Thermodynamic Description of the Interactions between Lipids in

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Thermodynamic Description of the Interactions between Lipids in Ternary Langmuir Monolayers: the Study of Cholesterol Distribution in Membranes Paweł Wydro*,† and Katarzyna Ha¸ c-Wydro‡ Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30-060 Krako´ w, Poland, and Department of General Chemistry, Faculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30-060 Krako´ w, Poland ReceiVed: October 23, 2006; In Final Form: December 17, 2006

The aim of this work was to get insight into cholesterol distribution between two leaflets of a phospholipids bilayer. In this order, the thermodynamic analysis of the interactions between membrane lipids in binary (cholesterol/phospholipid) and ternary (phospholipid/ phospholipid/cholesterol) mixed Langmuir monolayers has been performed. For our investigation, phosphatidylcholine and phosphatidylethanolamine, which are the main types of phospholipids determining the distribution of cholesterol in membrane leaflets, were chosen and mixed in proportions corresponding to their molar ratios in the inner and outer layers of the natural human erythrocyte membrane. Into these mixed systems, various amount of cholesterol were incorporated. It has been found that despite strong differences in the phospholipid composition of both investigated ternary mixed systems, the influence of cholesterol is very similar, which indicates that cholesterol is symmetrically distributed between the inner and outer leaflets of the human erythrocytes membrane.

Introduction The cellular membrane is a natural permeable barrier that not only separates and protects the cell from external environment but is also the site for various cellular processes, such as active transport, signal and energy transductions, or enzyme and protein activity.1 Since the membrane is crucial for cell functioning, its structure and composition have been systematically studied, and the knowledge of the membrane properties has changed and widened within the years.1,2 It is known that the structural backbone of the membrane is a bilayer formed spontaneously by phospholipid molecules, which, due to their amphiphatic structure, are able to organize specifically in an aqueous environment. Not attracted to water, hydrophobic tails of phospholipids are directed to the membrane interior, while polar heads face the aqueous environment. Thus, the membrane consists of only two layers of phospholipids in which sterols and protein molecules are built. In human membranes, phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids; however, phosphatidylserine and phosphatidylinositol are also present.3 Apart from phospholipids, percentage-wise, cholesterol is the second most abundant lipid membrane component. Moreover, in the membrane lipid mass significant amount covers sphingomyelin.1,3,4 The structure of the polar head and the length as well as the unsaturation of hydrocarbon tails of membrane lipids define the specific properties of particular molecules and, as a consequence, affect the membrane parameters.5 Thus, the membrane’s composition determines its properties (such as fluidity, rigidity, and permeability) and affects the membrane’s functioning. Therefore, the phospholipid and protein types and their mutual proportion * Corresponding author. Phone: +48 0-12 633-20-79. Fax: +48 0-12634-05-15. E-mail: [email protected]. † Department of Physical Chemistry and Electrochemistry. ‡ Department of General Chemistry.

as well as the cholesterol content in a membrane depend on the organism, cell, and membrane type.1,5,6 There are also differences in composition between the inner (cytosolic) and outer (exoplasmic) layers of a membrane. It has been proved that in the outer leaflet, mainly phosphatidylcholines and sphingomyelins are present, whereas in the inner layer, phosphatidylethanolamines and phosphatidylserines are localized.5,7,8 The phospholipid asymmetry in the membrane and the location of a particular type of molecules seem to be known; however, there are still controversies regarding the distribution and movement of cholesterol between the inner and outer layers of a membrane. The results of the investigations presented in the literature are often contradictory. Although it is known that cholesterol is present in both membrane leaflets and can exchange between them, some authors suggest its asymmetrical distribution and location is mainly in the outer layer.9-11 On the other hand, other authors have proposed that cholesterol is concentrated mainly in the inner layer12 or symmetrically distributed within the phospholipid bilayer.13 It is also known that cholesterol, in the outer leaflet of a membrane tends to form specific domains with sphingo- and phospholipids called “rafts”. It is suggested that these rafts play an important role in protein activity; however, their composition, structure, and role in membrane and cell functioning are still being elucidated.1,14-16 The aim of this work was to verify the influence of cholesterol addition on phospholipid Langmuir monolayers, which served as a simple model of a cellular membrane, and to examine the cholesterol distribution in the phospholipid bilayer. Although the Langmuir monolayers technique is frequently applied to building up the model of a natural membrane, the interactions between biomolecules are usually studied in binary mixed systems (e.g., cholesterol/phospholipid,17-20 phospholipid/phospholipid,21,22 drugs/membrane lipids,23-25 hormones/phospholipids26). In this work, the thermodynamic analysis of the interactions between membrane lipids in ternary Langmuir monolayers (cholesterol/phospholipid1/ phospholipid2) has been

10.1021/jp066950+ CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007

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Wydro and Ha¸ c-Wydro

Figure 1. (a) The surface pressure (π)-area (A) isotherms for DPPC/cholesterol mixed monolayers. (b) The surface pressure (π)-area (A) isotherms for DPPE/cholesterol mixed monolayers.

performed. The phospholipids investigated herein, namely, phosphatidylcholine and phosphatidylethanolamine, have been found to be decisive regarding cholesterol distribution in the membrane.27 Moreover, phosphatidylcholines and phosphatidylethanolamines represent the main class of phospholipids in the outer and inner membrane leaflets, respectively. The abovementioned phospholipids have been mixed in the proportions corresponding to their molar ratios in the outer (the proportion of DPPC/DPPE is equal to 4.036 (see ref 3)) and inner layer (DPPC/DPPE ratio, 0.319 (see ref 3)) of human erythrocytes, respectively. Then, into binary PC/PE monolayers, various amounts of cholesterol have been incorporated. We believe that the analysis of the interactions between the investigated lipids in ternary sterol/PC/PE monolayers allows us to draw conclusions regarding the influence of cholesterol on the molecular organization of model leaflets of the phospholipid bilayer as well as cholesterol distribution in the membrane. Experimental Cholesterol and dipalmitoyl L-R-phosphatidylcholine, DPPC (purity >99%), was purchased from Sigma, whereas dipalmitoyl L-R-phosphatidylethanolamine, DPPE (purity >99%), was purchased from Fluka. The spreading solutions were prepared by dissolving the investigated compounds in a mixture of freshly distilled chloroform and methanol, p.a. (POCh) (4:1 v/v). Mixed solutions were prepared from the respective stock solutions. Spreading solutions were deposited onto the water subphase with a Hamilton microsyringe, precise to 2.0 µL. After spreading, the monolayers were left to equilibrate for ∼10 min before the compression was initiated with a barrier speed of 20 cm2/min. π-A isotherms were recorded with a NIMA (U.K.) Langmuir trough (total area ) 300 cm2) placed on an antivibration table. Surface pressure was measured at an accuracy of (0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The subphase temperature (20 °C) was controlled thermostatically to within 0.1 °C by a circulating water system. Results To perform a precise analysis of the interactions between the molecules in the ternary mixtures, examination of the interactions between cholesterol and the respective phospholipids as well as between both phospholipids in the binary mixed monolayers is required. Therefore, in the first step of our investigations, the surface pressure (π)-area (A) isotherms for

mixed films of cholesterol/DPPC, cholesterol/DPPE, and DPPC/ DPPE of various compositions, formed at the air/water interface, have been recorded (Figures 1a, b and 2). The characteristic of the isotherms recorded for a one-component lipid monolayer (cholesterol, DPPC, and DPPE films, respectively) are in agreement with those presented in the literature (see, e.g., refs 18, 28, 29). The shape of the π-A curves for the cholesterol film indicates a solid (untilted) state of the monolayer, with a collapse surface pressure at 45 mN/m. In the case of the DPPC monolayer, a well-known phase transition between the LE and LC states as a plateau on the isotherm can be observed. In addition, the π-A curves for DPPE agree with those obtained by other authors concerning the shape of the isotherm, the liftoff area, and the collapse surface pressure. As is seen, π-A isotherms for mixed cholesterol/phospholipid monolayers (Figure 1a, b for cholesterol/DPPC and cholesterol/ DPPE mixed monolayers, respectively) lie between those obtained for the respective one-component films, and their shape changes systematically with the sterol content, becoming similar to cholesterol monolayer isotherm. This effect is especially noticeable for isotherms for cholesterol/DPPC monolayers, for which with the increase in cholesterol content the plateau region (characteristic for DPPC curve) systematically disappeared. The addition of cholesterol into the phospholipid film makes the π-A isotherms steeper and provokes their shift toward smaller areas. This strong influence of cholesterol on phospholipid monolayers is well-known as a condensing effect.30 It is also evident that the collapse surface pressure for mixtures changes with the monolayer’s composition, indicating miscibility of the monolayer components.31-33 Concerning mixtures consisting of phospholipids (Figure 2), the incorporation of DPPE into the DPPC monolayer causes a shift in the isotherm toward smaller areas and a vanishing of the plateau region, which is characteristic for a π-A curve for a pure DPPC monolayer. The state of the monolayers formed by cholesterol and investigated phospholipids can be concluded from the shape of their isotherms; however, for a precise definition and for a better description of the effect of sterol on the phospholipid monolayers, the compression modulus values, defined as follows,34 have been calculated.

CS-1 ) -A(dπ/dA)

(1)

The changes in the compression modulus (CS-1) with the surface pressure (π) for mixed systems of cholesterol/DPPC and cholesterol/DPPE of various monolayer compositions are pre-

Cholesterol Distribution in Membranes

J. Phys. Chem. B, Vol. 111, No. 10, 2007 2497 tions from ideality for cholesterol/DPPC and DPPC/DPPE suggest stronger attractions between molecules in these mixed films, as compared to pure cholesterol and the respective phospholipid films. The interaction in the mixed monolayer can be interpreted more quantitatively by analyzing the excess of the thermodynamic functions.31,35 On the basis of isotherm data points, the excess Gibbs energy of mixing (∆Gexc) for all of the investigated binary mixed systems have been calculated according to eq 333,35

∆Gexc ) N

Figure 2. The surface pressure (π)-area (A) isotherms for DPPC/ DPPE mixed monolayers.

sented in Figure 3a, b. Maximal values of the compression modulus prove a solid state for the cholesterol monolayers and liquid condensed state for the DPPE film. A characteristic minimum visible in the CS-1 vs π dependency for the DPPC film reflects a phase transition between the liquid expanded (LE) and liquid condensed (LC) states of the monolayer. The addition of cholesterol into the phospholipid monolayers causes an increase in the compression modulus values with respect to the pure phospholipid film. This indicates that the mixed cholesterol/ phospholipid monolayers are stiffer in comparison with DPPC and DPPE films. The dependence of the compression modulus vs the surface pressure has been plotted also for DPPC/DPPE mixed films (Figure 3c). The strongest effect of DPPE on the DPPC monolayers can be observed in the region of lower surface pressures. Namely, with an increase in the DPPE content in a mixed monolayer, a shift in the minimum toward higher pressures and its systematic vanishing can be observed. Interestingly, the addition of DPPE into the DPPC monolayer influences the compression modulus values at a higher pressure region (LC region for pure DPPC film) only at XDPPE ) 0.9. The interaction between molecules in mixed monolayers can be interpreted considering the miscibility of monolayer components, on the basis of the additivity rule proposed by Costin and Barnes.31 According to this rule, as a consequence of the interaction between molecules, the mixed monolayers show nonideal behavior, which is reflected in nonlinear dependencies between simple functions (e.g., area or surface pressure) and the monolayer composition. The interactions in mixed films investigated herein have been qualitatively evaluated on the basis of the excess areas of mixing (Aexc) calculated according to eq 233 at various surface pressures.

Aexc ) A12 - (A1X1 + A2X2)

(2)

A12 is the mean area per molecule in a mixed monolayer at a constant surface pressure, A1 and A2 are the molecular area of a single component at the same surface pressure, and X1 and X2 are the mole fractions of components 1 and 2 in the mixed film. The Aexc values for cholesterol/DPPC, cholesterol/DPPE, and DPPC/DPPE mixed systems have been presented as a function of the monolayer composition in Figure 4a-c. It is evident that the Aexc-vs-monolayer composition plots show deviation from linearity in the whole range of the monolayer composition, which indicates nonideal behavior of the mixed film and miscibility of their components and proves the existence of the interactions between the molecules. The negative devia-

∫0π (A12 - X1A1 - X2A2) dπ

(3)

where A12, A1, and A2 are the mean area per molecule in a binary mixed monolayer and in one-component films, respectively, and X1 and X2 are the mole fractions of components 1 and 2 in mixed film. The ∆Gexc is a measure of the interactions between components in mixed monolayers with respect to the interactions between molecules in the respective one-component films. The negative values of ∆Gexc indicate that the interactions between the components are more attractive (or less repulsive) as compared to those in their respective pure films. On the other hand, the positive values of the excess Gibbs energy of mixing (∆Gexc) indicate that the interactions in mixed film are less attractive (or more repulsive) than those in one-components monolayers. Moreover, the lower the ∆Gexc values, the more stable the mixed monolayer is. The values of the free energies of mixing for cholesterol/ DPPC and cholesterol/DPPE mixed films calculated at various surface pressures (5, 10, 20, and 30 mN/m) are presented as a function of the monolayer composition in Figure 5a, b. The course of both the Aexc and ∆Gexc values vs monolayer composition proves nonideal behavior of the cholesterol/ phospholipid monolayers. This nonideality is a consequence of a strong influence of cholesterol on phospholipid monolayer (a condensing effect30) and formation of the condensed complexes between the cholesterol and phospholipid molecules present in the mixed films. The incorporation of cholesterol molecules into the phospholipid monolayer or bilayer causes increased ordering of the acyl chains of the phospholipids.36 A brief description of the cholesterol/phospholipid interactions, liquid-liquid immiscibility, and formation of the condensed complexes is provided in an excellent review article by H. M. McConnell and M. Vrljic.37 As can be seen, the course of the ∆Gexc-vs-monolayer composition plots is strongly dependent on the kind of phospholipid that is mixed with the cholesterol. For cholesterol/DPPC mixed monolayers, the values of ∆Gexc are negative in nearly the whole range of monolayer composition, with a minimum at Xchol ) 0.5 (∆Gexc ≈ -1500 J/mol). On the other hand, for cholesterol/DPPE monolayers, the values of the free energy of mixing are positive for the composition of all the investigated monolayers. Since the length and the saturation of both DPPC and DPPE acyl chains are the same, it is clear that the interactions in mixed systems are influenced by the structure of the phospholipid polar head. In order to explain the differences in the interactions between cholesterol and the respective phospholipid, the DPPCDPPC and DPPE-DPPE interactions in their pure films should be elucidated. Due to their zwitterionic character, both phosphatidylcholines and phosphatidylethanolamines interact in their one-component films via electrostatic forces; however, in the DPPE film, between the ammonium and phosphate groups of



Figure 3. (a-c) The compression modulus CS-1 values vs surface pressure plots for mixed monolayers: cholesterol/DPPC (a), cholesterol/DPPE (b), and DPPC/DPPE (c).

Figure 4. (a-c) Excess area (Aexc) vs composition plots for mixtures of cholesterol/DPPC (a), cholesterol/DPPE (b), and DPPC/DPPE (c) at different constant surface pressures.

the neighboring molecules, additionally, hydrogen bonds exist,27 which makes the monolayer more condensed as compared to the DPPC film. The insertion of cholesterol into the phospholipid monolayer causes separation of the zwitterionic molecules and a decrease in the electrostatic repulsion between them. However, the existence of hydrogen bonds between PE molecules makes the separation of molecules difficult, and therefore, the influence

of sterol on the DPPE monolayer is opposite that in the DPPC/ cholesterol system. Thermodynamically less favorable mixing of cholesterol and phosphatidylamine in the monolayers reflects in positive values of the free energy of mixing for the cholesterol/DPPE mixed systems. The ∆Gexc values vs the monolayer composition plots for DPPC/DPPE mixed system are presented in Figure 5c. It is


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Figure 5. (a-c) Excess free energy of mixing (∆Gexc) vs composition plots for mixtures cholesterol/DPPC (a), cholesterol/DPPE (b), and DPPC/ DPPE (c) at various constant surface pressures.

Figure 6. (a) The surface pressure (π)-area (A) isotherms of ternary mixed monolayers of DPPC/DPPE/cholesterol (DPPC/DPPE proportion mimics inner leaflet of membrane). (b) The surface pressure (π)-area (A) isotherms of ternary mixed monolayers DPPC/DPPE/cholesterol (DPPC/ DPPE proportion mimics outer leaflet of membrane).

evident that for monolayers containing lower mole fractions of phosphatidylethanolamine, the free energy of mixing values are positive and suggest that the interactions between molecules in the mixed film are less attractive (or more repulsive) than those in one-component monolayers. With a further increase of DPPE in the mixed film, the ∆Gexc values decrease and are the most negative for XDPPE ) 0.5 ÷ 0.9. In the next step of our investigation, the influence of cholesterol on binary DPPC/DPPE mixed monolayers was studied. Since the aim of this work was to examine the distribution of cholesterol between the leaflets of the membrane bilayer, the effect of mammalian sterol on two DPPC/DPPE mixed systems of various proportions of the respective phospholipids was investigated. In the first of them, the DPPC/DPPE molar ratio was maintained to mimic the proportion of these phospholipids in the inner layer of the human erythrocyte membrane, and the second one, that in the outer layer of the

membrane cell.3 Into these binary mixtures various amounts of cholesterol were incorporated. Thus, in fact, the interactions between molecules in ternary mixed monolayers have been studied. It is worth stressing that the proportion of phospholipids in ternary mixtures always corresponds to the PC/PE molar ratio in the inner and outer layers of the human erythrocytes membrane. The π-A isotherms for cholesterol and DPPE/DPPC monolayers and their mixtures of various cholesterol content are presented in Figure 6a and b (for monolayers of DPPC/DPPE proportion corresponding to inner and outer layer, respectively). It is evident that with the cholesterol content in the DPPC/DPPE monolayer, the isotherms for ternary mixtures shift systematically toward the cholesterol curves. Moreover, the addition of sterol affects the collapse surface pressure for all of the investigated monolayers, suggesting miscibility of the components of the investigated monolayers. The incorporation of



Figure 7. (a-b) The compression modulus CS-1 values vs surface pressure plots for ternary mixed monolayers of DPPC/DPPE/cholesterol: (a) DPPC/DPPE proportion mimics inner leaflet of membrane; (b) DPPC/DPPE proportion mimics outer leaflet of membrane

Figure 8. (a-b) Excess area (Aexc) vs composition plots for for ternary mixed monolayers of DPPC/DPPE/cholesterol at various constant surface pressures: (a) DPPC/DPPE proportion mimics inner leaflet of membrane and (b) DPPC/DPPE proportion mimics outer leaflet of membrane.

cholesterol makes both investigated DPPC/DPPE model membranes generally less fluid, which is reflected in increasing values of the compression modulus (Figure 7a, b for inner and outer layer-imitating mixtures). The interactions between the components of the ternary mixed systems were analyzed on the basis of the values of the excess area and the free energy of mixing values calculated according to the following equations (eq 4 and 5, respectively):

Aexc ) A123 - ((X1 + X2)A12 + X3A3) ∆Gexc ) N

∫0π (A123 - (X1 + X2)A12 - X3A3) dπ

(4) (5)

wherein A123, A12, and A3 are the mean areas per molecule in ternary DPPC/DPPE/cholesterol, binary DPPC/DPPE, and onecomponent (cholesterol) monolayers, respectively. X1, X2, X3 are the mole fractions of components 1 (DPPC), 2 (DPPE), and 3 (cholesterol) in the ternary mixture, respectively. The values of Aexc and ∆Gexc, calculated for given surface pressures (5, 10, 20, 30 mN/m), have been plotted as a function of the cholesterol mole fraction in a mixed monolayer (Figure 8 and 9; for model inner (a) and outer layers (b), respectively). The course of Aexc vs Xcholesterol plots proves nonideal behavior of the investigated mixed systems resulting from the existence of the interactions among the mixed film components. The analysis of the ∆Gexc values allows for more quantitative interpretation of these interactions. As can be seen, the course of the ∆Gexc vs Xcholesterol dependencies is similar for both of the investigated model membranes. The minimal values of the free energy of mixing differ insignificantly (-1000 J/mol for the model inner layer and -1200 J/mol for the model outer

layer) and appear at nearly the same cholesterol content (Xcholesterol ≈ 0.3). Taking into account that the mixtures mimicking outer layer contain a much higher amount of phosphatidylcholine as compared to mixtures imitating the inner layer and, as proved by our studies for binary cholesterol/phospholipids mixtures, cholesterol exhibits a stronger affinity toward DPPC as compared to DPPE, thermodynamically more favorable interactions can be expected for monolayers mimicking the outer layer. However, upon the analysis of the results for ternary mixtures, the interactions between phospholipids (DPPC/DPPE) should also be considered. The course of the ∆Gexc vs XDPPC curves (Figure 5 c) proves that the interactions between both phospholipids depend on the monolayer composition; namely, for XDPPC e 0.8, the free energy of mixing values are negative, while for a higher phosphatidylcholine mole fraction, ∆Gexc is positive and the DPPC/DPPE interactions become thermodynamically less favorable. These results seem to be crucial to understanding the results obtained for ternary monolayers. In the mixed system imitating the outer layer, the proportion of DPPC/DPPE is equal to 4.036,3 which corresponds to XDPPC ≈ 0.8 in the binary DPPC/DPPE monolayer. For this DPPC/DPPE monolayer composition, ∆Gexc is positive (∆Gexc ≈ 200 J/mol at π ) 30 mN/m). Regarding ternary monolayers mimicking the inner layer, the phospholipids (DPPC/DPPE) ratio (0.319, see ref 3) corresponds to XDPPC ≈ 0.24 (in binary DPPC/DPPE monolayer) and ∆Gexc is negative (∆Gexc ≈ -350 J/mol at π ) 30 mN/m). Unfavorable mixing of DPPC with DPPE in the outer layer weakens the effect resulting from the strong attractions between the DPPC and cholesterol, while thermodynamically favorable mixing of DPPC with DPPE in the inner layer introduces an additional contribution to the total value of


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Figure 9. (a-b) Excess free energy of mixing (∆Gexc) vs composition plots for ternary mixed monolayers of DPPC/DPPE/cholesterol at various constant surface pressures: (a) DPPC/DPPE proportion mimics inner leaflet of membrane and (b) DPPC/DPPE proportion mimics outer leaflet of membrane.

∆Gexc. Therefore, despite a higher content of DPPC in the outer layer and a stronger affinity of cholesterol toward phosphatidylcholine than phosphatidylethanolamine, the resultant thermodynamic effect is very similar for both investigated ternary mixtures. The Correlation between Monolayers and Bilayers. The behavior of the lipids in a membrane is studied usually on model systems, such as a monolayer or bilayer, mimicking the natural membrane. As is known, the natural membrane consists of two layers of different composition, and the Langmuir monolayer technique allows modeling both membrane leaflets independently. However, since lipids molecules in biological membranes are organized into a bilayer, it is of great importance to know if the monolayer properties reflect bilayer behavior. Therefore, many scientists have performed detailed studies concerning the correlation between monolayers and bilayers consisting of membrane components.38-43 The thermodynamic analysis provided by Marsh38 proves that the lipid monolayers and bilayers are in corresponding states when the monolayer surface pressure is equal to the hydrophobic free energy density. Thus, the surface pressure in a lipid monolayer is related directly to the effective lateral pressure in a lipid bilayer. It has been found38 that the monolayer properties correspond to the properties of bilayers at a surface pressure between 30 and 35 mN/m. Moreover, in the above-mentioned surface pressure range, the area per lipid molecule in the monolayer corresponds to that in a bilayer, and the elastic compressibility modulus for a monolayer is comparable to that for a bilayer. Additionally, from the comparison of the characteristics of the phase transitions in bilayer and monolayer results,44 both the area per molecule and its change at the monolayer phase transition correspond to those in a bilayer at π ) 30 mN/m. Other experimental results regarding, for example, partitioning of amphiphiles into a lipid monolayer and bilayer,45,46 phosholipase activity45,47 and phospholipase hydrolysis of the erythrocyte membrane48 prove that monolayer and bilayer properties closely correlate at a surface pressure in the range of 30-35 mN/m. In addition, the results of our experiments correlate well with those presented in the literature for respective lipid bilayer. As has been found by Nagle et al.,39 the area per lipid molecule in a pure DPPC bilayer (20 °C) is equal to 0.48 nm2, whereas our results indicate that in a DPPC monolayer at π ) 30 mN/m (20 °C), the area per lipid molecule is 0.45 nm2. Concerning cholesterol, the area per molecule in the monolayer is equal to 0.37 nm2 (our results) (at π ) 30

mN/m), whereas Ro´g et al.49 and Smondyrev et al.50 adopted the area of 0.39 and 0.32 nm2, respectively, for bilayer experiments. Interestingly, the elastic compressibility modulus values for the investigated by us lipid monolayers (at π ) 30 mN/m) are also in good agreement with the results for the respective bilayers (166 ( 20 mN/m for DPPC monolayer (our experiments) and 140 ( 16 mN/m for egg-DPPC bilayer;51 530 ( 20 mN/m for 1:1 cholesterol/DPPC monolayer (our results) and 685 mN/m for 1:1 cholesterol/DMPC bilayer52). For cholesterol/DPPC mixed systems, the decrease in the area per molecule (at π ) 30 mN/m) in the monolayer has been observed, and the same effect has been found by Smondyrev et al.50 for cholesterol/DPPC and by Ro´g et al.49 for a cholesterol/ DMPC bilayer. The results of monolayer experiments for DPPC/DPPE mixed systems have been compared with those resulting from a molecular simulation study of mixed DPPC/DPPE bilayers.53 Both monolayer and bilayer studies proved that the values of the area per lipid molecule decrease with the addition of DPPE. Moreover, on the basis of the results obtained by Leekumjorn et al.,53 we have calculated the excess area per lipid molecule, and we found that for both mono- and bilayers, these values are negative in the investigated range of lipid proportion. Conclusions In order to get insight into the possible distribution of cholesterol between inner and outer layers of the human erythrocyte membrane, the interactions between cholesterol and phospholipids (phosphatidylcholine and phosphatidylethanolamine, the main type of phospholipids in outer and inner membrane leaflets, respectively) in ternary mixed Langmuir monolayers have been investigated. To link the obtained results to the situation in a natural membrane, a simultaneous analysis of the interactions in binary mixed systemsscholesterol/PC, cholesterol/PE, and PC/PEshave been performed. It has been found that despite a significantly stronger affinity of cholesterol toward phosphatidylcholine (as compared to PE), which is a main type of the phospholipids in the outer leaflet of a natural membrane, the influence of sterol on both outer- and innerlayer-mimicking mixed systems is very similar (the course of the ∆Gexc vs Xcholesterol plots is similar for both of the investigated model membranes). However, the analysis of the interaction in DPPC/DPPE mixed monolayers allows understanding the results

2502 J. Phys. Chem. B, Vol. 111, No. 10, 2007 obtained for ternary mixtures. In the mixed system imitating an outer layer, the DPPC/DPPE proportion corresponds to the composition of a binary DPPC/DPPE monolayer where positive values of ∆Gexc have been found. Positive values of the free energies indicate unfavorable mixing of DPPC and DPPE molecules, resulting in weakness of the effect of strong attractions between PC and cholesterol molecules in the outer layer. On the other hand, thermodynamically favorable mixing (negative values of of ∆Gexc) of molecules in a DPPC/DPPE monolayer of composition corresponding to the phospholipid proportion in ternary monolayers mimicking an inner layer introduces an additional contribution to the total value of ∆Gexc obtained for a ternary monolayer (inner leaflet). As a result, the thermodynamic effect in both ternary mixed systems is very similar. This allows us to conclude that cholesterol can be distributed symmetrically between the inner and outer layers of the human erythrocyte membrane. The obtained results agree with those obtained by Blau and Bittman,13 who investigated the binding of filipin to cholesterol in ghosts prepared from human erythrocytes. The results of our experiments for ternary mixtures also provide information concerning the influence of cholesterol on membrane fluidity and molecular packing. On the basis of the analysis of the isotherms and the values of the compression modulus at a surface pressure of π ) 30 mN/m, it can be concluded that the incorporation of cholesterol into the DPPC/DPPE model membrane causes a decrease in both the area per lipid molecule and membrane fluidity. Moreover, as proved by our results, the areas per molecule in ternary monolayers mimicking an inner layer are lower as compared to those in the mixed system imitating outer layer. This suggests that in a natural membrane, the inner leaflet is packed more densely than the outer leaflet. References and Notes (1) Karp, G. Cell and molecular biology: concepts and experiments, 4th ed.; Wiley & Sons: New York, 2004; Chapter 4. (2) Edidin, M. Nat. ReV. Mol. Cell. Biol. 2003, 4, 414. (3) Virtanen, J. A.; Cheng, K. H.; Somerharju, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4964. (4) Ohvo-Rekila, H.; Ramstedt, B.; Leppimaki, P.; Slotte, J. P. Prog. Lipid Res. 2002, 41, 66. (5) Boesze-Battaglia, K.; Schimmel, R. J. J. Exp. Biol. 1997, 200, 2927. (6) Anderson, J. M. Org. Biomol. Chem. 2005, 3, 201. (7) Op den Kamp, J. A. F. Annu. ReV. Biochem. 1979, 48, 47. (8) van Deenen, L. L. M.; Op den Kamp, J. A. F.; Roelofsen, B.; Witz, K. W. A. Pure Appl. Chem. 1982, 54, 2443. (9) Chabanel, A.; Flamm, M.; Sung, K. L.; Lee, M. M.; Schachter, D.; Chien, S. Biophys. J. 1983, 44, 171. (10) Fisher, K. A. Proc. Nat. Acad. Sci. 1976, 73, 173. (11) Krylov, A. V.; Pohl, P.; Zeidel, M. L.; Hill, W. G. J. Gen. Physiol. 2001, 118, 333. (12) Brasaemle, D. L.; Robertson, A. D.; Attic, A. D. J. Lipid Res. 1988, 29, 481. (13) Blau, L.; Bittman, R. J. Biol. Chem. 1978, 253, 8366.

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