A C-13 AND H-2 NUCLEAR-MAGNETIC-RESONANCE STUDY OF PHOSPHATIDYLCHOLINE CHOLESTEROL INTERACTIONS - CHARACTERIZATION OF LIQUID-GEL PHASES

A detailed study on the structure, dynamics, and thermodynamic behavior of phosphatidylcholine/cholesterol (PC/CHOL) mixtures was undertaken using differential scanning calorimetry (DSC) and solid-state nuclear magnetic resonance (NMR) spectroscopy. DSC thermograms of mixtures of cholesterol (CHOL) with 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-phosphatidylcholine (DSPC), and 1,2-diarachidoyl-sn-phosphatidylcholine (DAPC) showed a broadening of the first-order gel --> liquid crystalline transition and a decrease in the transition enthalpy, indicating a gradual loss of cooperativity for high CHOL concentrations. DPPC and DSPC were labeled with C-13 at the carbonyl group of the sn-2 chain and H-2 was introduced into the middle of the sn-2 chain at the 6- and 12-position for DPPC and DSPC, respectively. The C-13 and H-2 NMR spectra of each labeled lipid were studied as a function of temperature and CHOL concentration. The residual quadrupole splitting in the H-2 NMR spectra, DELTAnu(Q perpendicular-to), was analyzed as a function of temperature and composition. For CHOL concentrations less than 30 mol %, a precipitous change in DELTAnu(Q perpendicular-to) occurs near the chain melting temperature of the phospholipid. Further increases in CHOL concentration broaden the transition and shift the midpoint to higher temperature, indicating the presence of a new phase at higher CHOL contents. Moreover, at a given temperature, DELTAnu(Q perpendicular-to) increases with increasing cholesterol content, which indicates a more ordered structure. The C-13 NMR spectra in the gel state consisted of a superposition of two components which can be attributed to both gel-like and fluid phospholipid domains in the bilayer. This two-component spectrum can be simulated quantitatively with a two-parameter chemical exchange model, which permits the fraction of each form and the exchange rate to be determined as a function of temperature and composition. At high CHOL contents the line width of the fluid component broadens, suggesting an increase in the exchange rate between the domains. These results were interpreted in terms of a temperature composition diagram with one region L(beta'), two regions LG(I) and LG(II), and one liquid crystalline region L(alpha), with LG denoting ''liquid-gel'' type phases. Liquid-gel phases correspond to phases with increased order in the hydrocarbon chains (in comparison to that of the pure PC bilayer in the L(alpha) phase) combined with fast limit axial diffusion that averages the C-13 NMR spectrum to a ''fluidlike'' line. These phases are similar to those found in phosphatidylethanolamine/cholesterol (PE/CHOL) mixtures [Blume, A., & Griffin, R. G. (1981) Biochemistry 24, 6230] and are in agreement with the results of Vist and Davis [(1990) Biochemistry 29, 451].