8512
J. Phys. Chem. B 2000, 104, 8512-8517
Influence of Cholesterol on Domain Shape and Lattice Structure in Arachidic Acid Monolayers at High pH R. Johann,* C. Symietz, D. Vollhardt, G. Brezesinski, and H. Mo1 hwald Max-Planck-Institute of Colloid and Interface Science, D-14476 Golm, Germany ReceiVed: April 4, 2000; In Final Form: June 26, 2000
Monolayer mixtures of arachidic (eicosanoic) acid with cholesterol are investigated on a subphase of pH 12.0 at 25 °C using grazing incidence X-ray diffraction (GID) and Brewster angle microscopy (BAM). GID is applied in the low concentration range from 0 to 5 mol % cholesterol and reveals significant nonmonotonic changes in the dependence of the mean molecular area and the crystallinity (expressed by the position correlation length) of the monolayer on the cholesterol concentration. The condensation and lattice order strongly depend on the surface pressure. At zero pressure, condensation and lattice order are relatively large below 1 mol % cholesterol and decrease with increasing cholesterol concentration. At high pressure, this behavior is reversed. These cholesterol effects are compared with those observed in phospholipid bilayers, which are not yet completely understood. With BAM, the formation of domains and the phase separation of arachidic acid and cholesterol is observed with 1-20 mol % cholesterol. The shape of the domains is different from that in the absence of cholesterol, which is attributed to the “line activity” of cholesterol rather than to structural changes. The isolation of the domains embedded in a condensed matrix allows the observation of the recovery of the inner domain structure with variation of the surface pressure.
Introduction Cholesterol is a fundamental part of mammalian membranes, where its concentration can range from less than 1 to 50 mol %. It has a significant influence on the degree of order and condensation, as well as the permeability and viscosity, of phospholipid membranes. Furthermore, cholesterol can affect the function of membrane proteins probably either through the propagation of changes in the packing and ordering of the lipids or by direct binding to the proteins.1 The cholesterol molecule consists of three parts: a planar and rather rigid steroid skeleton, which reaches up to 9 or 10 carbon atoms of an extended alkyl chain; a flexible isooctyl chain; and a hydroxy group in 3β position (Figure 1). Only 4% of the surface area of cholesterol is hydrophilic, although it is an amphiphile with OH as the polar headgroup. Cholesterol forms stable monolayers with a limiting molecular area of about 0.38 nm2.2 The rigidity of the steroid skeleton causes a reduction in the trans-gauche isomerization of neighboring alkyl chains and enhances the chain order over the length of the steroid ring system, so that, with very long chains, ordering of the methyl ends is no longer noticeable.3 The degree of ordering increases as more cholesterol is incorporated between the hydrocarbon chains, and whereas in the pure phospholipids the chains are significantly disordered by rapid trans-gauche isomerization, the chains are nearly in the all-trans conformation in the presence of large amounts of cholesterol.4 The effect of increasing cholesterol concentration on the chain ordering is analogous to that of decreasing temperature.3 Through the trans ordering, the chain segments concerned straighten, which can lead to a stretching of all of the chains and an increase in the bilayer thickness. In mixtures with phospholipids, the cholesterol molecules were found to be located such that the steroid ring with the chain is confined to the hydrophobic part of the bilayer and the OH groups lie in * Author to whom correspondence should be addressed. Telephone: 49331-5679461. Fax: 49-331-5679202. E-mail:
[email protected].
Figure 1. Sketch of a cholesterol molecule and of arachidic acid in the all-trans conformation.
the same plane as the lipid ester carbonyl groups.5 The question as to whether the hydroxy groups contribute to the condensation by forming hydrogen bonds with the carbonyl oxygens in their immediate vicinity, possibly mediated by water,6 is not yet resolved. The ordering effect described is displayed by cholesterol in membranes in the liquid-crystalline state that is commonly adopted under physiological conditions. In the gel state, however, cholesterol breaks the structure and disturbs the translational order of the membranes (is a “crystal breaker”) and the orientational order of the hydrocarbon chains, which is accompanied by an increase in the mean molecular area. This blurs the difference between the two phases, which is reflected by a decrease in the gel to liquid-crystalline phase melting transition enthalpy with increasing cholesterol concentration.7 The lipids of the outermost layer of skin (stratum corneum) are assumed to form a gel phase, which, because of its water impermeability and mechanical stability, contributes to the protective function of the skin.8 The lipid layer mainly consists of ceramides, free fatty acids that differ in chain length and degree of saturation, and cholesterol. It has been proposed to be built up of a mosaic of domains in the gel state that are
10.1021/jp001293l CCC: $19.00 © 2000 American Chemical Society Published on Web 08/11/2000
Influence of Cholesterol on Arachidic Acid Monolayers separated by a thin region of a liquid-crystalline phase8 constituting a structure that is reminiscent of the two-phase coexistence in monolayers at the air-water interface. Langmuir monolayers serve as model systems for membranes, and the effects of cholesterol on the lipid interactions should be very similar for both systems. The gel phase in membranes corresponds to the liquid-condensed (LC) phase in monolayers, and the liquid-crystalline phase is comparable to the liquid-expanded (LE) phase. It is known that minute amounts of cholesterol on the order of 1 mol % can have a marked influence on the shape of LC domains that are produced when the lipid mixed with cholesterol is compressed into the two-phase region. The boundary, i.e., the LE-LC interface, of DPPC domains was found to increase by a factor of 10-100 or more when some cholesterol was added.9 Cholesterol is assumed to preferentially adsorb to the two-dimensional LE-LC interface and to reduce the line tension, behaving as a “line-active” agent. The question arises whether and how much cholesterol is incorporated into the LC phase domains and to what extent the structural changes induced by cholesterol affect the domain morphology. In the present work, especially the latter aspect of this question is related to arachidic acid, which forms smooth domains at pH 12.0 and facetted ones after the addition of cholesterol. Experimental Section Arachidic acid (eicosanoic acid) (>99%) was purchased from Merck, and cholesterol (99+%) from Sigma. The substances were dissolved in a mixture of n-heptane (for spectroscopy, Merck) with 5% ethanol (p.a., Merck) without further purification. Arachidic acid and the mixtures of arachidic acid with cholesterol were spread on a subphase of pH 12.0. Whereas most of the fatty acid molecules are dissociated at this pH, with a pKs ≈ 16 for alcohols, the number of dissociated cholesterol molecules is negligible. This holds even more so, as the pH at a negatively charged interface is lower than that of the bulk phase. The pH of the subphase was adjusted with NaOH (1 M Titrisol, Merck) to which 10-4 M EDTA (99.9995%, Aldrich) was added in order to prevent traces of polyvalent cations from binding to the charged monolayer and altering its properties. A BAM2 Brewster angle microscope from NFT (Go¨ttingen, Germany) fixed on a homemade thermostated trough with a continuous Wilhelmy-type pressure-measuring system allowed simultaneous imaging of the surface with a resolution of 4 mm and recording of the isotherms. With image processing software, the brightness and contrast of the images were improved. Grazing incidence X-ray diffraction (GID) experiments were performed using the liquid-surface diffractometer on the undulator beamline BW1 at HASYLAB, DESY, Hamburg, Germany. The trough was placed in a housing that was filled with helium gas. Air was kept out during measurements by maintaining an excess pressure of the helium gas. The time for the gas exchange at the beginning of the experiments was about 30 min, so that the reduction in pH due to atmospheric carbon dioxide is less than 0.1.10 A monochromatic synchrotron beam, deflected by a beryllium crystal, strikes the air-water interface with an angle of incidence Ri ) 0.85Rc, where Rc ≈ 0.14° is the critical angle for total reflection. Ri was chosen lower than Rc in order to keep the proper balance between the intensity of the diffracted beam and the penetration depth of the transmitted wave. The diffracted intensity is detected by a linear positionsensitive detector (PSD) (OED-100-M, Braun, Garching, Germany) and resolved vertically by a multichannel analyzer. A Soller collimator is placed in front of the PSD, providing the resolution of the horizontal scattering angle 2θ. The scattering vector Q is defined as Q ) Qxy + Qz ) kf - ki, where Qxy and
J. Phys. Chem. B, Vol. 104, No. 35, 2000 8513 Qz are, respectively, the in-plane (horizontal) and out-of-plane (vertical) components of Q and kf and ki are, respectively, the wave vectors of the incident and diffracted beams, with k ) 2π/λ and λ as the wavelength. The intensities as a function of Qxy and Qz were corrected for polarization, effective area, and Lorentz factor and fit with a Lorentzian in the x-y plane and with a Gaussian in the z direction. From the scattering geometry, one obtains for Qxy
Qxy ) 2π 4π sin θ cos2 Ri + cos2 Rf - 2 cos Ri cos Rf cos 2θ = λ x λ (1) The term on the right-hand side gives the Bragg formula with Qxy ) 2π/d, where d denotes a lattice spacing, dhk, from which the lattice parameters are determined. The monolayer can be regarded as a 2D powder, where some lattice planes of an (hk0) set are always oriented relative to the incident beam such that the Bragg condition is fulfilled. The position correlation is shortranged and decays exponentially with distance. The approximate value of the position correlation length x is obtained from the in-plane peak width at half-maximum, fwhm.
ξ)
2
xfwhm
2
(2)
- 0.00822
With the second term under the root, the resolution of the Soller collimator is taken into account. The out-of-plane component of the scattering vector provides information about the orientation of the molecules hk Qhk z ) Qxy cos ψhk tan t
(3)
where t is the tilt angle and ψhk is the angle between the projection of the molecule onto the x-y plane and the normal to the lattice lines hk. The cross-sectional area per molecule is determined from the area per molecule in the monolayer plane, Axy, by
A0 ) Axy cos t
(4)
The results of GID, BAM, and isotherm measurements refer to (25 ( 1)°C. Results and Discussion Structure. A surprising and unexpected result is the condensing effect of cholesterol in the crystalline phase at very low concentrations (Figure 2). The same concentration dependence is displayed by the tilt angle extrapolated to zero surface pressure and the compressibility of the condensed phase (Table 1). Above 1 mol %, expansion occurs with increasing cholesterol content, and saturation is reached at about 3%, which may represent the upper limit of miscibility. Below 1%, however, condensation is significant, reaching a maximum at approximately 0.4 mol %. Because of the lack of experimental data, one cannot exclude an even deeper minimum between 0 and 1%, but the considerations that follow are nevertheless correct, at least qualitatively. The position correlation [given for the (11) reflections], which is a measure of the crystallinity and lattice order, behaves opposite to the change in area, as illustrated in Figure 2. The crystallinity, i.e., the order of the lattice, is highest when the molecules are closest to each other, and the translational order of the lattice decreases as the lattice expands with growing amount of cholesterol. The curves in Figure 2 may reflect the two opposing effects of cholesterol on the lattice order. There
8514 J. Phys. Chem. B, Vol. 104, No. 35, 2000
Johann et al.
Figure 2. Variation of the in-plane area Axy (solid line, circles) and the position correlation length ξ of the (11) reflections (dotted line, diamonds) with the cholesterol concentration. The data were obtained by linear extrapolation to zero surface pressure.
TABLE 1: Concentration Dependence of Arachidic Acid Monolayer Characteristics % chol.a
tilt (π ) 0)b
dAxy /dπc
Axy (π ) 30)d
ξ d,e (π ) 30)
A0 (π ) 0)
0.0 0.4 1.0 3.0 5.0
33.2° 31.2° 33.0° 36.5° 35.7°
-0.1138 -0.1000 -0.1325 -0.1413 -0.1363
20.00 19.94 19.30 19.47 19.48
81 125 187 199 189
19.59 19.62 19.52 19.06 19.14
a Concentration of cholesterol in mol %. b π is the surface pressure in mN/m. c A unit of area is 0.01 nm2/molecule. d The Axy and ξ values linearly extrapolated to π ) 30 are not the real values at 30 mN/m, as the molecules are untilted above about 28 mN/m. e ξ is the position correlation length for the (11) reflections in 0.1 nm.
is a mismatch between the size of a lattice site and the dimension of a cholesterol molecule (which is larger), so that the lattice expands and becomes more and more disordered as an increasing number of cholesterol molecules is accommodated in the lattice. At low cholesterol concentration, however, the disturbance of the lattice is small, and the condensing effect of cholesterol prevails, which leads to an increase in the lattice order. There is a strong dependence of the position correlation length on the surface pressure, as shown in Figure 3 for the (11) reflections. The behavior is the same for the (02) reflections, the values of which are smaller than for the (11) reflections and the slopes are always positive. The slopes, i.e., the increases in the lattice order with pressure, continuously grow with increasing cholesterol concentration up to 3 mol %, which has the consequence that the concentration dependence of the position correlation length at zero pressure (Figure 2) is reversed at high pressures (Table 1) where the highest crystallinity is found above 1%. The reason for this might be that the pressure produces an ordering of the lattice and compensates for the disturbance caused by the incorporation of cholesterol molecules, so that the ordering by cholesterol becomes much more effective. With 0.4%, although the crystallinity is much higher, there is almost no difference in slope compared to that with 0%, as the lattice disorder is very small and the effect of cholesterol is optimal already at zero pressure. The extrapolated values for the cross-sectional areas of the chains at zero pressure, A0 (Table 1), seem to decrease with increasing cholesterol concentration. Usually, the values for the cross section of tilted chains are, compared to the in-plane parameters, associated with a relatively large error, as they depend on the accuracy of the tilt angle (eq 4) which is about 1°. In the present case, however, the areas were extrapolated
Figure 3. Pressure dependence of the position correlation length ξ of the (11) reflections for 0 mol % (solid line), 0.4 mol % (long-hatched line, circles), 1.0 mol % (medium-hatched line, diamonds), 3.0 mol % (short-hatched line, crosses), and 5.0 mol % (dotted line) cholesterol. The lines represent linear fits. For clarity, only the data for 0.4, 1.0, and 3.0 mol % are shown.
from three pressure values, and they vary almost continuously with the cholesterol concentration by more than 0.005 nm2 in a way that does not look like scattering. The values indicate a continuous reduction in the trans-gauche isomerization with increasing amounts of cholesterol. This indicates that at least at higher cholesterol concentrations, there is no correlation between the order of the lattice and that of the chains in the arachidic acid monolayers. Furthermore, these values show that at low cholesterol concentrations where the lattice order is highest, the order of the chains is not much different from that at 0% cholesterol. The pressure dependence of the crystallinity, however, suggests that the amount of cholesterol does not need to be very high to cause significant ordering of the lattice, but that the effectiveness of the ordering is heavily affected by the disturbance of the lattice. It is interesting to note that the reduction in the in-plane area at low cholesterol concentration is accomplished merely by a reduction in tilt angle, whereas the contribution by the reduction in the chain cross section must be considered only at higher concentrations. The main part of the condensing effect of cholesterol, which is related to the ordering of the lattice, thus consists of forcing the alkyl chains of arachidic acid into a more upright position. This is also seen with higher cholesterol concentrations at high surface pressure, where the assumed enhanced efficiency of the cholesterol molecules causes a decrease in the area and hence in the tilt angle (Table 1). It is, however, not clear why and how cholesterol makes the hydrocarbon chains stand more upright. It is unlikely that the increased ordering of the chains is the reason for that, as the reduction in the cross section of a chain due to ordering relative to the cross section of the headgroup, which remains unchanged, is expected to cause an increase in the tilt.11 This effect is most conspicious at very low cholesterol concentrations, where the disturbance of the lattice is small. Larger disturbances, however, hinder the effect of cholesterol and cause the lattice to expand and the molecules to increase their tilt. The point at which the decrease in the tilt angle on the addition of cholesterol is compensated by an increase in the tilt because of the expansion and increasing disorder of the lattice is represented by the minimum at about 0.4 mol % cholesterol. The strong effect of cholesterol already at such a low concentration indicates significant cooperativity. There is experimental
Influence of Cholesterol on Arachidic Acid Monolayers
Figure 4. (a) Smooth domains of arachidic acid in the two-phase coexistence region with 0% cholesterol. (b) Polygonal domains grown from a mixture of arachidic acid with 1 mol % cholesterol at 25 °C, pH 12.0. Image size is 290 × 290 µm2.
evidence that the cholesterol molecules tend to keep maximal distance from each other, arranging in a hexagonal superlattice.12 Assuming maximal distance between the cholesterol molecules, with 4 cholesterol molecules in 1000 molecules total, there are 15 molecules of arachidic acid between 2 neighboring cholesterol molecules. This implies that only a very small number of cholesterol molecules is able to cause marked changes in the tilt angle and lattice order for many of fatty acid molecules. Morphology. Domains of arachidic acid with rounded boundary lines at pH 12.0 become cornered and polygonal with g1 mol % cholesterol (Figure 4). What is the reason for the change in domain shape, as illustrated in the figure, when some cholesterol is added? It must be a consequence of either a variation in
J. Phys. Chem. B, Vol. 104, No. 35, 2000 8515 the structure and the intermolecular interactions by cholesterol molecules that are incorporated in the LC phase domains or a manipulation of the energetic situation at the LE-LC interface by cholesterol molecules adsorbed to the domain boundary. The shape of domains under equilibrium conditions is determined by the line tension, which can be anisotropic, i.e., the magnitude of which can depend on the position at the domain boundary line. In ref 13, it was suggested that the characteristic shapes in the form of polygons are due to a decrease in the isotropic component of the line energy in the presence of cholesterol. This probably occurs by the attachment of cholesterol molecules to the domain boundary. An effect on the line energy, however, should also be expected by a change in the lattice structure and, correspondingly, in the intermolecular interactions. The line energy is higher the stronger the attractive interactions between the molecules are, and it should, therefore, become smaller when the average distance between the molecules is increased and become larger with increasing crystallinity and decreasing mean molecular area. For the extrapolation into the two-phase region to about 8 mN/m, the lattice is slightly more condensed and more ordered with 1% cholesterol than with 0%, so that no reduction in the line energy is expected from the cholesterol in the bulk of the domains.Thus, the change in domain shape appears to be merely due to the “line activity” of cholesterol. The formation of domains of arachidic acid in a mixture with up to 20 mol % cholesterol is illustrated by means of BAM in Figure 5a-h. The discussion of the images will address two aspects: first, the development of domains of arachidic acid, i.e., phase separation in the mixture, and second, the recovery
Figure 5. Representative images for mixtures of arachidic acid with 5-20 mol % cholesterol. (a) The monolayer is heterogeneous with small aggregates (bright) that are not observed with pure arachidic acid, at a mean molecular area of 0.7 nm2 in the expanded part of the isotherm. (b) Dark areas free of aggregates and regions with a domain seed of almost pure arachidic acid in the center emerge in the plateau region of the isotherm. (c) Domains in an advanced state of growth with a “halo” of aggregate free area. (d) In a monolayer compressed to the limiting area, the domains are surrounded by a homogeneous matrix consisting of arachidic acid and cholesterol. The molecules in the domains are untilted, as revealed with an analyzer. (e) When the monolayer is expanded slightly below the point at which the molecules start to tilt, irregular patches with different molecular tilt directions appear in the domains. (f) On further expansion, the patches arrange to form the inner domain structure. White arrows point to the irregular boundary between two regions of different molecular tilt directions in two selected domains. (g) and (h) These lines slowly straighten and form the boundaries between two wedge-shaped segments with different tilt directions of the molecules. Compression rate 0.1-0.2 10-2 nm2/(min molecule), image size 290 × 230 µm2.
8516 J. Phys. Chem. B, Vol. 104, No. 35, 2000
Figure 6. Surface pressure-area isotherm of arachidic acid with 5.0 mol % cholesterol at 25 °C, pH 12.0, compression rate 0.01 nm2/(min molecule).
of the inner domain structure, when the tilted phase is entered from the untilted phase on lowering of the surface pressure. The monolayer of the arachidic acid/cholesterol mixture, spread on a subphase at pH 12.0 with an area per molecule of 0.8 nm2, appears inhomogeneous (Figure 5a), with small grains (bright) that indicate the formation of aggregates. The images presented do not differ for the cholesterol concentrations of 5, 10, and 20 mol % investigated, except in the area fraction occupied by the domains, which becomes smaller as the amount of cholesterol increases. Also, the features of the isotherm at 5% (Figure 6) are representative for all concentrations. Hence, the cholesterol concentrations are not distinguished in the discussion. When the area is decreased and the pressure of the expanded phase increases, the grains segregate and become larger. In the twophase coexistence region, represented by the plateau in the isotherm, initially regions appear that are free of aggregates and that look like dark holes in the monolayer. On further compression, eventually bright seeds form in the center of these holes and grow at the expense of the surrounding aggregates (Figure 5b). The dark areas obviously represent depleted regions where a diffusion gradient provides material for the growth of the domain. The content of cholesterol in the domains is probably very small and invariable to the cholesterol concentration in the mixture, as the domains look the same for all cholesterol concentrations. The aggregates, the size of which no longer changes and which are possibly small domains of arachidic acid with cholesterol that are hindered to grow, are dissolved at the edge of the dark areas. During the growth process, the concentration of cholesterol in the depletion region will be enriched. Figure 5c shows some domains in an advanced state of growth. The presence of the “halos” indicates that the domains are still growing. With an analyzer, an inner structure is observed like that in Figure 4b. The formation of depletion regions around each growing domain prevents the nuclei from emerging in too close proximity to each other and provides their regular distribution. During growth, however, the domains aggregate, as is shown in the Figure 5d-h. The tendency for aggregation is much more pronounced than without cholesterol. It has been discussed that domains form a superlattice and repel each other when the difference between the dipole densities of the domains and the surrounding fluid phase is large.14 The cholesterol, almost all of which is in the fluid phase, may reduce the repulsive dipole forces between the domains by dielectric screening or by a change in the dipole density of the fluid phase. In time, the domains become rounded as atmospheric carbon dioxide penetrates into the subphase and reduces the pH. In Figure 5d, the monolayer is compressed to the limiting area, and the molecules in the domains are untilted, so that no inner
Johann et al. structure is observed with an analyzer. At high pressure, the aggregates fuse, and the domains are enclosed in a homogeneous matrix of arachidic acid and cholesterol. When the monolayer is now expanded slightly below the pressure where the molecules become untilted, irregularly distributed patches with different shades of gray appear on the domains, each shade representing a region where the molecules are uniformly tilted in a certain direction in the monolayer plane (Figure 5e). On further decrease of the surface pressure, i.e., on increase of the tilt angle, some of these patches grow in area and arrange such that a pattern is formed that already indicates the typical segmented inner structure of the domains (Figure 5f). The process of the arrangement of the molecular orientations is very rapid, which points to a high mobility of the hydrocarbon chains in this stage. In Figure 5f, one can see that the orientation of the molecules and the segments with respect to the domain boundary corresponds to that of an unimpaired domain, while the boundaries between the segments are strongly curved and not well defined. This suggests that the subdivision of the domains into a certain number of segments and the direction of the molecular tilt with respect to the domain boundary, which characterizes each segment, are governed by forces originating at the domain boundary.13 The straightening of the boundaries between the segments, which proceeds as the monolayer is expanded, occurs more slowly than the formation of the segments, which indicates that the driving force for this process is relatively weak. The driving force for the straightening of the boundary may stem from the fact that, at the boundary line between two segments, a certain angle between the two different molecular tilt directions of both segments is energetically favored, which is 60° in a domain with 6 segments. Conclusion The object of the investigation has been to investigate the structural changes in arachidic acid monolayers at pH 12.0 with increasing content of cholesterol from 0 to 5 mol % and the influence on the shape and inner structure of the LC phase domains in the range from 1 to 20 mol % cholesterol. For zero pressure, a strong condensation of the monolayer by cholesterol below 1 mol % and an expansion of the lattice above this concentration are revealed. The magnitude of condensation and the position correlation length, which expresses the degree of lattice order, are found to vary proportionally. The lattice order increases with increasing condensation, and the expansion of the lattice leads to a reduction in the translational correlation. The expansion of the lattice with increasing cholesterol concentration at zero pressure is interpreted as indicating that the cholesterol molecules fail to properly fit into the lattice of the fatty acids and act as “crystal breakers”. At high pressures, however, the situation reverses, and the largest reduction in area and increase in lattice order is found for the higher cholesterol concentrations. This is interpreted such that the expansion of the lattice and the concomitant decrease in its order because of the bad fit of the cholesterol molecules into the lattice of arachidic acid chains are reversed and compensated for by the increasing surface pressure. The closer contact between the molecules and the enhanced lattice order again increase the condensing and ordering efficiency of cholesterol. The decrease in mean molecular area is mainly caused by the reduction in the tilt angle of the molecules, and the contribution due to the reduction in the chain cross section by chain ordering grows with increasing amount of cholesterol. The results demonstrate that even traces of cholesterol can cause conspicious changes in the structure of lipid monolayers and that the study of lipid
Influence of Cholesterol on Arachidic Acid Monolayers cholesterol mixtures with very low cholesterol concentrations may contribute to the understanding of the cholesterol interactions at the molecular level, the knowledge of which is still incomplete. The change in the shape of the LC phase domains that are smooth without cholesterol and that grow facetted when g1 mol % cholesterol is added appears to be the consequence of the adsorption of cholesterol onto the LE-LC phase boundary rather than of the structural modifications caused by cholesterol. The formation of domains, whose appearance does not change irrespective of the cholesterol concentration, demonstrates that the arachidic acid and cholesterol phases separate. This offers the possibility of investigating the formation of the inner structure of isolated domains that are captured and fixed in a condensed matrix of arachidic acid and cholesterol. After the molecules commence tilting, first the domains divide into segments, in each of which the orientation of the molecules is uniform but different from that in the other segments, and finally, on further lowering of the pressure, the boundary lines between the segments straighten.
J. Phys. Chem. B, Vol. 104, No. 35, 2000 8517 References and Notes (1) Yeagle, P. L. Biochim. Biophys. Acta 1985, 822, 267-287. (2) Lafont, S.; Papaport, H.; So¨mjen, G. J.; Renault, A.; Howes, P. B.; Kjaer, K.; Als-Nielsen, J.; Leiserowitz, L.; Lahav, M. J. Phys. Chem. B 1998, 102, 761-765. (3) Morrow, M. R.; Singh, D.; Lu, D.; Grant, C. W. M. Biophys. J. 1995, 68, 179-186. (4) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451-464. (5) Karolis, C.; Coster, H. G. L.; Chilcott, T. C.; Barrow, K. D. Biochim. Biophys. Acta 1998, 1368, 247-255. (6) Huang, C. Nature 1976, 259, 242-243. (7) Schofield, M.; Jenski, L. J.; Dumaual, A. C.; Stillwell, W. Chem. Phys. Lipids 1998, 95, 23-36. (8) Forslind, B. Acta Derm.-Venereol. 1994, 74, 1-6. (9) Weiss, R. M.; McConnell, H. M. J. Phys. Chem. 1985, 89, 44534459. (10) Johann, R.; Vollhardt, D. Mater. Sci. Eng., C 1999, 8-9, 35-42. (11) Peters, G. H.; Toxvaerd, S.; Olsen, O. H.; Svendsen, A. Langmuir 1995, 11, 4072-4081. (12) McMullen, T. P. W.; McElhaney, R. N. Curr. Opin. Colloid Interface Sci. 1996, 1, 83-90. (13) Johann, R.; Vollhardt, D.; Mo¨hwald, H. Colloid Polym. Sci. 2000, 278, 104-113. (14) Lo¨sche, M.; Duwe, H. P.; Mo¨hwald, H. J. Colloid Interface Sci. 1988, 126, 432-444.
