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Film Balance and Fluorescence Microscopic Investigation of the Effects of Ca2+ on Mixed DMPC/DMPG Monolayers C. K. Park,† F. J. Schmitt,‡ L. Evert,§ D. K. Schwartz,| J. N. Israelachvili,*,† and C. M. Knobler⊥ Department of Chemical Engineering, University of California at Santa Barbara, Santa Barbara, California 93106, Institute of Polymer Research Dresden, P.O. Box 120411, D-01005 Dresden, Germany, Guidant Corp., 26531 Ynez Road, Temecula, California 92589-9018, Department of Chemistry, Tulane University, New Orleans, Louisiana 70118, and Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095 Received May 11, 1998. In Final Form: October 15, 1998 Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) mixed monolayers at the air-water interface, with and without 5 mM calcium in the subphases, were investigated by pressurearea measurements and simultaneous fluorescence microscopy. Condensed-phase domains were observed over both types of subphases and quantified by analyzing the microscope images. The domains’ shape changed and area increased with the mole fraction of DMPG, and hysteresis effects in the compressiondecompression cycles were more prevalent on the PG-rich side of the phase diagram. The origin of hysteresis and other nonequilibrium effects in monolayers is discussed, and the equilibrium pressure-composition phase diagram for this binary system is presented which shows a measured liquidus curve and calculated solidus curve. The overall effect of calcium is to facilitate formation of condensed-phase domains.
Introduction From a physical chemistry point of view, complex multicomponent surfactant membranes and their interactions can be simplified by studying systems with fewer molecular species. However, even this can be difficult when applied to understanding their equilibrium phase behavior. As many researchers have found, even the isotherm of a one-component surfactant hides much of the true nature of the phases and transitions. In the absence of a force field to macroscopically separate the two phases, apparently stable domains of many shapes and mosaics can appear.1,2 Much work has been done with single-component surfactant and lipid monolayers in the hope of gaining insight into biological membrane functionality. Thermodynamics and kinetic studies of these systems continue to reveal interesting physics.3,4 However, living cells typically have a large number of amphiphilic components in their membranes, including proteins. Therefore, increasing the complexity of the system by the addition of components, both lipid and ionic, takes a small step toward understanding more biologically relevant systems. Two of the more prominent mammalian cell lipid headgroups are the uncharged but zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG-), and many studies have identified interesting physics and biology involving PG--Ca2+ interactions.5-9 Calcium is * Corresponding author. † University of California at Santa Barbara. ‡ Institute of Polymer Research Dresden. § Guidant Corp. | Tulane University. ⊥ University of California at Los Angeles. (1) Klingler, F. F.; McConnell, H. M. J. Phys. Chem. 1993, 97, 29622966. (2) Heckl, W. M.; Miller, A., Mo¨hwald, H. Thin Solid Films 1988, 159, 125-132. (3) McConnell, H. M.; De Koker, R. Langmuir 1996, 12 (20), 48974904. (4) Miller, A.; Mo¨hwald, H. J. Chem. Phys. 1987, 86 (7), 4258-4265.
prevalent in many biological systems and cells. There are many examples from biology that include, depend, or otherwise involve the presence of calcium: it is involved in cell adhesion and membrane fusion; it is found in “ionophores” that are carriers of information; it is essential for proper bone growth and the prevention of diseases. In this work, we are primarily concerned with the phase state of the lipid molecules in our mixed monolayer and in the role of calcium. The phase state of a monolayer determines many of its properties, such as permeability, and interactions, such as adhesion. Frequently, due to nonidealities, mixtures have properties that differ greatly, both quantitatively and qualitatively, from either of the pure substances.10 The study of binary mixtures also adds another degree of freedom in the design of technologically useful “biomimetic” membranes. Dimyristoyl lipids were chosen because their main chain phase transition, Tc, is conveniently close to room temperature.11 Materials and Methods L-R-Dimyristoylphosphatidylcholine
(DMPC) and L-R-dimyristoylphosphatidylglycerol (DMPG) were used as provided in the powdered form from Avanti Polar Lipids, Inc. The spreading solutions were prepared at ≈1 mg/mL in 9:1 chloroform:methanol (5) Rand, R. P.; Kachar, B.; Reese, T. S. Dynamic Morphology of Calcium-Induced Interactions between Phosphatidylserine Vesicles Biophys. J. 1985, 47, 483-489. (6) Madden, T. D.; Tilcock, C. P. S.; Wong, K.; Cullis, P. R. Spontaneous Vesiculation of Large Multilamellar Vesicles Composed of Saturated Phosphatidylcholine and Phosphatidylglycerol Mixtures Biochemistry 1988, 27, 8725-8730. (7) Du¨zgu¨nes, N.; Nir, S.; Wilschut, J.; Bentz, J.; Newton, C.; Portis, A.; Papahadjopoulos, D. J. Calcium- and Magnesium-Induced Fusion of Mixed Phosphatidylserine/Phosphatdiylcholine Vesicles: Effect of Ion Binding, Membr. Biol. 1981, 59, 115-125. (8) Nag, K.; Rich, N. H.; Keough, K. M. W. Thin Solid Films 1944, 244, 841-844. (9) Bonte´, F.; Taupin, C.; Puisieux, F. J. Colloid Interface Sci. 1987, 115 (1), 268-270. (10) Leckband, D. E.; Helm, C. A.; Israelachvili, J. Biochemistry 1993, 32, 1127-1139. (11) Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990.
10.1021/la980552j CCC: $18.00 © 1999 American Chemical Society Published on Web 12/16/1998
Effects of Ca2+ on Mixed Monolayers
Figure 1. Pure lipid monolayer isotherms over calcium-free and calcium-containing subphases. Isotherms in calcium-free buffer were measured at T ) 26 °C, while in the calciumcontaining subphases T ) 17 °C. solutions. The pH 7.0 control subphase was prepared as 10 mM NaCl (Fluka), 0.1 mM TES (N-tris[hydroxymethyl]methyl-2aminoethanesulfonic acid, Sigma), and 1 mM EDTA (Sigma) in doubly distilled and filtered MilliQ water. The calcium-containing subphase had, in addition to the above, 5 mM CaCl2 (Fisher). All glassware was cleaned in a sulfuric acid bath containing “NoChromix” brand oxidizing agent and thoroughly rinsed in doubly distilled and filtered MilliQ water prior to use. A Joyce-Loebl Langmuir-Blodgett trough of area ≈500 cm2 was used for measuring the surface pressure-area (Π-A) isotherms. Surface pressures were measured with the Wilhelmy plate method. The barrier speeds used were typically ≈1 Å2 molecule-1 sec-1 in these experiments. The temperature, T, of the subphase was set at one of four values between 10 and 25 °C, which spans the Tc of DMPC and DMPG. Fluorescence microscopy at the air-water interface was done with the home-built apparatus of Knobler et al. at UCLA. This equipment was an epifluorescent microscope with a small (≈25 cm2) Langmuir trough mounted on the stage.12 The fluorescent probe used was (1-myristoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine) NBD-PC at ≈1 mol %. This hydrophilic probe is excluded from densely packed areas of lipid, providing contrast between lipid phases.
Results Pure Lipids on Calcium-Free Subphase (10 mM NaCl, pH 7.0). Figure 1 shows representative Π-A isotherms for pure DMPC and DMPG over the calciumcontaining and calcium-free subphases. The isotherms on calcium-free subphases show liquid-expanded (LE) behavior for both lipids up to large pressures, with DMPG being more expanded than DMPC, probably due to the additional repulsive electrostatic forces between the charged PG- headgroups. Fluorescence microscopy showed that only at low temperatures and high surface pressures (T ) 10 °C, Π ≈ 40 mN/m) were domains observable in (12) Moore, B. G.; Knobler, C. M.; Akamatsu, S.; Rondelez, F. J. Phys. Chem. 1990, 94, 4588.
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Figure 2. Isotherms of pure DMPG (4), pure DMPC (O), and their equimolar mixture (0) over the 5 mM CaCl2 subphase at 17 °C. The inset fluorescence micrographs show the shape and concentrations of DMPG-rich domains as darkened areas (the fluorescent probe molecules remain desegregated in the more fluid, DMPC-rich phase). The arrows indicate which area of the isotherm each micrograph was taken from.
the absence of calcium. These small, circular domains were observable at all mixtures of DMPC and DMPG. Pure Lipids on Calcium-Containing Subphase (5 mM CaCl2, pH 7.0). While the isotherms on calcium-free water were similar for DMPC and DMPG, they were markedly different for the monolayers spread over Ca2+containing subphases: The isotherms of the two lipids are shifted in different directions, and a phase transition is induced in DMPG. The kink and plateau in the isotherm for DMPG are generally associated with onset of a firstorder phase transition between the LE and LC (liquid condensed) states. It is exactly at this surface pressure, Πt, that phase-separating domains are first visualized with fluorescence. Pure and Mixed DMPC-DMPG Monolayers on CaCl2: Domain Growth. Figure 2 shows the Π-A isotherms and what is observed with epifluorescence microscopy over a Ca2+-containing subphase for the pure lipids and their equimolar mixture. Transition pressures Πt were taken as the pressures where domains were first observed on compression. These pressures agreed with those of the onset of the plateau-like regions. Fluorescence microscopy shows that the domains nucleate at a critical pressure and then grow on further compression. Significantly, very few new domains appear/nucleate after the initial nucleation. Even though the film was sweeping past the field of view of the objective, observations could be made by observing one domain for the time it remained visible. During compression, these domains grew in size while the surrounding fluidlike area typically remained devoid of any new nucleation sites. It was also noticed that the domains are not circular, but exhibit protrusions characteristic of fractal-like diffusion-limited growth. This occurred to greater extents (more nonequilibrium finger-
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solidus points are hidden by the isotherms’ curvature at tighter packings. There are several ways of calculating the solidus points. First, we tried using the well-known lever rule. For each fluorescent image we can calculate the relative areas for each phase by measuring the fraction of dark pixels in the image, FLC. This can be related to other parameters by the “lever rule”
FLC )
Xsystem - XLE XLC - XLE
(1)
where X is the mole fraction and the subscripts refer to the phase described. If we use NLC and NLE to describe the number of molecules in each phase, we can further write this as
Figure 3. Liquidus phase boundary lines deduced from visualization of fluorescent domains (filled symbols) and isotherm plateaus (open symbols). (]) points are from fluorescence visualization over calcium-free subphases at 10 °C. All other points are for 5 mM calcium-containing subphases: 10 °C (O), 17 °C (4), 21 °C (3), and 25 °C (0). The continuous lines are guides to the eye.
ing) for monolayers with greater amounts of DMPG. This type of domain is in contrast to the rounded domains observed in the monolayers without calcium. Reversibility. Upon retraction of the barrier (decompression or increasing the area per molecule), melting of the solid-like domains could be observed. Thermal melting (by heating the trough a few degrees) and finger-softening (observed by waiting a few minutes) with time were also observed with fluorescence. As the domain melts, the smallest features dissolve first. When this occurs on fingerlike domains, the sharp edges of the fractal-like fingers begin to soften. While the domain loses area to the preferred LE phase, it attains a rounder shape. “Control” experiments were performed by injecting a concentrated calcium solution into the calcium-free subphase behind the barrier of the epifluorescence Langmuir trough and observing the formation of domains. This condensation effect was seen to be reversible by adding EDTA chelating ions to the subphase in a similar manner and observing the “solvation” or shrinking and disappearance of the domains. This process occurs on the order of minutes but depends on the packing of the monolayer. Analysis of the Data. We now look further into the LE/LC transition. Figure 3 shows the liquidus lines (Πt composition curves) at the four temperatures studied, T ) 10, 17, 21, and 25 °C. The liquidus lines were obtained from where domain formation starts, as well as from the onset of the plateau region of the isotherm. The diamond symbols show Πt measured on a Ca2+-free subphase, where only at 10 °C were domains observed; at higher temperatures, the monolayers collapse before a phase transition is observed. The rest of the data of Figure 3 refer to the Ca2+-containing subphase. These data show a decrease in Πt as the fraction of DMPG increases. At the other end of the phase diagram, at low DMPG fractions, the transition pressures Πt become impossible to measure due to monolayer collapse. To construct a Π-X phase diagram, it is necessary to know the partitioning of the two amphiphiles in the LE and LC phases. The liquidus line is easily constructed as the locus of (Πt, molecular area) at different compositions. These points are easily read from an isotherm because the beginning of the transition is sharp. However, the
FLC )
NLC NLC ) NLC + NLE N
(2)
The average molecular area of the system, Asystem, is measured in the monolayers’ isotherm and can be calculated as
Asystem ) FLCALC + (1 - FLC)ALE
(3)
where ALC and ALE are defined by equations of the type PC ALC ) XLCAPG LC + (1 - XLC)ALCALG ) PC XLGAPG LG + (1 - XLG)ALG (4)
It is necessary to assume something about the molecular areas of the components in their phases. This information can be obtained from the isotherms of the pure components. This technique ultimately fails due to the large amount of scatter in the calculated data. The second way of calculating the phase diagram of a system does not depend on analyzing fluorescence microscope images. If we break up the Gibbs free energy into an ideal and excess part, we can examine the sign of the latter to determine where phase separation should occur.
∆Gexcess ) Gmix - Gideal
(5)
and components
Gideal(xi, P, T) )
∑i
X iG i
(6)
We can calculate our free energies by integrating the appropriate isotherm.
G(xi, P, T, Π) )
Π A dΠ ∫Π)0
(7)
Putting this all together we can write
Gexcess ) XPC
Π APC dΠ + ∫Π)0 Π Π XPG ∫Π)0 APG dΠ - ∫Π)0 Amix dΠ
(8)
The fourth figure is an example of the excess Gibbs free energies that can be calculated for the system from isotherm data for Ca2+ subphases. This energy construction lets us infer the positions of the liquidus and solidus lines by invoking an equilibrium stability argument. Thermodynamically, regions of Figure 4 which are concave
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Figure 5. Phase diagram for DMPC-DMPG monolayer mixtures over 5 mM calcium-containing subphase. Liquidus data (symbols) are taken from Πt of isotherms. The line through the liquidus data is a guide to the eye. The solidus line is calculated from eq 9. Dashed lines represent rough estimations of where the phase boundary may lie.
Figure 4. A Gibbs excess free energy diagram for DMPCDMPG mixtures at 25 °C. Negative energies reflect phase stability while positive energies show phase instability and phase separation. Π values are as follows: 0 mN/m, O; 14 mN/ m, 0; 15 mN/m, 4; 20 mN/m, 3; 25 mN/m, ]; 30 mN/m, +; 34 mN/m, ×.
up, or ∂∆G/∂x > 0, are unstable and imply a two phase region. The inverse condition, ∂∆G/∂x < 0, implies stability and a one-phase region. The resolution of the phase diagram depends, ultimately, on the number of mole fractions investigated. This was done for 10 different solutions giving limits of (0.10 mole fraction uncertainties in the phase boundaries’ position. The final and easiest method of constructing a solidus line is to turn to a well-known thermodynamic equation derived from the Gibbs-Duhem relation. In two dimensions it is written
(∂ ln∂y Π)
T
)
(y - x) y(1 - y)
(9)
The equation assumes that the ratio of the solid phase molar area to that of the liquid phase is small.13 This ratio can be estimated from isotherm data as ≈1/3. The above equation was applied by fitting a curve to ln Πt vs y and solving for x. The results are plotted in Figure 5. If we include the estimated ratio of solid to liquid phase molar areas, then the separation between liquidus and solidus lines increase by 50%. Discussion The observed decrease in area A with Ca2+ in PG-containing layers is expected and commensurate with previously measured Langmuir isotherms and X-ray crystallographic structure.11,14 The Ca2+ has several effects. The divalency of calcium greatly reduces the interheadgroup electrostatic repulsion. Most importantly, calcium binds two DMPG lipids, presumably making “dimers”, (13) Denbigh, K. The Principles of Chemical Equilibrium, Cambridge University Press: Cambridge, U.K., 1981. (14) Evert, L. L.; Leckband, D.; Israelachvili, J. N. Langmuir 1994, 10, 303-315.
with decreased lipid headgroup area.14,15 These dimers are then electrically neutral and able to more easily aggregate. Additionally, domains of these aggregates are more thermally stable.15 DMPC, however, binds at most one Ca2+ per headgroup, thus becoming charged and electrostatically dispersed. The domains formed under these conditions are reminiscent of diffusion-limited growth crystallites seen by other researchers in other systems.16,17,20 The other monovalent ions affect the electrostatic decay between charged lipids. They are included to stabilize the pH at 7.0. The EDTA is present to chelate trace multivalent ions. The large errors present in the image-analysis-based solidus lines are due to the flatness of the liquidus line at large DMPG mole fractions (not shown). Small errors in Πt correspond to large errors in XLE giving unreasonable mole fractions for XLC. Interestingly, eq 9 above gives reasonable results for the solidus lines shown in Figure 5. Importantly, the equations for generating the solidus curve are written for equilibrium systems. The increasing fractalness of the domain shapes are indicative of nonequilibrium structures. However, it is assumed that the phase coexistence described here is an equilibrium phenomenon. The larger amount of fractal growth occurs at the highest DMPG concentrations. The accuracy of this calculation can be further increased by including measurements of the liquidus and solidus phase molar areas. Figure 4 may represent a more sensible way to obtain phase diagram data. In such a scheme, many isotherms at different concentrations would have to be acquired to gain reasonable accuracy in mole fraction measurement of the phase boundaries. What has been presented shows that above a certain pressure, ∆Gexcess > 0 for all mixed isotherms, implying strong demixing. In contrast, at low (15) Silvius, J. R. Lipid-Protein Interactions; J. Wiley & Sons: New York, 1982. (16) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441-76. (17) Miller, A.; Knoll, W.; Mo¨hwald, H. Phys. Rev. Lett. 1986, 56 (24), 2633. (18) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171. (19) Pethica, B. A. Colloids Surf., A 1994, 88, 147-155. (20) Losche, M.; Mohwald, H. J. Colloid Interface Sci. 1989, 131, 56-67.
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Π, ∆Gexcess < 0, the system favors mixing. At intermediate Π (20 mN/m) two-phase coexistence regimes are present. This fact implies an eutectic point between 15 and 20 mN/m. Conclusion DMPC-DMPG binary mixtures at an air-solution interface have been shown to exhibit interesting calciumdependent phase behavior. In the absence of calcium, the lipids show only liquid-expanded phases. Calcium binds to DMPG headgroups making the overall lipid-ion complex charge neutral. Liquidus lines on a Πt-XDMPG diagram are measured from isotherm features as well as from fluorescence microscopic observation. Solidus lines, calculated from equilibrium relations and the microscopically measured area phase fraction, show reasonable trends, but large errors. Calculating the phase diagram
Park et al.
from ∆Gexcess ultimately appears more reasonable, but is labor intensive. Using a thermodynamic relation derived from the Gibbs-Duhem equation leads to the easiest construction of a solidus line. Previous work in this lab has focused on the effect of calcium on mixtures of 3:1 DMPC/DMPG. Experiments by Evert14 showed the importance of calcium in the LELC phase transition and how it affects domain formation on free and solid surfaces. The surface forces apparatus has been used by Leckband10 to show the importance of calcium in the adhesion and fusion of PG-containing bilayers. Future work is planned along these lines. Acknowledgment. This work was funded by National Science Foundation grant award CTS-9634050. LA980552J
