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Surface Potential of Lipid Interfaces Formed by Mixtures of Phosphatidylcholine of Different Chain Lengths M. del C. Luzardo,† G. Peltzer, and E. A. Disalvo* Laboratory of Physical Chemistry of Lipid Membranes, Quı´mica General e Inorga´ nica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Buenos Aires, Argentina Received November 20, 1997. In Final Form: April 23, 1998 The dipole and ζ potentials of mixtures of dimyristoylphosphatidylcholine /dipalmitoylphosphatidylcholine (DMPC/DPPC), dioleoylphosphatidylcholine/ distearoylphosphatidylcholine (DOPC/DSPC), and dioleoylphosphatidylcholine /dimyristoylphosphatidylcholine (DOPC/DMPC) were measured as a function of the mole fractions at temperatures below and above the phase transition. The dipole potentials of lipid films were determined in monolayers by means of the constant-area method proposed by MacDonald and Simon (Proc. Natl. Acad. Sci. USA 1987, 84 4089). The ζ potentials were obtained by measuring the electrophoretic mobility of MLVs for similar mixtures at the same temperature. Discontinuities in the dipole potential and the ζ potential for 0.33 and 0.66 molar fractions were observed for DPPC/DMPC mixtures in the gel state well above the experimental error. These singularities in the values of dipole and ζ potentials were less noticeable or absent in DSPC/DOPC and DMPC/DOPC. The results can be explained as a consequence of the organization of the polar head groups at the lipid interface for peculiar molar ratios when the lipids are miscible in the whole range of molar fractions. It is concluded that the organization of lipids in bilayers gives similar arrangements in the surface to those in monolayers. The surface properties seem to be a consequence of the type and phase state of the lipids in the mixture rather than of the supramolecular organization such as monolayer or bilayer. Thus, monolayers and bilayers are intrinsically similar model systems in regard to their surface properties.
Introduction Lipid monolayers and bilayers are well-known experimental model systems to study membrane properties. Monolayers at the air-water interface provide data on the area per phospholipid and the dipole potential.2-4 Bilayer systems, such as liposomes and vesicles, are useful to measure the surface and the permeability properties.5-7 Several works have been addressed in order to compare the properties of bilayers and monolayers of the same composition.2,8 An important question regards the packing and phase properties in both model systems. The packing in a bilayer has been indirectly determined to be 32-33 dyn/cm by comparing the action of phospholipase on monolayers and bilayers.9 More recently, MacDonald and Simon1 suggested that the organization of molecules in liposomes corresponds closely to that in monolayers in equilibrium with those liposomes. They showed that the dipole potential in a monolayer decreases abruptly at the phase transition congruent with the changes in the refractive index or turbidity observed in a liposome * To whom correspondence should be addressed. Phone: 54 (1) 9648249. Fax: 54 (1) 9648274. E-mail:
[email protected]. † Present address: Facultad de Biologı´a, Universidad de La Habana, Cuba. (1) MacDonald, R. C.; Simon, S. A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4089. (2) Blume, A. Biochim. Biophys. Acta 1979, 557, 32. (3) Simon, S. A.; Lis, L. J.; Kauffman, J. W.; McDonald, R. C Biochim. Biophys. Acta 1975, 375, 317. (4) Pethica, B. A. Faraday Trans. Soc. 1955, 51, 1402. (5) Bangham, A.; de Gier; J.; Greville, G. D. Chem. Phys. Lipids 1976, 1, 225. (6) Reeves, J. P.; Dowben, R. M. J. Membr. Biol. 1970, 3, 313. (7) McLaughlin, S. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 113. (8) Wolfe, D. H.; Brockman, H. L. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4285. (9) Demel, R. A.; Geurts van Kessel, W. S. M.; Zwaal, R. F. A.; Roelofsen, B.; van Deenen, L. L. M. Biochim. Biophys. Acta 1975, 406, 10.
dispersion.10 These inferences have been done by comparing a surface property, such as the dipole potential, with optical properties of liposome dispersions that involve changes in other regions of the bilayer.11 However, despite its relevance in the interpretation of the biophysicochemical properties of a lipid membrane, no attempt has been made, to our knowledge, to check this correspondence in more complex systems such as binary lipid mixtures. The properties of different binary mixtures of phospholipids have been obtained from measurements of fluorescence anisotropy and calorimetry.12-14 Phase diagrams have been interpreted in terms of the ideality of the mixing of the different species. Different techniques have been able to show the coexistence of gel and liquid crystalline states near the transition temperature. Gel domains have been detected in regions of the phase diagrams corresponding to liquid, and liquid domains were found in the region corresponding to the solid state, in both monolayer and bilayer systems.15 Moreover, also in single lipids the sharp transition observed at the transition temperature does not correspond to an “all or none” process delimiting only gel phase on one side and only liquid crystalline phase on the other. Thus, miscibility may not mean a priori a structural homogeneity in all the regions of the membrane. The lipid mixtures may have a structural organization at the interface not reflected in the phase diagrams obtained with bulk properties. These arrangements could affect the electrical surface properties, and in addition, (10) Yi, P.; McDonald, R. C. Chem. Phys. Lipids 1973, 11, 114. (11) Disalvo, E. A. Chem. Phys. Lipids 1991, 59, 199. (12) Lee, A. G. Biochim. Biophys. Acta 1977, 472, 237. (13) Parente, R. A.; Hochli, M.; Lentz, B. R. Biochim. Biophys. Acta 1985, 812, 493. (14) Marsh, D. Handbook of Lipid Bilayers, 1st ed.; CRC Press: Boca Raton, FL, 1991; Section II.13. (15) Bergelson, L. O.; Gawrish, K.; Ferretti, J.; Blumenthal, R. Mol. Membr. Biol. 1995, 12, 1.
S0743-7463(97)01273-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/12/1998
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they should be independent of the lipid model system in which it is measured to be relevant for biophysical studies. For these reasons, the purpose of this paper is to verify the existence of similarities between the structural organizations of a monolayer and a bilayer of binary mixtures by directly comparing their surface electrical properties. For this, we determined the dipole potential of lipid films composed of DMPC/DPPC, DOPC/DMPC, and DOPC/DSPC by means of the constant area method proposed by MacDonald and Simon.1 In parallel, data of the ζ potential were obtained by measuring the electrophoretic mobility of MLVs for similar mixtures at the same temperature. Material and Methods Dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC) were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL) and used as received after checking their purity by thin-layer chromatography. Mixtures of the lipids were prepared in chloroform and kept under nitrogen in stock. For the monolayer experiments, analytical grade KCl was roasted for 3 h at 350 °C. All the solutions were prepared in bidistilled water. Multilamellar vesicles (MLVs) for electrophoretic mobility were prepared by dispersing a lipid film in 1 mM KCl by vortexing above the transition temperature. The lipid film was obtained by evaporating an aliquot of the respective chloroformic solution under vacuum during at least 5 h. The ζ potential was calculated from the electrophoretic mobility using the Helmholtz-Smoluchowsky equation:
ζ ) ηu/0 where u is the electrophoretic mobility, and η are the dielectric permittivity and the dynamic viscosity of the suspension (assumed to be equal to water), respectively, and 0 the permittivity of free space.16 The electrophoretic mobilities of the different samples suspended in 1 mM KCl were determined in a capillary H-cell with Ag/AgCl electrodes. The electrodes were connected to a continuous variable direct current source. The effective electrical distance was calculated by using KCl solutions of known conductivity at 25 °C. The conditions were standardized by determining the ζ potential of phosphatidylserine liposomes whose value was around -120 mV in 0.01 M NaCl at pH 7.4. The rate of migration was determined by microscopic observation of the displacement of individual liposomes along a regular lattice (length, 1 mm). A total of 10 measurements (5 in each direction) were done by applying a voltage of 40 V and alternatively changing the polarity of the electrodes to avoid polarization. All mobilities are the average obtained with three different batches of samples prepared as described. Dipole potential measurements were determined in monolayers spread on the air-water interface of a 1 mM KCl solution. A Teflon trough with a surface area of 16 cm2 was used. The dipole potential was measured at an air-water interface by determining the drop of electrical potential between a Ag/AgCl electrode in the subphase and an aluminum grid (as counter electrode) placed 1 or 2 cm above the surface. The conductivity link was obtained with ionized air, accomplished by means of a 210Po plate, which is an R emissor, sealed between a silver and a gold layer. The foil is protected by the aluminum grid, which prevents direct contact of the foil with the air-water interface. The aluminum grid was connected to a specially designed UHIP (ultrahigh-impedance probe, Z > 1017 Ω). The UHIP and the Ag/AgCl electrode are connected to a data acquisition system. The whole setup was carefully noise-filtered, grounded ,and shielded. Electronic samples were collected at a rate of one sample per second by means of the data acquisition setup. Briefly, the monolayer experiments consist of adding aliquots of the chloroformic solution of lipids to the air-water interface, (16) McLaughlin, S.; Harary, H. Biochemistry 1976, 15, 1941.
Figure 1. Typical variation of the surface potential of the airwater interface with the addition of a chloroform solution of a DSPC/DOPC mixture. (A) After each addition, the solvent was allowed to evaporate before another aliquot was added. The spikes shown by the arrows correspond to the subsequent additions. When the potential showed a constant value, the difference between those obtained without and with lipids was taken as a measure of the dipole potential for that temperature and lipid mixture. (B) Magnitude of the dipole potential obtained for different surface lipid concentrations. previously cleaned exhaustively by suction, until the potential was unchanged. Under these conditions, a constant potential is reached above 20 mmol of lipids. A rough calculation of the area per lipid under these conditions results in a much lower area than that corresponding to pure DMPC, DPPC, DSPC, and DOPC, indicating that the amount of lipids present at the moment of the dipole potential determination was in excess of that needed to saturate the monolayer. Thus, an equilibrium between monolayers and liposomes at the subphase can be reasonably assumed. Temperature was measured with a calibrated thermocouple within (0.5 °C. The values of dipole potential were determined by using the following expression:
Vdipole ) VAg/AgCl - Vgrid ) Vsolution - Vgrid within an error of (50 mV.
Results Figure 1 shows a typical trace obtained with the sequential addition of lipids dissolved in chloroform onto the surface of a 1 mM KCl solution at 20 °C. In this case, aliquots of a solution containing DSPC in DOPC in a 0.33 molar fraction were added at constant time intervals long
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Figure 3. Electrokinetic potential of MLVs of mixtures of (2) DPPC in DMPC at 20 °C, (O) DSPC in DOPC at 20 °C, and (b) DMPC in DOPC at 27 °C.
Figure 4. Dipole potential of monolayers and the ζ potential for MLVs formed by mixtures of DPPC in DMPC at 20 °C.
Figure 2. Dipole potential of monolayers formed by different molar fractions of (A) DPPC (Tc ) 45 °C) and DMPC (Tc ) 24 °C) measured at 20 °C, (B) DSPC (Tc ) 52 °C) and DOPC (Tc ) -5 °C) measured at 20 °C, or (C) DMPC (Tc ) 24 °C) and DOPC (Tc ) -5 °C) measured at 27 °C. Tc ) transition temperature of the pure lipid.
enough to allow the solvent evaporation. The dipole potential measured under these conditions is the difference between the stationary lines with lipids and without lipids, as denoted in Figure 1A. Part B of Figure 1 indicates the change in the dipole potential with the amount of lipids added to the surface. The monolayer becomes saturated at the surface concentration 0.125 mmol/Å2, and collapse occurs at the lateral pressure at which it is in equilibrium with liposomes in the subphase.1 The dipole potentials corresponding to mixtures of different molar fractions of DPPC in DMPC, DSPC in DOPC, and DMPC in DOPC are shown in Figure 2. In part A, both lipids are below the phase-transition temperature at 20 °C. The data show marked oscillations with the increase of DPPC mole fraction in DMPC. The values for pure DMPC are lower than that obtained for
pure DPPC at the same temperature (340 and 460 mV, respectively). This is congruent with data previously published.17 In part B of Figure 2, the dipole potentials obtained at 20 °C by adding lipid solutions with different DSPC/DOPC ratios are shown. At the temperature of measurement, pure DOPC is fluid and pure DSPC is in the gel state. In this case, the dipole potential of the mixed monolayers increases from 380 to 540 mV and shows variations between 500 and 560 mV above the molar ratio 0.2. When a mixed solution of DMPC and DOPC, both fluid at 27 °C in the pure state (part C), is spread on the surface, the dipole potentials of the different mole fractions are between 340 and 440 mV. These variations are practically within the experimental error ((50 mV). In Figure 3, the ζ potentials of MLVs prepared with the same mixtures analyzed in Figure 2 are shown. It is observed that the mixtures of DMPC/DPPC show high oscillations with the increase of the mole fraction of DPPC. The other two lipid mixtures show a continuous decrease from -50 to -10 mV when the DSPC or DMPC molar fraction is respectively increased in DOPC. A comparison of the data of dipole potential in monolayers and ζ potentials in MLVs for the DMPC/DPPC mixtures in the gel state is shown in Figure 4. It is observed that the dipole potential shows two minima: one at 0.33 molar fraction and another at 0.66 molar fraction, respectively. At these ratios, the ζ potential shows the more positive values. (17) Smaby, J. M.; Brockman, H. L. Biophys. J. 1990, 58, 195.
Lipid Interfaces Formed by Mixtures of Phosphatidylcholine
Discussion Discontinuities in the dipole potential and the ζ potential for 0.33 and 0.66 molar fractions are observed for DMPC/ DPPC mixtures in the gel state (part A of Figure 2, and Figure 3). These singularities in the values of dipole and ζ potentials are less noticeable or absent in DOPC/DSPC and DOPC/DMPC. DMPC and DPPC are miscible in the range of molar fractions. At the temperature of measurement, all the mixtures are solid. Then, a continuous change in the surface properties would be expected. In contrast, sharp discontinuities at definite ratios of the lipids are found with both the dipole and ζ potentials. The other two mixtures are composed of lipids having very low miscibility.14 In this case, at the temperature of measurement, one lipid in the system DOPC/DSPC or the two lipids in the system DOPC/DMPC are in the fluid state. The surface properties of these mixtures show a more continuous change with the molar fractions of DSPC in DOPC and DMPC in DOPC. The dipole potential shows an increase from 380 to 520 mV at 0.2 mole fraction, which corresponds to pure DSPC (Figure 2B). This value remains between 500 and 560 mV in the range of composition, which is within the experimental error. In the data of Figure 2C, the dipole potential values are between 340 mV for 0.2 mole fraction and 440 mV. The values for pure DOPC (384 mV) and pure DMPC (340 mV), both corresponding to fluid states, are similar within the experimental error. The disappearance of the singular points at 0.33 and 0.66 mole fractions is even more noticeable when the ζ potentials of the liposomes of those mixtures are measured (Figure 3). The curve corresponding to DOPC/DSPC reaches the value for pure DSPC at around 0.6 mole fraction. The curve for the DOPC/DMPC system shows a shallow maximum at this mole fraction. The behavior of the dipole and ζ potentials observed in the mixture in which both lipids are miscible in the gel state (Figures 2A and 3) denotes peculiar features. In this case, the dipole potential value for pure DMPC is 340 mV and that for pure DPPC is 460 mV. The value increases to 500 mV at 0.2 mole fraction and decreases again to 340 mV at 0.33 mole fraction. The differences in the dipole potential found in the DMPC/DPPC mixtures are larger than the experimental error (140 mV). These results indicate that the lipids mixed at the gel state present a characteristic organization at the interface for defined compositions. This organization is washed out when lipids do not mix, as inferred from the results with the mixtures DOPC/DMPC and DOPC/DSPC. This behavior is independent of whether the lipids form a bilayer or a monolayer. The fact that MLVs are formed spontaneously by the dispersion of lipids in water and that the monolayers in these experiments are obtained in the absence of external forces (no lateral pressure is applied) suggests that this is the spontaneous organization of the lipids in a binary mixture at a given temperature independent of whether they form monolayers or bilayers. This conclusion is congruent with the proposal that monolayers are similarly structured to bilayers, as inferred from the coincident transition temperatures obtained by means of light scattering, refractive index, and dipole potential changes.1,10,11 The singular values of dipole and ζ potentials at definite molar ratios imply a particular bidimensional organization, that may be a consequence of next-neighbor interactions that expand in a lateral lattice, for specific mole fractions. Rearrangements of the lateral structure have
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been reported in mixtures of DPPC and dibehenoyl PC, leading to long-lived heterogeneous interfacial regions.18 Although the experimental information derived only from the electrical measurements does not allow to modelize the molecular array, some comments may be made on the dipole and ζ potentials in mixtures in relation to those for the models proposed for pure lipids in the literature.19,20 The surface dipole moment µ is a complex quantity consisting of three components
µ ) µ 1 + µ2 + µ3 where µ1 is the contribution to the dipole potential of the polar groups, µ2 is the contribution due to the orientation of the subphase water molecules, and µ3 is the dipole moment of the terminal methyl group of the fatty acyl chains.21 For PC molecules, µ1 is a collective value representing the phosphocholine head group and the glycerol backbone with two negatively charged carboxyl ester linkages. It seems that water orientation and sn2 carbonyls contribute to a great extent to the dipole potential.22 1 H NMR experiments in pure lipids have shown that the head group is oriented parallel to the bilayer plane, contributing in a negligible form to the dipole potential.19 In contrast, the sign of the dipole and ζ potentials has been proposed by Makino et al.,20 assuming a change in the orientation of the phospholipid head group. In a mixture of lipids of different lengths, the regular array of the chains in a pure phospholipid may be changed. Matching of the fatty acid chains of different lengths would result in phospholipid head groups located in different planes. This possibility would allow the phospholipid head group to rotate around the C-O-P bond, in accordance with Makino’s model. Further support that the lipidlipid interaction may have effects on the topological properties is given by the finding that the mixing of lipids with different chain lengths results in vesicles of different size.23 The ζ potential regards the orientation of chemical groups of the monolayer constituents at the interface and their interactions with water, ions, and polar molecules at the aqueous subphase.7,24,25 According to Nicklas et al.,26 to create a favorable site for the adsorption of a cation on the membrane, the COO groups in the membrane must be oriented. The higher positive values for the dipole potential could be explained by the orientation of the positive end toward the membrane phase and the negative end toward the water phase. The ζ potential reaches +15 mV at the ratio 0.33, for which the dipole potential is a minimum. The negative end of the carbonyl at the interface of this ratio seems to be less oriented to the water phase than that at 0.2 or 0.4 mole fraction according to the values of the dipole potential. (18) Jørgensen, K.; Klinger, A.; Braiman, M.; Biltonen, R. J. Phys. Chem. 1996, 100, 2766. (19) Seelig, J. Biochim. Biophys. Acta 1978, 515, 105. (20) Makino, K.; Yamada, T.; Kimura, M.; Oka, T.; Ohshima, H.; Kondo, T. Biophys. Chem. 1991, 41, 175. (21) Standish, M. M.; Pethica, B. A. Trans. Faraday Soc. 1968, 64, 1113. (22) Gawrisch, K.; Ruston, D.; Zimmerberg, J.; Parsegian, V. A.; Rand, R. P.; Fuller, N. Biophys. J. 1992, 61, 1213. (23) Massariu, S.; Colonna, R. Biochim. Biophys. Acta 1986, 863, 264. (24) Davies, T. J.; Rideal, E. K. Interfacial Phenomena, 2nd ed.; Academic Press: New York, 1963. (25) Mozaffary, H. Chem. Phys. Lipids 1991, 59, 39. (26) Nicklas, K.; Bocker, J.; Schlenkrich, M.; Brickman, J.; Bopp, P. Biophys. J. 1991, 60, 261.
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Thus, this does not explain the positive ζ potential at 0.33 mole fraction, since the adsorption of positive ions would be less favored at this ratio than at 0.2 or 0.4 mole fraction. However, if the model of Makino is assumed to be valid, the positive end of the choline group would be oriented toward the membrane phase, thus allowing the adsorption of cations to the phosphates located in a plane near the water phase. These speculations should be confirmed by NMR measurements of these mixtures. In conclusion, a nonrandom orientation of the polar groups could be present at a definite ratio in a mixed lipid membrane in the gel state. These surface structures are formed when lipids stabilize in monolayers as well as in
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bilayers. These peculiar organizations are not formed or are least are less noticeable when lipids do not mix. Acknowledgment. Authors are grateful to Dr. H. Chaimovich and D. Bernik for comments and reading of the manuscript. This work was supported by funds from CONICET (PIA 7235/96) and UBACyT (FA024). M.C.L. is a recipient of a MUTIS fellowship. E.A.D. is a member of the Career of Investigator of the CONICET (R. Argentina). LA971273J
