European Biophysics Journal

, Volume 48, Issue 8, pp 757–772 | Cite as

Electrostatic interactions of alkaline earth cations with 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) model membranes at neutral and acidic pH

  • Patrick Garidel
  • Alfred BlumeEmail author
Original Article


The binding of alkaline earth cations Mg2+, Ca2+, and Sr2+ (M2+) to unilamellar 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) vesicles was analysed by pH potentiometry, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and FT-IR spectroscopy. The binding of alkaline earth cations induces deprotonation of the DMPA headgroup even at very low concentration of divalent cations (~ 100 µM). The amount of deprotonated DMPA was measured by pH potentiometry as a function of divalent cation concentration. The thermotropic phase behaviour of DMPA:M2+ complexes was studied by DSC and FT-IR as a function of pH of the dispersion (pH 7 and pH 3-5). The formation of metastable phases was observed, especially for Ca2+ and Sr2+ at pH 3–5. In unbuffered solutions, the divalent cations bind to single and/or double negatively charged DMPA, leading to the formation of different complexes and changes in the mixing behaviour of the two complexes. At pH 7, all three equimolar lipid/cation mixtures form a very stable, highly ordered 1:1 DMPA:M2+ complex. At lower divalence, the presence of a mixture of 2:1 and 1:1 complexes was observed. FT-IR spectroscopy experiments indicated an ordering of the acyl chains of DMPA after ion binding even in the liquid-crystalline phase and the induction of the dissociation of the second proton from the headgroup induced by Ca2+ or Sr2+ binding at pH 7. With ITC, the binding enthalpy ΔH of Mg2+, Ca2+, and Sr2+ to DMPA model membranes in the gel and in the liquid-crystalline phase was measured. Evidence for dehydration of hydrophobic surfaces due to cation binding was derived from changes in heat capacity.


Phosphatidic acid Divalent cation binding Potentiometry DSC ITC FT-IR 



We thank B. Fölting for her excellent technical assistance and Wigand Hübner for fruitful discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bl-182/7-3), the Max-Planck-Gesellschaft zur Förderung der Wissenschaften, and the Fonds der Chemischen Industrie.

Supplementary material

249_2019_1402_MOESM1_ESM.docx (291 kb)
Supplementary material 1 (DOCX 291 kb)


  1. Asher IM, Levin IW (1977) Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles. Biochim Biophys Acta 468:63–72PubMedGoogle Scholar
  2. Bellemare F, Lesage R (1991) Mg2 + , Ca2+, and Mn2+ bound on anionic phospholipids resist desalting dialysis: evaluation of binding parameters using stern adsorption isotherms. J Colloid Interface Sci 147:462–473Google Scholar
  3. Blume A (1983) Apparent molar heat capacities of phospholipids in aqueous dispersion. Effects of chain length and head group structure. Biochemistry 22:5436–5442Google Scholar
  4. Blume A (1988) applications of calorimetry to lipid model membranes. In: Hidalgo C (ed) Physical properties of biological membranes and their functional implications. Plenum Press, New York, pp 71–121Google Scholar
  5. Blume A (1996) Properties of lipid vesicles: FT-IR spectroscopy and fluorescence probe studies. Curr Opin Colloid Interface Sci 1:64–77Google Scholar
  6. Blume A (2016) Temperature induced and isothermal phase transitions of pure and mixed lipid bilayer membranes studied by DSC and ITC. In: Bastos M (ed) Biocalorimetry: foundations and contemporary approaches. CRC Press, Boca Raton, pp 103–136Google Scholar
  7. Blume A, Eibl H (1979) The influence of charge on bilayer membranes calorimetric investigations of phosphatidic acid bilayers. Biochim Biophys Acta 558:13–21PubMedGoogle Scholar
  8. Blume A, Garidel P (1999) Lipid model membranes and biomembranes. In: Kemp RB (ed) Handbook of thermal analysis and calorimetry, vol 4. From macromolecules to man. Elsevier Press, Amsterdam, pp 109–173Google Scholar
  9. Blume A, Tuchtenhagen J (1992) Thermodynamics of ion binding to phosphatidic acid bilayers. Titration calorimetry of the heat of dissociation of DMPA. Biochemistry 31:4636–4642PubMedGoogle Scholar
  10. Blume A, Huebner W, Messner G (1988) Fourier transform infrared spectroscopy of 13C=O labeled phospholipids hydrogen bonding to carbonyl groups. Biochemistry 27:8239–8249PubMedGoogle Scholar
  11. Boughriet A, Ladjadj M, Bicknell-Brown E (1988) Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies. Biochim Biophys Acta 939:523–532PubMedGoogle Scholar
  12. Brockerhoff H, Yurkowski M (1965) Simplified preparation of l-α-glyceryl phosphoryl choline. Can J Biochem 43:1777PubMedGoogle Scholar
  13. Buehler LK (2016) Cell membranes. Garland Science, New YorkGoogle Scholar
  14. Butko P, Ogawa R, Nagao K, Taniuchi K, Tsuchiya M, Kato U, Hara Y, Inaba T, Kobayashi T, Sasaki Y, Akiyoshi K, Watanabe-Takahashi M, Nishikawa K, Umeda M (2015) Development of a novel tetravalent synthetic peptide that binds to phosphatidic acid. PLoS One 10:e0131668Google Scholar
  15. Casal HL, Mantsch HH (1984) Polymorphic phase behaviour of phospholipid membranes studied by infrared spectroscopy. Biochim Biophys Acta 779:381–401PubMedGoogle Scholar
  16. Cevc G (1990) Membrane electrostatics. Biochim Biophys Acta 1031:311–382PubMedGoogle Scholar
  17. Cockcroft S, Frohman M (2009) Special issue on phospholipase D. Biochim Biophys Acta 1791:837–838PubMedGoogle Scholar
  18. Conway BE (1981) Ionic hydration in chemistry and biophysics, vol 12. Studies in physical and theoretical chemistry. Elsevier Scientific Pub. Co., AmsterdamGoogle Scholar
  19. Desormeaux A, Laroche G, Bougis PE, Pezolet M (2002) Characterization by infrared spectroscopy of the interaction of a cardiotoxin with phosphatidic acid and with binary mixtures of phosphatidic acid and phosphatidylcholine. Biochemistry 31:12173–12182Google Scholar
  20. Eibl H, Blume A (1979) The influence of charge on phosphatidic acid bilayer membranes. Biochim Biophys Acta 553:476–488PubMedGoogle Scholar
  21. Faraudo J, Travesset A (2007a) Electrostatics of phosphatidic acid monolayers: insights from computer simulations. Colloids Surf Physicochem Eng Asp 300:287–292Google Scholar
  22. Faraudo J, Travesset A (2007b) Phosphatidic acid domains in membranes: effect of divalent counterions. Biophys J 92:2806–2818PubMedPubMedCentralGoogle Scholar
  23. Frank C, Keilhack H, Opitz F, Zschörnig O, Böhmer F-D (1999) Binding of phosphatidic acid to the protein-tyrosine phosphatase SHP-1 as a basis for activity modulation. Biochemistry 38:11993–12002PubMedGoogle Scholar
  24. Garidel P (1997) The negatively charged phospholipids phosphatidic acid and phosphatidylglycerol. University of Kaiserslautern, KaiserslauternGoogle Scholar
  25. Garidel P, Blume A (1999) Interaction of alkaline earth cations with the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol: a differential scanning and isothermal titration calorimetric study. Langmuir 15:5526–5534Google Scholar
  26. Garidel P, Blume A (2000a) Calcium induced nonideal mixing in liquid-crystalline phosphatidylcholine–phosphatidic acid bilayer membranes. Langmuir 16:1662–1667Google Scholar
  27. Garidel P, Blume A (2000b) Miscibility of phosphatidylethanolamine-phosphatidylglycerol mixtures as a function of pH and acyl chain length. Eur Biophys J 28:629–638PubMedGoogle Scholar
  28. Garidel P, Blume A (2005) 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) monolayers: influence of temperature, pH, ionic strength and binding of alkaline earth cations. Chem Phys Lipids 138:50–59PubMedGoogle Scholar
  29. Garidel P, Blume A, Hübner W (2000a) A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol. Biochim Biophys Acta 1466:245–259PubMedGoogle Scholar
  30. Garidel P, Forster G, Richter W, Kunst BH, Rapp G, Blume A (2000b) 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) divalent cation complexes: an X-ray scattering and freeze-fracture electron microscopy study. Phys Chem Chem Phys 2:4537–4544Google Scholar
  31. Gennis RB (1990) Biomembranes: molecular structure and function. Springer, New YorkGoogle Scholar
  32. Hübner W, Blume A (1998) Interactions at the lipid–water interface. Chem Phys Lipids 96:99–123Google Scholar
  33. Jang J-H, Lee CS, Hwang D, Ryu SH (2012) Understanding of the roles of phospholipase D and phosphatidic acid through their binding partners. Prog Lipid Res 51:71–81PubMedGoogle Scholar
  34. Jung Y-J, Lee T-H, Lee J-Y, Kim J-H, Park J-B (1999) Phosphatidic acid is important to the translocation of Rab3A from the cytosol to phospholipid membranes. NeuroReport 10:2859–2863PubMedGoogle Scholar
  35. Kooijman EE, Burger KNJ (2009) Biophysics and function of phosphatidic acid: a molecular perspective. Biochim Biophys Acta 1791:881–888PubMedGoogle Scholar
  36. Kooijman EE, Carter KM, van Laar EG, Chupin V, Burger KNJ, de Kruijff B (2005) What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special? Biochemistry 44:17007–17015PubMedGoogle Scholar
  37. Kouaouci R, Silvius JR, Graham I, Pezolet M (2002) Calcium-induced lateral phase separations in phosphatidylcholine-phosphatidic acid mixtures. A Raman spectroscopic study. Biochemistry 24:7132–7140Google Scholar
  38. Laroche G, Dufourc EJ, Dufourcq J, Pezolet M (1991) Structure and dynamics of dimyristoylphosphatidic acid/calcium complexes by deuterium NMR, infrared, and Raman spectroscopies and small-angle X-ray diffraction. Biochemistry 30:3105–3114PubMedGoogle Scholar
  39. Lehrmann R, Seelig J (1994) Adsorption of Ca2+ and La3+ to bilayer membranes: measurement of the adsorption enthalpy and binding constant with titration calorimetry. Biochim Biophys Acta 1189:89–95PubMedGoogle Scholar
  40. Lipowsky R, Sackmann E (1995) Structure and dynamics of membranes, vols 1A and 1B. From cells to vesicles. Elsevier Science B.V., AmsterdamGoogle Scholar
  41. McPhail LC, Waite KA, Regier DS, Nixon JB, Qualliotine-Mann D, Zhang W-X, Wallin R, Sergeant S (1999) A novel protein kinase target for the lipid second messenger phosphatidic acid. Biochim Biophys Acta 1439:277–290PubMedGoogle Scholar
  42. Nichols N, Sköld R, Spink C, Suurkuusk J, Wadsö I (1976) Additivity relations for the heat capacities of non-electrolytes in aqueous solution. J Chem Thermodyn 8:1081–1093Google Scholar
  43. Ohki K (1988) Ca2+-induced lateral phase separation in ternary mixtures of phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine inferred by calorimetry. J Biochem 104:14–17PubMedGoogle Scholar
  44. Pittler J, Bu W, Vaknin D, Travesset A, McGillivray DJ, Lösche M (2006) Charge inversion at minute electrolyte concentrations. Phys Rev Lett 97:046102PubMedGoogle Scholar
  45. Pokotylo I, Kravets V, Martinec J, Ruelland E (2018) The phosphatidic acid paradox: too many actions for one molecule class? Lessons from plants. Prog Lipid Res 71:43–53PubMedGoogle Scholar
  46. Silvius JR, Gagne J (2002) Calcium-induced fusion and lateral phase separations in phosphatidylcholine-phosphatidylserine vesicles. Correlation by calorimetric and fusion measurements. Biochemistry 23:3241–3247Google Scholar
  47. Stace C, Ktistakis N (2006) Phosphatidic acid- and phosphatidylserine-binding proteins. Biochim Biophys Acta 1761:913–926PubMedGoogle Scholar
  48. Takahashi H, Yasue T, Ohki K, Hatta I (1995) Structural and thermotropic properties of calcium-dimyristoylphosphatidic acid complexes at acidic and neutral pH conditions. Biophys J 69:1464–1472PubMedPubMedCentralGoogle Scholar
  49. Tamm LK, Tatulian SA (1997) Infrared spectroscopy of proteins and peptides in lipid bilayers. Q Rev Biophys 30:365–429PubMedGoogle Scholar
  50. Tatulian SA (1992) Ionization and ion binding. In: Cevc G (ed) Phospholipid handbook. Marcel Dekker, Inc., New York, pp 511–552Google Scholar
  51. Träuble H, Eibl H (1974) Electrostatic effects on lipid phase transitions: membrane structure and ionic environment. Proc Natl Acad Sci USA 71:214–219PubMedGoogle Scholar
  52. Träuble H, Teubner M, Woolley P, Eibl H (1976) Electrostatic interactions at charged lipid membranes. Biophys Chem 4:319–342Google Scholar
  53. Tuchtenhagen J (1994) Kalorimetrische und FT-IR-spektroskopische Untersuchungen an Phospholipidmodellmembranen. Department of Chemistry, vol Ph.D. University of KaiserslauternGoogle Scholar
  54. Tuchtenhagen J, Ziegler W, Blume A (1994) Acyl chain conformational ordering in liquid-crystalline bilayers: comparative FT-IR and 2H-NMR studies of phospholipids differing in headgroup structure and chain length. Eur Biophys J 23:323–335Google Scholar
  55. Vaknin D, Krüger P, Lösche M (2003) Anomalous X-ray reflectivity characterization of ion distribution at biomimetic membranes. Phys Rev Lett 90:178102PubMedGoogle Scholar
  56. van Dijck PWM, de Kruijff B, Verkleij AJ, van Deenen LLM, de Gier J (1978) Comparative studies on the effects of pH and Ca2+ on bilayers of various negatively charged phospholipids and their mixtures with phosphatidylcholine. Biochim Biophys Acta 512:84–96PubMedGoogle Scholar
  57. Wang X, Devaiah S, Zhang W, Welti R (2006) Signaling functions of phosphatidic acid. Prog Lipid Res 45:250–278PubMedGoogle Scholar
  58. Wang W, Anderson NA, Travesset A, Vaknin D (2012) Regulation of the electric charge in phosphatidic acid domains. J Phys Chem B 116:7213–7220PubMedGoogle Scholar
  59. Zhang Y, Du G (2009) Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases. Biochim Biophys Acta 1791:850–855PubMedPubMedCentralGoogle Scholar
  60. Ziegler W, Blume A (1995) Acyl chain conformational ordering of individual components in liquid-crystalline bilayers of mixtures of phosphatidylcholines and phosphatidic acids. a comparative FTIR and 2H NMR study. Spectrochim Acta Part A 51:1763–1778Google Scholar

Copyright information

© European Biophysical Societies' Association 2019

Authors and Affiliations

  1. 1.Institute of Chemistry-Physical ChemistryMartin-Luther-University Halle-WittenbergHalle (Saale)Germany

Personalised recommendations