Langmuir 1991, 7,975-977
975
Interaction Energies of Cholesterol, Phosphatidylserine, and Phosphatidylcholine in Spread Mixed Monolayers at the Air-Water Interface M. A. Alsina, C. Mestres, J. M. Garcia Antbn, M. Espina, I. Haro, and F. Reig'pt Physicochemistry Unit, Faculty of Pharmacy, Barcelona, Spain, and Laboratory of Peptides, CID, CSIC, Barcelona, Spain Received February 26,1990. In Final Form: July 2, 1990
By use of the Pagan0 and Goodrich equations, the mixing excess free energies, interaction energies, and interaction parameters of ternary mixtures of cholesterol/phosphatidylcholine/phosphatidylserine have been calculated. The effects of Na and Ca ions on this system at the air-water interface were studied. Introduction Phospholipidshave been involved in the action of opiate receptors.' Some of them, such as phosphatidylserine, phosphatidylinositol, and phosphatidic acid (all acidic), increase the binding of dihydromorphine to the receptor, and this process is reversed by the presence of phospholipases.2 Nevertheless, it is not clear if these phospholipids are a part of the opiate receptor or if they only act as passive modulators, modifying the microviscosity of the receptor surroundings. On the other hand, it is wellknown that the presence of cholesterol in mono- and bilayers of phospholipids changes drastically the ordered state of molecules and influences the permeability and fluidity of the whole system.3 In previous studies, we have found that the interactions between phosphatidylserine and morphine or naloxone are different and modified by the presence of ions.4 Moreover, when studying mixed monolayers of phosphatidylcholine (PC)/phosphatidylserine(PS)interactions, we found a clear deviation from linearity that became more significant in the presence of Na and Ca ions for a PC/PS molar ratio of 0.2/0.85 (mixed monolayers are more expanded than the corresponding to ideal mixtures). We have chosen this molecular relationship to study the influence of increasing amounts of cholesterol in this mixtures, on the thermodynamic parameters. In this paper the interactions among molecules in these three-component mixtures are calculated by means of the mixing excess free energy, interaction energy, and interaction parameters. Moreover, the influence of Na and Ca ions on this system has also been studied. Materials and Methods Bovine phosphatidylserine was purchased from Supelco. Purity was checked by HPLC using C18-coated silica gel 60 plates (Merck). The developing system was chloroform/ methanol/ammonia (4 N) (9:7:2) (v/v/v). A single spot was obtained after spraying with sulfuric/chromic acid and charring. The content of phosphorus was determined + Physicochemistry
Unit.
(1) Cho,T. M.;Hanegawa,J.4.; Ge, B.-L.; Loh, H. H. Proc.Natl. Acad. Sci. 1986, 83, 4138-4142. (2) Loh, H. H.; Hitzeman, R. J. Proceedings of the European Society for Neurochemistry;Neuhoff, V., Ed.;Verlag Chemie: Berlin, 1978; Vol. 1, pp 404-424. (3) Nakagaki,M.;Tomita, K.; Handa, T.Biochemistry 1985,24,461* 4624. (4) Reig, F.; Meetree, C.; Garcia Anthn, J. M.; Valencia, G.; Alsina, M. A. Znt. J. Pharm. 1988,44,269-272. (5) Abina, M. A.; Mestres, C.; Valencia, G.; Garcia AnMn, J. M.; Reig, F.Colloids Surf. 1989-89, 34, 151-158.
0743-7463/91/2407-0975$02.50/0
after perchloric acid digestioq6assuminga molecular mass of 790 daltons. Egg yolk lecithin was obtained from Merck. It was purified by column chromatography on alumina by using CHCls/MeOH (9:l)(V/V)' as eluent. Its estimated molecular mass was 745 daltons. Cholesterol, sodium chloride, and calcium chloride were purchased from Sigma. Water for the Langmuir balance was prepared by distillation over potassium permanganate of single distilled water in an all-glass apparatus. The resistitivity was always greater than 16 MO/cm, the pH was 5.5-6, and it was distilled every day. Chloroform (Merck, pro analysi) was used as spreading solvent. Phosphatidylserine/phosphatidylcholine (0.80.2), phosphatidylserine/phosphatidylcholine/cholesterol, and cholesterol films were prepared from chloroform solutions of 1mg/mL. The PC/PS ratio was maintained constant in all the mixtures. Compression isotherms were performed on a Langmuir film balance equipped with a Wilhelmy platinum plate, as described by Verger and de Ham8 The output of the pressure pickup (Beckmann LM 600 microbalance) was calibrated by recording the well-known isotherm of stearic acid. This isotherm is characterized by a sharp phase transition at 25 "am-' on pure water at 20 "C. The Teflon trough (surface area, 495 cm; volume, 309.73 mL) was regularly cleaned with hot chromic acid; moreover, before each experiment it was washed with ethanol and rinsed with double-distilledwater. Before each run, the platinum plate was cleaned with chromic acid and rinsed with double distilled water. Films were spread on the aqueous surface with a Hamilton microsyringe, and at least 10 min was allowed for solvent evaporation. Films were compressed continuously at a rate of 4.2 cm/min; changes in the compression rate did not alter the shape of the isotherms. All the isotherms were run at least 3 times in the direction of increasing pressure with freshly prepared films. The accuracy of the system under the conditions in which the bulk of the reported measurements were made was 0.5 nN-m-l for surface pressure. Stability of phosphatidylserine/ phosphatidylcholine/cholesterol films was assessed by compressing monolayersto a pressure of 25 mN-m-l, stopping the barrier, and observing the pressure decay. No pressure changes were observed after 30 min. All measurements were made at 21 f 1 "C and at a pH in the range 5.5-6. As we previously determined, no further pH adjustement was necessary because phosphatidylserine, phosphatidyl(6) Barlett, G.R. J. Biol. Chem. 1969, 234 (31, 466-468. (7) Singleton, W. S. J. Am. Oil Chem. SOC.1965,42-53. ( 8 )Verger, R.; de Haaa, G. H. Chem. Phys. Lipids 1973,10,127-135.
0 1991 American Chemical Society
Alsina et al.
976 Langmuir, Vol. 7, No. 5, 1991
choline, and cholesterol films are not significantly influenced by pH changes in this range. This fact is in agreement with published d a h g
AG; -a -
Rssults The mean area per molecule for the lipid mixtures under study has been given previously.1° It is clear that the presence of cholesterol induces a compression of the mixed monolayer all over the lipidic compositions assayed and this behavior is not highly modified by the presence of Na or Ca ions. This condensing effect of cholesterol is usual when this molecule interactswith lipids that give expanded monolayers and can be explained by two theories. Shah and Shulmanll suggested that the area decreases observed for mixed monolayers of phospholipids and cholesterol are not due to hydrophobic interactions but to the presence of intermolecular cavities, between the conical shapes of phospholipids molecules. This situation could allow the cholesterol molecules to be introduced or to occupy the room between adjacent phospholipid molecules, thus producing a more condensed monolayer. The dynamic model proposed by Cadenhead', was that the presence of cholesterol reduces the free motion of hydrocarbon chains, and this results in a condensing effect. The condensing effect of cholesterol is manifested in a negative deviation from linearity (ideal behavior). The values of excess free energy of mixing (Figure 1) can be calculated from the difference between areas under the isotherms of the experimental and ideal films for a specified surface pressure. The sign they have gives information about the stability of these mixtures. In the present case, negative excess free energy of mixing indicates that the interactions are more favored than in the ideal state. The values of AGm" have been calculated by applying eq 1. This equation has been obtained by following the Goodrich13 and Pagano and Gershfeld14 approaches. Numerical values were calculated according to the mathematical method of Simpson. The region below the lowest reproducible pressure was assumed to go to zero at the lift-off print. For this reason the two-phase liquid-gas and phase gaseous regions are not included in the integration.
T
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-4-
0
U
S
a
2
-6
9
-10 8
(9) Bangham, A.; Papahadjopouloe,D. Biochim. Biophys. Acta 1966, 126,181-184. (10) b i g , F.; Meetres, C.; Valencia, G.; Garch AnMn, J. M.; k i n a , M. A. Colloid Polym. Sci. 1989,267, 139-144. (11) Shah, D. 0.; Shiao, S. Y. Monolayers; Advances in Chemistry Series 144;Goddard,E. D., Ed.;AmericanChemical Society Washington, DC, 1978; pp 153-176. (12) Cadenhead, D. A.; Muller-Landau, F. J. Colloid Interface Sci. 1980, 78, 26W270. (13) Goodrich, F. C. Proceedings of the 11th International Congress on Surface Actioity; Butterworthe: London, 1957. (14) Pagano, R. E.; Gerehfeld, N. L. J. Colloid Interface Sci. 1972,41 (21, 311.
ao
a4
ao
a4
ao
a4
AG;
-
-a
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h
-4-
0 U S
a
2s
-6
- 0 1 -
8
ia
16
mNm' c
01
-1
the mean molar area in the mixed film, A1 and A2 are the molar areas of the two pure components, and N1 and N2 are the molar fractions of monolayers components 1 and 2. In this work component 1is a PS/PC mixture of constant molar composition (0.8/0.2) and component 2 is cholesterol. Graphics of Figure 1 show that at high pressures the interactions among molecules increase, thus rendering higher absolute values for AGmex. This trend is attenuated when the cholesterol content of the mixtures increase due to the fact that cholesterol diminishes drastically the compressibility of the monolayer and thus the ability of
16
mN.m-'
AG: A12 is
ia
-
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a
: s
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-101
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ia
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Figure 1. Excess free energy of mixing (AG,Jsmol-') in mixed
monolayers of PS/PC/Chol: ( 0 )PS/PC/Chol (0.64/0.16/0.2) (+) PS/PC/Chol (0.48/0.12/0.4) (*) PS PC/Chol (0.32/0.08/ 0.6) (0) PS/PC/Chol (0.16/0.04/0.8). he subDhaees were (a) pure'water,' (b)h a c 1 0.1 hd,and (c).CaCl 1 mhi.
4
molecules to interact with each other. Moreover, as AGm" varies linearly with respect to the surface pressure, phase changes can probably be discarded. The presence of ions affects the compressibility of the
Interaction Energies
Langmuir, Vol. 7, No. 5, 1991 977
monolayer, the effect being greater for Ca2+than for Na+. On comparison of the changes in the AGme' values, as a function of cholesterol content, the regularity observed in AGm" over water is modified when there are ions in the subphase. Ca and Na ions seem to interact in a different degree with phosphatidylserine/phosphatidylcholine/ cholesterol monolayers as a function of the initial composition of the monolayer. But in general it is not possible to find significant differences among the three subphases, only the fact that Ca2+ions expand the mixed monolayer and compensate partly for the condensing effect of cholesterol specially at lower contents of cholesterol. If we examine the effect of ions on monolayers of pure cholesterol, we can observe that they do not modify the area/molecule, but ita presence reduces the collapse pressure of monolayers.s That is why the values of AG,", Ah, and a for subphases of NaCl 0.1 M could not be calculated for mixtures containing a high percentage of cholesterol. The values of interaction parameter (a)at different pressures and the energy corresponding to these interactions, Ah,have been calculated by applying eq 2 and 3 derived from Joos et al.l"17 and Margulesls
Table I. Interaction Parameters and Energies in PS/PC/ Chol Mixed Monolayers, Measured at Different Pressures*
r
8 12 16 20 24
r
8 12 16 20 24
a=
RT(X,X,~+ x2x,2)
Ah = RTa/Z (3) X1 and X2 are molar fractions (1 and 2 being the same components as defined for eq 1). For the determination of the coordination number (Z)we , followed the model of Quikenden and Tam,l9 by considering that in a closely packed monolayer (collapse),each molecule is surrounded by six neighbors. For lower pressures, we applied eq 4 to calculate the packing fraction (PF). This value was used to obtain the correspondingZ according to the equivalences given in ref 19.
PF = 0.907Am,/A, (4) A,, is the area/molecule of the mixture at the collapse point and Am the area molecule of the mixture at each of the intermediate pressures. The numerical values for all these parameters are given in Table I. The variation of Ah values is not as regular as for other parameters because in its calculation one must take into account the coordination numbers. As packing fractions are calculated according to eq 4 and 2 is derived from these values, if the areas at different pressures are not different from the collapse area, the PF will be close to unity and in consequence the corresponding 2 number will be 6. For water containing subphases, isotherms from 12to 16mN-m-l soon reach the solid state and this explains the lack of regularity observed in the sequence values up to 16 "om-1 (indicated with an asterisk). In summary (15)Jm, P.Bull. SOC.Belg. 1969,78, 207. (16)J m , P.;Demel, R. A. Biochem. Biophys. Acta 1969,183, 447. (17)J m , P.;Ruywen, R.; Miilonee, J.; Garcia Femhdez, S.;Sanz
Pdrrro, P. J. Chim. Phy6. Phyricochim. Eiol. 1969, 66 (lo), 1666. (18)ThermodymmicsforChemists;Glasstone, S., Ed.;Aguilar (epanish version), 1972;Chapter XIV. (19)Quickenden,T.I.; Tan,G. K.J. Colloid Interface Sci. 1974,48, (31, 382-393.
CaCl2 -2682.72 -4222.8 -5713.93 -7290.58 -8842.83
(b) PS/PC/Chol: 0.48/0.12/0.4 a Ah, J-mol-' HzO NaCl CaCl2 HzO NaCl -3.47 -4.26 -1.5 -2120.5 -3472.6 -5.22 -6.37 -2.43 -3190.0 -3892.79 -6.91 -8.44 -3.28 -2813.80; -5157.70 -8.50 -10.49 -4.11 -3464.51 -6110.44 -10.06 -12.52 -4.88 -4100.37* -7651.15
CaClz -916.67 -1484.98 -2007.77 -2511.67 -2982.22
(c) PS/PC/Chol: 0.32/0.08/0.6 Ah, J-mol-' NaCl CaCl2 H20 NaCl -2.85 -1.7 -1435.83 -2323.03 -4.27 -2.51 -2145.0 -2609.44 -5.51 -3.23 -1883.04* -3367.24 -7.06 -3.94 -2315.09* -4314.47 -8.48 -4.58 -2739.4+ -5182.12
CaCh -1038.89 -1408.49 -1973.87 -2407.76 -2798.88
HzO -6.7 -10.07 -13.18 -16.37 -19.37
a
I
AG,"
(a) PS/PC/Chol: 0.64/0.16/0.2 Ah, J-mol-' NaCl CaClz Ha0 NaCl -7691.6 -6.29 -4.39 -4094.31 -9.84 -6.91 -6153.79 -8021.37 -9.35 -8054.44 -8253.70 -13.5 -17.36 -11.93 -6672.24. -10608.84 -21.2 -14.47 -7890.16* -12955.49 a
8 12 16 20 24
H2O -2.35 -3.51 -4.62 -5.68 -6.72
(d) PS/PC/Chol: 0.16/0.04/0.8 a Ah, Jamol-1 NaCl CaClz HzO NaCl -2.10 -1.53 -1100.0 -1283.34 -3.14 -2.30 -1619.45 -1918.87 -4.15 -3.09 -1414.30* -2536.13 -3.94 -1733.82* -4.76 -2042.01*
HzO CaC12 8 -1.8 -934.98 12 -2.65 -1406.66 16 -3.47 -1888.31 20 -4.25 -2407.76 24 -5.01 -2908.90 0 HzO stands for experimenta carried out on a water subphase, NaCl for experimenta carried out on a 100 mM sodium chloride solution, and CaClz for experiments carried out on a 1 mM calcium chloride eubphaee. T
one can say that calcium ions interact strongly with phosphatidylserine ionic groups and clearly inhibit the condensing effect of large amounts of cholesterol, whereas the effect of sodium ions in this system is negligible. The influence of ions in the binding of opiates to their receptors is highly dependent on the agonist-antagonist character of the molecules under study. As a general rule monovalent cations inhibited the binding of agonists and have no effect in the binding of antagonists. Divalent cations activate the binding of 6 agonists and inhibit the binding of K and p agonists. To explain these factsan allostericchange in the receptor has been suggested. Following this assumption, as the influence of sodium ions in our system is not important, this should be the model for the allosteric state of interaction with antagonists. Acknowledgment. This work was supported by agrant (No. PA85-0110-C02-02 from DGICYT (Spain). Registry No. CaZ+, 7440-70-2;Na+, 7440-23-5;cholesterol, 57-88-5.
