individual linewidths are about 4Hz. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phosphatidylinositol and phosphatidic acid. 6. In mixtures of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55-58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. It is concluded that both charge and the packing properties of the head group are major factors in determining the distribution of phospholipids in mixed vesicles.