The Proton nuclear magnetic resonance (PMR) spectroscopy has been applied extensively in recent years to elucidate the structural or conformational properties of phospholipids and biomembranes (1). Most of these studies, however, are concerned primarily with the mobility of fatty acyl chains of the bilayer (2, 3). As reported several times (4-6), paramagnetic ions such as europium (Eug+) added to the exterior of lipid vesicles shift the field position of the -N+(CHI3)3protons of phosphatidylcholine (PC) molecules that reside on the outer surface of lipid vesicles, which are closed shelllike spheres of about 250 X in diameter, each comprised of a single, continuous, lipid-bilayer membrane enclosing a volume of aqueous solution (7). Using these paramagnetic ions as shift markers, the head groups in the two halves of vesicle bilayers can be spectrally identified. Consequently, the application of PMR technique has now been extended to investigate the structural properties of the outer and (or) inner polar head regions of the bilayer. The interaction of phospholipid with cholesterol in the bilayer structure has been studied recently by a variety of physical techniques (8-10). On the basis of these studies, it has been proposed that the effect of cholesterol on phospholipids in the bilayer structure is to restrict differentially the number of steric conformations accessible to the segments of the fatty acyl chains (11). However, more recent studies have shown that the 3,8-OH group of cholesterol is also of crucial importance for phospholipid-sterol interaction (12, 13), and, as a consequence, interactions in the polar head regions of the bilayer should be examined further.In this preliminary communication, we report the effects of cholesterol content on the -N+(CH3)3 proton resonance signal of the vesicle bilayer in the presence of a paramagnetic lanthanide ion, praseodymium ion (Pr'+). We chose Pr'+ rather than Eu8+ as a shift marker to study differentially the two polar head regions of the vesicle bilayer because, at a lower concentration, the Pr'+ ions produced at greater spectral splitting of the -N+(CH3)3 resonance signal upon coordination with the polar head groups of the bilayer.
EXPERIMENTAL MATERIALS AND PROCEDURESCholesterol was obtained from Sigma Co., St. Louis, Mo., and further purified through bromination according to the method of Schwenk and Werthessen (14). L-a-Dipalmitoyl PC was purchased from Calbiochem, La Jolla, Calif. Egg PC was isolated and purified as described previously (7)