Building up of the liquid-ordered phase formed by sphingomyelin and cholesterol

Abstract

The long-range and molecular orders and dynamics in codispersions of egg sphingomyelin-cholesterol have been investigated by synchrotron x-ray diffraction and electron spin resonance using phosphatidylcholine spin-labeled at several positions on the sn-2 chain. Mixtures containing 0, 17, 33, 41, 50 mol% cholesterol exhibited a single phase by x-ray diffraction methods. The temperature dependence of the d-spacing between 20 and 50 degrees C is attenuated with increasing proportions of cholesterol, becoming invariant for cholesterol contents of 41 and 50 mol% on completion of the liquid-ordered phase. Electron spin resonance revealed two sites for 17 and 33 mol% cholesterol. One site is highly ordered and the other is less ordered than the fluid phase of pure sphingomyelin as shown by the molecular and the intramolecular order parameters reflecting the segmental motions of the probe. The two-sites exchange rate indicates a mean lifetime of the sites of approximately 0.1 micros during which the lipid displacement is approximately 1 nm. The short lifetime of the sites probed by ESR and the single phase detected by x-ray diffraction support in this binary mixture, the building up of the Lo phase by a progressive accumulation of randomly distributed sphingomyelin-cholesterol condensed complexes rather than by diffusional exchange between extended domains.

FIGURE 1

ESR spectra of the PC-16 spin probe recorded at 17°C. (A) Egg sphingomyelin (SM) without cholesterol, Lβ phase. The spectral fittings yield (B) SM-CHOL 17 mol%. The outermost features indicated by arrows show the presence of two components. The fitting of the two-component spectrum yields for a site, and for the other site. (C) SM-CHOL 50 mol%, Lo phase. The spectral fittings yield

FIGURE 2

Experimental (solid line) and computed (dots) spectra of PC-5–16 probes in pure SM (left panel) and SM-CHOL 50 mol% (right panel) at 41°C. The fittings have been performed by means of the LQCF or NLSL programs with and as the main adjustable parameters. For these fittings, we have taken () for the g tensor and () for the hyperfine coupling tensor A. These principal values are means of values given in Ge et al. (1999) for the spin probes PC-5 and PC-16 in DPPC and SM. Owing to the local polarity, the hyperfine coupling constant decreases from ∼1.55 to ∼1.4 mT from PC-5 to PC-16 and depends also on the CHOL concentration. The principal values of A have therefore been corrected by a factor which is adjusted in spectral fittings using the LQCF program.

FIGURE 3

Experimental (solid line) and computed (dots) spectra of PC-5–14 spin probes in pure sphingomyelin (left panel) and SM-CHOL 41 mol% (right panel) samples at 41°C. The simulations have been performed using the DOXFIT program that takes into account the overall molecular as well as the segmental orderings and motions using the parameters given in Table 3.

FIGURE 4

Fittings of the two spectral components of PC spin probes in the SM-CHOL 17 mol% sample at 41°C. The solid lines represent the component A (the most ordered site) and the component B simulated with parameters from the fitting of experimental spectra by means of the LQCF program. The dots correspond to the simulation of the A and B components by means of the DOXFIT program with parameters given in Table 3.

FIGURE 5

Small-angle (SAXS, left panel) and wide-angle (WAXS, right panel) x-ray scattering intensity recorded for multilamellar aqueous dispersions of egg SM mixed with increasing CHOL concentrations (A) egg SM; (B) SM-CHOL 17 mol%; (C) SM-33 mol%; (D) SM-CHOL 50%; (E) ternary mixture equimolar SM-PC-CHOL mixture. The sequence of diffractograms is recorded during the initial heating scan from 20 to 50°C (1 frame per °C, scan rate 1°C/min). Tick marks identify the values cited in Results for the reciprocal spacing (S, given in nm⁻¹) and temperature. Fig. 5 E (ternary equimolar mixture SM-PC-CHOL) shows the coexistence of two phases (tick marks).

FIGURE 6

Variation as a function of the temperature of the d-spacing of the lamellar phase identified by SAXS in multilamellar aqueous dispersion of SM-CHOL mixtures. For each concentration (A, egg SM; B, SM-CHOL 17 mol%; C, SM-CHOL 33 mol%; D, SM-CHOL 41 mol%; and E, SM-CHOL 50 mol%) the reciprocal d-spacing (S in ) is plotted as a function of the temperature at the rate of ±1°C/min.

FIGURE 7

Dependence of the order parameters on temperature and cholesterol concentration for PC-7 and PC-14 spin probes. For clarity, the order parameter of the spin probes for SM-CHOL 17 mol% and SM-CHOL 33 mol% are given as the weighted average of values for sites A and B.

FIGURE 8

Experimental spectra (solid line), simulated spectra (dots), and difference spectra amplified five times (×5) of PC-14 spin probe in SM-CHOL 17 mol% at 37°C. The arrow denotes a feature suggesting the existence of two sites. The fittings yield the following parameters: (a) assuming a single site, ; (b) assuming two sites and no intersite-exchange, fraction site A 0.34; site A, ; site B, ; (c) assuming two sites and intersite-exchange, fraction site A 0.52, exchange rate A↔B ; site A, ; site B, The standard deviations between the experimental and computed spectra are (a) σ = 1%, (b) σ = 0.65%, (c) σ = 0. 35%.

FIGURE 9

(Top panel) Fractional population of the most ordered site A for SM-CHOL 17 mol% (○) and SM-CHOL 33 mol% (•) calculated from the fittings of PC-12, PC-14, and PC-16 ESR spectra. (Bottom panel) Exchange rate between the sites A and B calculated from the spectral fittings for mixtures comprising SM-CHOL 17 mol% (○) and SM-CHOL 33 mol% (•). The solid line represents the least-squares fit for an exponential dependence of on 1/T yielding and an activation energy of 43 kJ/mol. From the diagram it is estimated that the lifetime of site A between 17 and 53°C varies from 0.44 to 0.02 μs and from 0.54 to 0.03 μs for CHOL 17 mol% and CHOL 33 mol%, respectively.

FIGURE 10

Temperature dependence of for PC-14 and PC-16 spin probe in site A (most ordered site, •) and B (○) of SM-CHOL 17 mol% (left panels) and SM-CHOL 33 mol% (right panels).

FIGURE 11

Model of a SM-CHOL leaflet containing the spin probe (vertical arrow). The stiff or curved lines represent SM and the ellipses CHOL. During its displacement (a few nanometers), the probe experiences successively highly ordered and poorly ordered environments due to the creation and disruption of short-life SM-CHOL condensed complexes.