Langmuir 1992,8, 1388-1391
1388
Miscibility of Dipalmitoylphosphatidylcholine and Sulfatides in Mono- and Bilayers C. Mestres,? M. A. Alsina,+M. Espina,t L. Rodriguez,$and F. Reig'ls Physicochemical Department, Faculty of Pharmacy, Barcelona, Spain, Faculty of Medicine, Barcelona, Spain, and Laboratory of Peptides, CSIC-CID, Barcelona, Spain Received May 24,1991. In Final Form: January 29, 1992 The miscibility of dipalmitoylphosphatidylcholinelsulfatidemonolayerswas studied by using compression isotherms and differential scanning calorimetry. The mixing excess free energies, interaction energies, and interaction parameters of these mixtures were calculatedfrom the compressionisotherms. The influence of pH and Ca2+ ions in the miscibility was determined. The results show that these parameters have a very small influence in the miscibility energies of this binary system.
Introduction Sphingolipids are involved in the synaptic transmission, and play an important role modulating the movement of Ca2+ions across the plasma Compared to phospholipids, their geometry, bulkiness, and polarity introduce the possibility of different statesof aggregation and order.3 For this reason it is interesting to know how this type of lipid interacts with the main lipidic component of biological membranes (phosphatidylcholine). In this work, the miscibility characteristics of sulfatides and dipalmitoylphosphatidylcholine were studied using the differential scanning calorimetry (DSC) of multilamellar liposomes and the compression isotherms of mixed monolayers. The influence of calcium ions and pH in this system was determined. The thermodynamic parameters associated with this process, the excess free energy of mixing AG%, interaction parameters a and E, and interaction energies Ah and hE, were calculated a t different surface pressures and at the collapse pressures.
Materials and Methods Sulfatides and DPPC were purchased from Supelco. All reagents and solvents were of analytical grade. Water for the Langmuir film balance was prepared by distillation of distilled water over potassium permanganate in an all-glass apparatus. The pH of water ranged from 5.5 to 6 and ita resistivity was always greater than 16 MQ/cm. Water was freshly distilled every day. Spreading solvent was chloroform (Merck). Lipid films were prepared from chloroform solutions of approximate concentrations of 1 mg/mL. Monolayers were spread on the following subphases: water pH = 5.5; M CaC12, pH = 5.5 and pH = 7.4.
Compression Isotherms. Compression isotherms were performed on a Langmuir film balance equipped with a Wilhelmy platinum plate, as described by Verger and de Haas.' The output of the pressure pickup (Beckmann Sartorius LM 600 microbalance) was calibrated by recording the well-known isotherm of stearic acid, which is characterized by a sharp phase transition at 25 mN.m-'on pure water at 20 "C. The Teflon trough (surface area, 495 cm*;volume, 309.73mL) was regularly cleaned with hot + Faculty of
Pharmacy. 8 Faculty of Medicine. 8 CSIC-CID. (1) Cumar, F.
A.; Maggio, B.; Caputto, R. Biochim. Biophys. Acta 1980,597, 174-182. (2) Blow, A. M.; Botham, G. M.; Way, J. A. Biochem. J. 1979, 182, 555-563. (3) Beitinger, H.; Schifferer, F.; Sugita, M.; Araki, S.; Satake, M.; M6bius, D.; Rahmann, H. J. Biochem. 1989,105,664-669. (4) Verger, R.; de Haas, G. H.Chem. Phys. Lipid8 1973,10,127-135. 0743-746319212408-1388$03.0010
chromic acid; moreover, before each experiment it was washed with ethanol and rinsed with twice distilled water. Films were spread on the aqueous surface using 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 increasingpressure with freshly prepared films. The accuracyof the system under the conditions in which the bulk of the reported measurements were made was f0.5 mN-m-' for surface pressure. Stability of sulfatides and DPPC films was assessed by compressing monolayers to a pressure of 25 mN-m-l,stoppingthe barrier,and observingthe pressuredecay. No pressure changes were observed after 30 min. All measurementa were made at 21 f 1 "C. Calorimetric Study. Lipid sampleswere prepared by mixing adequate volumes of mother solutions of 20 mM DPPC and sulfatides in chloroform and chloroform/methanol, respectively. After evaporation of the solvent,the lipid mixtures were hydrated by addition of 150 rL of water (pH = 5.5) or CaClz 10 mM (pH = 5.5 and 7.4). The three seriesof samples,containinglodifferent molar compositions of DPPC/sulfatides, were heated at 60 "C for 1 h and equilibrated at approximately 4 "C for over 1week. The thermal analyseswere carried out in a Perkin-ElmerDSC2 calorimeter with a heating speed of 5 "Clmin and an argon atmosphere.
Results and Discussion Compression Isotherms. The miscibility of DPPC/ sulfatide was determined as monolayers spread on three different subphases, water at pH = 5.5 and M CaC12 at pH = 7.4 and 5.5. In a previous study using PC/sulfatide mixtures, we found that maximal deviation from ideality appeared at CaC12 pH = 7.4 subphases; on the contrary a nearly ideal miscibility was found on water at pH = 5hW5For this reason, these subphases were selected in the present study. The pressure-area curves for pure DPPC and DPPC/ sulfatide monolayers show a great similarity independently on the subphase composition. The isotherms corresponding to a subphase of CaCl2 at pH = 5.5 are given in Figure *I.
There are not great differences in the compressibility of the monolayers, being all are in the liquid-expanded state, but the maximum compression pressures achieved are dependent on the composition of the subphase. In Table I, the collapse pressures and the respective areas are shown. (5) Mestres, C.; Espina, M.; Haro, I.; b i g , F.; Alsina, M. A.; Garcia AnMn, J. M. Colloid Polym. Sci. in press.
0 1992 American Chemical Society
Miscibility of DPPC and Sulfatides
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Langmuir, Vol. 8, No. 5, 1992 1389 \
50
I
i
I
E co
W
II
E
I
130
E
n
8
9 L
I 2o
3 10
80 120 160 200
AREA (A? molec-') Figure 1. Surface pressure/area curves of mixed monolayers of DPPC and sulfatides. The DPPC molar fractions were as follows: ., 1.0; A, 0.8;*, 0.6;0 , 0.4;0,0.2; 0.0.
*,
Table I. CollaDse Pressures and the Reswctive Areas in DPPC/Sulfatide Mixed Monolayers SDread on Different SubDhases ~
~~
H20 (PH = 5.5)
~
CaCl2 (pH = 5.5)
CaCl2 (pH = 7.4)
sulfatide molar fraction
collapse measure. mN1m
area, 1\21 molecule
collapse Dressure, mN1m
area, 1\21 molecule
collapse Dressure, mN1m
area, 1\21 molecule
0 0.2 0.4 0.6 0.8
58.7 56 55.25 51 49.75 59.8
47.27 47.27 41.21 40 40
53.75 53.63 53.63 53.63 53.63 43.60
58.18 50.90 47.21 41.21 47.21
57 56.75 53.63 53.63 53.63 55.50
47.27 47.27 50.80 41.21 40
1
40
40
40
between the two types of molecules and is slightly dependent on the surface pressure. The values of excess free energy of mixing AGE^ were calculated from the differences between areas, under the isotherms, of experimental and ideal films for a specified surface pressure, following the mathematical method of Simpsod and according to the Goodrich7and Pagano and Gershfelda approaches
z
AGEM= JA ; 2l
0
0,2
0,4
0,6
0,8
1
SULFATIDE MOLAR FRACTION
Figure 2. Variation of the surface pressureof the phase transition of DPPC with the sulfatides content, for three subphases used.
The presence of increasing amounts of sulfatides lowers the pressure of the phase transition of DPPC. Moreover, the presence of calcium ions has a positive effect in this parameter, specially a t neutral pH values. This behavior is illustrated in Figure 2. The mean molecular areas of mixed monolayers represented as a function of the composition a t three surface and 40 "am-l) are given in Figure 3. pressures (10,20, There are small positive deviations from ideality, specially for subphases containing CaC12 at pH = 7.4. In this case the deviation at DPPC/sulfatide 0.6/0.4is maximum. This fact is indicative of the presence of repulsive interactions
dn - N I J L A l d n - N2JLA2dn
where A12 is the mean molar area in the mixed film, A1 and A2 are the molar areas in the pure films, and N1 and N2 are the molar fractions of monolayer components 1 and 2. The interaction parameters at different pressures (a) and at the collapse point ([) and the energies corresponding to these interactions (Ahand AJ3) were calculated by applying the equations given in ref 6 derived from Joos et al.+l1 and Margules12 (6) Alsina, M. A.; Mestres, C.; Valencia, G.; Garcia Anan, J. M.; b i g , F. Colloids Surf. 1989, 34, 151-158. (7) Goodrich, F. C. Proceedings of the 2nd International Congress Surface Activity; Butterworths: London, 1957. (8) Pagano,R. E.; Gershfeld, N. L. J. Colloid Interface Sci. 1972,41, 311. (9) Joos, P.Bull. SOC.Chim. Belg. 1969, 78, 207. (10) Joos, P.; Demel, R. A. Biochim. Biophys. Acta 1969,447. (11) Joos, P.; Ruyssen, R.; Miflones, J.; Garcia Fernhdez, S.; Sanz Pedrero, P. J . Chim. Phys. Chim. Biol. 1969,66, 1665. (12) Glasstone, S. In Thermodynamics for Chemists; Aguilar, Ed.; Madrid, 1972; Chapter 14 (Spanish version).
Mestres et al.
1390 Langmuir, Vol. 8,No. 5, 1992
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Table 11. Interaction Parameters and Energies in DPPC/Sulfatide Mixed Monolayers Spread on Different Subphases
40 mNlm 0
H20
* CsU2pHd.5
05
sulfatide AGE^, molar fraction J/mol a z 20 mN/m, CaClz Subphase (pH = 5.5) 0.2 -750.4 -1.9 4 0.4 -460.1 -0.7 4 0.6 155.9 0.2 4 0.8 528.8 1.3 4
+ CsC12pH-7.4
751 65
-
95
20 mNlm 0 H20 CaW2pH-5.5 .c CaC12 pH-7.4
*
85-
-
75
-
,
0.0
.
,
0,2
.
,
'
I
0.4
M
,
0.6
.
0,8
W FRACTIONS
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1
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95
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10 mNlm
1
H20 CaC12 pH-5.5 + CaC12 pH-7.4 0
*
65-
35 0,o
0.2
0,4
0,6
0.8
1 .O
MOLAR FRACTION S
Figure 3. (Area/molecule)/molarcomposition for the mixed monolayersspread on three differentsubphases. Measurements were done at 10, 20, and 40 "em-'.
where X a and Xb are the molar fractions, AI is the collapse pressure of component 1and rm the collapse pressure of the mixture, k is the Boltzmann constant, ~2 is the molar fraction of component 2,and A1 is the area per molecular of component 1a t the collapse point. The energies corresponding to the interactions were calculated by applying the corresponding equations
Ah = RTa/z AE = RTSIz The calculated values of these parameters at 20 mN0m-l
a,
J/mol
-279.3 -114.0 38.0 270.5
0.2 0.4 0.6 0.8
20 mN/m, Water Subphase (pH = 5.5) 52.6 0.1 4 0.2 4 129.1 -215.3 -0.3 4 -209.1 -0.5 4
73.3 134.4 -220.0 -324.0
0.2 0.4 0.6 0.8
20 mN/m, CaClz Subphase (pH = 7.4) -1098.7 -2.8 4 1698.4 2.8 2 1201.6 2.0 4 344.8 0.8 4
-1711.8 3533.8 1247.2 538.0
of surface pressure and at the collapse point are given in Tables I1 and 111. Their sign gives information about the stability of these mixtures. While the interaction effects, which have been observed, appear striking when plotted in terms of molecular areas, the excess free energies of mixing are relatively small. In all cases, the absolute values of these energies are much less than RT (which amounts 2474 J/ mol at 25 "C). Although it is not possible to compare a and [ because they have been calculated by two different methods. One can appreciate that the maximal interactions are found in both cases for CaC12 pH = 7.4 subphases at a sulfatide molar fraction of 0.4. The rest of the energies are so low that no other conclusions can be drawn. Calorimetric Studies. The DSC curves of DPPC give two peaks corresponding to the following transitions: a pretransition (309.5K)due to the change from hydrated gel phase (LBt) to ripple phase (PPI, and the main peak (314.5K)that represents the transition from Pg' to liquid crystalline lamellar phase (La). Sulfatides are a mixture of miscible compounds whose chemical structures are closely related, and their interval of transformation appears between 322 and 329.5 K. The DSC curves corresponding to the lipids aqueous dispersions in CaC12 10 mM, pH = 5.5, for the different lipid mixtures are given in Figure 4. Registers of pure DPPC and DPPC molar fraction 0.9 were recorded at 4 times lower sensitivity, and the sample containing a 0.8 molar fraction of DPPC was recorded at 2 times lower sensitivity than the rest of the measurements. For a qualitative point of view one can appreciate two, four, or three peaks depending on the sulfatide molar fraction. Samples having 10% and 20% sulfatides have two peaks, while when the amount of sulfatides is 30% or 40% the DSC curves show four peaks, and at higher percentages of sulfatides only three peaks can be noticed. These characteristics are shown in the phases diagram of Figure 5. One can observe (a) a total miscibility in the hydrated phase Lp and in the liquid crystalline lamellar phase Laand (b) the presence of a ripple phase (Pp3from 0 to 45 mol % of sulfatides. These facts promote the existence of three biphasic regions and a triphasic degenerated region at approximately 320 K. On the other hand, differences in pH values (5.5 or 7.4) and presence or absence of calcium ions have a limited influence in the transition temperatures of pure DPPC and on the miscibility of both components; although the broadening observed in the peak profile suggests a
Miscibility of DPPC and Sulfatides
Langmuir, Vol. 8, No. 5, 1992 1391
Table 111. Interaction Parameters, 4, and Energies, AE, in DPPC/Sulfatide Mixed Monolayers at the Collapse Point. sulfatide 5 AE,Jlmol H2O CaClz CaC12 molar H2O CaClz CaClz (pH = 5.5) (pH = 7.4) (pH = 5.5) (pH = 5.5) (PH = 7.4) fraction (pH = 5.5) 0.2 5.31 0.420 0.71 2191.5 173.30 293.0 1019.4 0.4 2.47 0.106 9.60 41.27 3962.2 0.6 2.45 0.047 1.07 1011.2 19.39 441.6 0.8 4.06 0.016 0.60 1675.7 10.73 247.6 0
The coordination number (Z) at the collapse point is 6.
T (Kl
330
325
320
315
310
Li?
20
40
60
80
I
s
DPPC I*O I
I00 I
I10 1
IIJ I
110
M a I (I1 I
Figure 4. DSC heating curves of DPPC/sulfatide mixtures in M CaC12, pH = 5.5.
reduction in the cooperativity of the transformation for liposomes containing calcium ions. Nevertheless, the sequence of DSC registers given in Figure 4 shows an important change for molar contents of sulfatides of 0.4 that are coincident with the results obtained in the mixed monolayers. Comparing the values of excess free energy of mixing of PC/S (ref 5 ) and DPPC/S, one can appreciate that the highest deviations from ideality appear when using PC. This behavior is due to the fluidity of PC monolayers, that at 21 "C are in liquid crystalline phase. In this
3 mol S --c
Figure 5. Phase diagram of DPPC/sulfatide mixtures in CaC12, pH = 5.5.
s M
situation repulsions among PC/S molecules can be freely manifested. On the contrary at this temperature DPPC molecules are in a tightly packed gel state, having thus a limited freedom of movement and being, as a consequence, less sensitive to distortions. The data obtained from the above cited techniques are coincident and show clearly that the energy involved in the interactions between DPPC and sulfatides is very small.
Acknowledgment. This work was supported by a g r y t (FAR 88-0692)from Ministerio de Industria y Energia (Spain)* Registry No. DPPC, 2644-64-6;Ca2+,7440-70-2.
