DIRECT NMR EVIDENCE FOR ETHANOL BINDING TO THE LIPID-WATER INTERFACE OF PHOSPHOLIPID-BILAYERS

The mechanisms behind the membrane-mediated effects of ethanol were examined via the interaction of ethanol with phospholipid bilayers at hydration levels of 10-12 water molecules per lipid. H-2 and P-31 nuclear magnetic resonance (NMR) spectroscopy was used to monitor deuterated water and ethanol and the headgroups and acyl chains of neutral phospholipids. Ethanol was found to interact strongly with both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bilayers, giving H-2 NMR quadrupolar splittings for CH3CD2OH between 6.3 and 9.4 kHz. The quadrupolar splittings for ethanol in gel-phase lipids remained well resolved and were not significantly larger than those in the L(alpha) phase, suggesting that little or no ethanol was bound in the hydrocarbon interior of the bilayer. Ethanol binding significantly altered the orientation of the lipid headgroups, as shown with headgroup-deuterated PC bilayers. The entire lengths of the acyl chains were significantly disordered by the ethanol interaction, evidenced by significant reductions in the H-2 NMR order parameters of the chains. The disordering corresponds to an increase in the area per lipid by an estimated 6% with one ethanol molecule per lipid, and a total of 18% with a second ethanol per lipid. This pronounced area increase is presumably caused by the disruption of lipid packing in the rigid region of the glycerol backbone rather than in the acyl chains, since the order of hydrocarbon chains is not affected to a significant degree by incorporation of alkanes and long-chain alcohols into the hydrocarbon interior. From these data it was concluded that ethanol interacts with phospholipid bilayers at the lipid-water interface (consisting of the headgroup, glycerol backbone, and uppermost chain methylene groups) rather than in the hydrocarbon interior. An interfacial binding of ethanol that is also capable of disordering the entire length of the acyl chains could explain the small ethanol-induced fluidization of membrane lipids that has been reported frequently in the literature.