HYDROPHOBIC BARRIERS OF LIPID BILAYER-MEMBRANES FORMED BY REDUCTION OF WATER PENETRATION BY ALKYL CHAIN UNSATURATION AND CHOLESTEROL

The hydrophobicity profiles across phosphatidylcholine (PC)-cholestero l bilayer membranes were estimated in both frozen liposome suspensions and fluid-phase membranes as a function of alkyl chain length, unsatu ration, and cholesterol mole fraction. A series of stearic acid spin l abels, with the probe attached to various positions along the alkyl ch ain, cholesterol-type spin labels (cholestane and androstane spin labe ls), and Tempo-PC were used to examine depth-dependent changes in loca l hydrophobicity, which is determined by the extent of water penetrati on into the membrane. Local hydrophobicity was monitored primarily by observing the z component of the hyperfine interaction tenser (A(z)) o f the nitroxide spin probe in a frozen suspension of the membrane at - 150 degrees C and was further confirmed in the fluid phase by observin g the rate of collision of Fe(CN)(6)(3-) with the spin probe in the me mbrane using saturation recovery ESR. Saturated-PC membranes show low hydrophobicity (high polarity) across the membrane, comparable to 2-pr opanol and 1-octanol, even at the membrane center where hydrophobicity is highest. Longer alkyl chains only make the central hydrophobic reg ions wider without increasing the level of hydrophobicity. Introductio n of a double bond at C9-C10 decreases the level of water penetration at all locations in the membrane, and this effect is considerably grea ter than the cis configuration than with the trans configuration. Inco rporation of cholesterol (30 mol %) dramatically changes the profiles; it decreases hydrophobicity (increases water penetration) from the po lar headgroup region to a depth of approximately C7 and C9 for saturat ed- and unsaturated-PC membranes, respectively, which is about where t he bulky rigid steroid ring structure of cholesterol reaches in the me mbrane. Membrane hydrophobicity sharply increases at these positions f rom the level of methanol to the level of pure hexane, and hydrophobic ity is constant in the inner region of the membrane. Thus, formation o f effective hydrophobic barriers to permeation of small polar molecule s requires alkyl chain unsaturation and/or cholesterol. The thickness of this rectangular hydrophobic barrier is less than 50% of the thickn ess of the hydrocarbon regions. Results obtained in dioleoyl-PC-choles terol membranes in the fluid phase are similar to those obtained in fr ozen membranes. These results correlate well with permeability data fo r water and amino acids in the literature.