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Langmuir 2000, 16, 1662-1667
Calcium Induced Nonideal Mixing in Liquid-Crystalline Phosphatidylcholine-Phosphatidic Acid Bilayer Membranes Patrick Garidel and Alfred Blume* Martin-Luther-University, Halle-Wittenberg, Institute of Physical Chemistry, Muehlpforte 1, D-06108 Halle/Saale, Germany Received July 12, 1999. In Final Form: October 15, 1999 The influence of increasing concentrations of calcium ions on the mixing properties of 1,2-dimyristoylsn-glycero-3-phosphatidic acid (DMPA) with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at pH 7 was investigated using differential scanning calorimetry (DSC). The composition of the mixture as well as the DMPA to calcium molar ratio (DMPA/Ca2+) was systematically changed. We particularly focused our attention on the question of binding of Ca2+ to fluid bilayers and whether low amounts of Ca2+ (the physiological relevant concentration is between 10 and 250 µM) can induce fluid-fluid immiscibility in liquid-crystalline bilayers. Therefore, we investigated mixtures with low calcium content, i.e., with DMPA/ Ca2+ ratios in the range of 1:0.01-1:0.1. The analysis of the DSC curves for DMPA/DMPC + Ca2+ (DMPA/ Ca2+ > 1:0.1) mixtures lead to the conclusion that with increasing Ca2+ content increased nonideal mixing in the liquid-crystalline phase with the tendency of domain formation for like lipids occurs. In this concentration regime Ca2+, binds to DMPA in a stoichiometry 2:1, the 2:1 complex being miscible with the remaining DMPC/DMPA mixture in the fluid bilayers. With higher Ca2+ content, the apparent pK of DMPA shifts to lower values and doubly charged DMPA is formed which forms a 1:1 complex with Ca2+ which is quasi-crystalline and no longer miscible with the remaining DMPC/DMPA mixture. A phase separation of a solidlike DMPA/Ca2+ complex is observed. The bilayers then consist of DMPA-Ca2+ rich domains with tightly packed DMPA hydrocarbon chains in a liquid-crystalline like DMPC rich phase.
Introduction In the last years, the question of cluster formation in lipid membranes1-7 and especially in the liquid-crystalline phase8-10 has attracted much attention, because domain formation can influence many properties of biological membranes.2,11-14 For example, nonideal mixing of phospholipids can be realized in systems composed of lipids with a large difference in the acyl chains of the components15-17 or by altering the charge of the headgroups of lipids in mixtures. In biological systems, all * Address correspondence to: Prof. Dr. A. Blume, Institut fu¨r Physikalische Chemie, Fachbereich Chemie, Martin-LutherUniversita¨t Halle-Wittenberg, D-06108 Halle/Saale, Germany; Tel.: +49-345-5525850. Fax: +49-345-5527157. E-mail: blume@ chemie.uni-halle.de. (1) Bloom, M.; Evans, E.; Mouritsen, O. G. Quart. Rev. Biophys. 1991, 24, 293-397. (2) Vaz, W. L. C. Biophys. Chem. 1994, 50, 139-145. (3) Vaz, W. L. C. Mol. Membrane Biol. 1995, 12, 39-43. (4) Welti, R.; Glaser, M. Chem. Phys. Lipids 1994, 73, 121-137. (5) Yang, L.; Glaser, M. Biochemistry 1996, 35, 13966-13974. (6) Denisov, G.; Wanaski, S.; Luan, P.; Glaser, M.; McLaughlin, S. Biophys. J. 1998, 74, 731-744. (7) Rietveld, A.; Simons, K. Biochim. Biophys. Acta 1998, 1376, 467479. (8) Hinderliter, A. K.; Huang, J.; Feigenson, G. W. Biophys. J. 1994, 67, 1906-1911. (9) Garidel, P.; Johann, C.; Blume, A. Biophys. J. 1997, 72, 21962210. (10) Garidel, P.; Johann, C.; Mennicke, L.; Blume, A. Eur. Biopys. J. 1997, 26, 447-459. (11) Carruthers, A.; Melchior, D. L. Biochemistry 1983, 22, 57975807. (12) Hoekstra, D. Biochemistry 1982, 21, 2833-2840. (13) Verkleij, A. J. Biochim. Biophys. Acta 1984, 779, 43-63. (14) Tocanne, J.-F.; Ce´zanne, L.; Lopez, A.; Piknova, B.; Schram, V.; Tournier, J.-F.; Welby, M. Chem. Phys. Lipids 1994, 73, 139-158. (15) Inoue, T.; Tasaka, T.; Shimozawa, R. Chem. Phys. Lipids 1992, 63, 203-212. (16) Inoue, T.; Nibu, Y. Chem. Phys. Lipids 1995, 76, 171-179. (17) Inoue, T.; Nibu, Y. Chem. Phys. Lipids 1995, 76, 181-191.
charged lipids are negatively charged. Changes in headgroup charge can be achieved by changing the pH of the system, which results in the protonation of the acidic lipid component.9,10,18,19 The addition of multivalent cations which bind to the negatively charged lipid headgroups of phosphatidic acid, phosphatidylglycerol, or phosphatidylserine leads to charge compensation and concomitant changes in mixing behavior.8,20-25 In this study we investigated the interaction of Ca2+ ions with mixed lipid membranes containing one zwitterionic lipid (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) and a negatively charged phospholipid (1,2dimyristoyl-sn-glycero-3-phosphatidic acid, DMPA) at pH 7. We systematically changed the composition of the mixtures (xDMPA ) 0.2, 0.4, 0.5, 0.6, 0.8) and the molar ratio of DMPA to Ca2+ from 1:0 to 1:10. The mixtures were investigated by differential scanning calorimetry (DSC). In most of the work published previously, the amount of Ca2+ added to bilayer systems containing phosphatidic acids was always in a 1:1 stoichiometric ratio or even in excess. In this case, a PA/Ca2+ 1:1 complex is formed which is quasicrystalline and separates from the rest of the mixture. In this study, we have focused our analysis on the DSC curves of mixtures containing low (18) Tokutomi, S.; Ohki, K.; Ohnishi, S.-I. Biochim. Biophys. Acta 1980, 596, 192-200. (19) Garidel, P.; Johann, C.; Blume, A. J. Liposome Res. 1998, 8, 58-59. (20) Hui, S. W.; Boni, L. T.; Stewart, T. P.; Isac, T. Biochemistry 1983, 22, 3511-3516. (21) Graham, I.; Gagne´, J.; Silvius, J. R. Biochemistry 1985, 24, 71237131. (22) Kouaouci, R.; Silvius, J. R.; Graham, I.; Pe´zolet, M. Biochemistry 1985, 24, 7132-7140. (23) Tilcock, C. P. S.; Cullis, P. R.; Gruner, S. M. Biochemistry 1988, 27, 1415-1420. (24) Feigenson, G. W. Biochemistry 1989, 28, 1270-1278. (25) Vincent, J. S.; Levin, I. W. Biophys. J. 1991, 59, 1007-1021.
10.1021/la990922j CCC: $19.00 © 2000 American Chemical Society Published on Web 12/11/1999
Ca Induced Nonideal Mixing in DMPC/DMPA Bilayers
Langmuir, Vol. 16, No. 4, 2000 1663 Table 1. Thermodynamic Data for the Main Phase Transition of DMPA and DMPC in the Presence of CaCl2 in Water at pH 7a lipid lipid/Ca2+ (molar ratio) 1:0 1:0.01 1:0.05 1:0.1 1:0.25
DMPA
DMPC
Tm/°C
∆Hc/kJ‚ mol-1
Tm/°C
∆Hc/kJ‚ mol-1
51.3 50.4, 51.8 50.6, 53.1 51.0, 54.0 51.3, 56.1
30.1 26.1 24.7 22.6 9.8
24.0 23.8 23.9 23.8 24.0
30.8 26.4 24.4 22.8 21.9
a T ) maxima of the c curve, ∆H ) calorimetrically determined m p c transition enthalpy for the total phase transition.
Figure 1. DSC heating curves of DMPA/Ca2+ and DMPC/ Ca2+ liposomal suspensions (pH 7) at various molar ratios as indicated.
amounts of Ca2+ where the formation of a quasi-crystalline DMPA/Ca2+ 1:1 complex is not observed and where, depending on the DMPA/Ca2+ ratio and the total amount of DMPA in the bilayers, Ca2+ binding will first occur in a 2:1 ratio. We will show that this complex is miscible with the remaining bilayer lipids but that low amounts of Ca2+ induce increasingly nonideal mixing in the fluid phase without the appearance of quasi-crystalline DMPA/ Ca2+ complexes. Experimental Section Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were obtained from Nattermann Phospholipid GmbH (Cologne, Germany) and Sygena Ltd (Liestal, Switzerland). The lipid purity was checked by thin-layer chromatography.26,27 Calcium chloride dihydrate of analytical grade was obtained from E. Merck, Darmstadt (Germany). Methods. The preparation of the DMPA/DMPC phospholipid mixtures (c ) 4-6 mM) at pH 7 in pure water was performed as described before.9,10 After addition of the corresponding CaCl2 solution to the lipid dispersions, the samples were sonicated for 15 min above 80 °C. The pH of the samples was checked and readjusted to pH 7. Differential scanning calorimetry (DSC) measurements were performed with a MicroCal MC-2 scanning calorimeter (MicroCal, Inc., Northhampton, MA). The heating rate was 1 °C‚min-1 and the measurements were performed in a temperature interval from 5 to 95 °C. The reproducibility of the DSC curves was checked by three consecutive scans of each sample. The accuracy of the DSC experiments was ( 0.5 °C for the main phase transition temperature Tm and ( 1 kJ‚mol-1 for the main phase transition enthalpy ∆Hm.
Results Pure Phospholipids. Figure 1 shows heat capacity curves (cp curves) of the third heating scan of the pure phospholipids with different lipid to calcium molar ratios (26) Hahn, F. L.; Luckhaus, R. Z. Anal. Chem. 1956, 149, 172-177. (27) Garidel, P. Diploma Thesis, University of Kaiserslautern, Germany, 1993.
at pH 7. The measurements were performed in a temperature range between 5 and 95 °C, but only the temperature range in which a phase transition was observed is shown. DMPA/Ca2+. The addition of small amounts of calcium ions to DMPA dispersions at pH 7 induces the appearance of an additional peak at higher temperature. The first transition peak occurs at a similar temperature as observed for pure DMPA at pH 7 (∼51 °C). With increasing amounts of calcium, the second transition peak is continuously shifted to higher temperatures (see Figure 1) while the transition enthalpy decreases. A plot of the phase transition enthalpy vs the molar ratio of DMPA/Ca2+ shows an almost linear decrease of the transition enthalpy28,29 with the molar ratio up to a value of 1:0.25. When the molar ratio of DMPA/Ca2+ becomes smaller (
