Langmuir 1994,lO,2267-2271
2267
Interactions of Calcium Ions with Phospholipid Membranes. Studies on n-A Isotherms and Electrochemical and Quartz-CrystalMicrobalance Measurements1 Yasuhito Ebara, Hiroshi Ebato, Katsuhiko Ariga,? and Yoshio Okahata* Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama 227, Japan Received July 27, 1993. In Final Form: April 22, 1994@ Interactions of calcium ions with a monolayer of synthetic phospholipid 1,3-dihexadecylglycer0-2phosphoethanolamine(2ClsPE)were investigated with three different methods: surface-pressure (n-A) isotherms, electrochemicalreactions on an electrode, and quartzcrystal microbalance(QCM)measurements. n-A isotherms showed that the 2ClsPE monolayer exists with the most condensed molecular packing at pH 5.8 probably due to intermolecular zwitterionic interactions. Ca2* ions destroy this interaction and expand the monolayer packing. This behavior can be examined on a bilayer-covered SnOz electrode in electrochemical methods. Oxidation peak currents of a maker ion (Fe(CN)63'4-)in the aqueous phase through the lipid films were increased by the addition of Ca2' ions only when the membrane was in a bilayer and in the fluid liquid crystalline state above the Tc. The binding behavior of Ca2' ions onto the 2C16PE LB films was followed directly from the frequency changes of the LB film-deposited QCM. Ca2* ions can penetrate into the fluid liquid crystalline LB films, but not into the solid films. From these findings, Ca2-ions can bind and penetrate into only the fluid liquid crystalline state of a bilayer and disturb or expand the membrane structures.
Introduction Biomembranes respond to chemical stimuli such as hormones and neurotransmitters, and express various function^.^-^ Ca2+ions play important roles in biomembrane systems such as cell fusion, active transport, and signal t r a n s m i s ~ i o n . ~Most - ~ of these events are occurring in proteins and in membranes. Responses oflipid matrices to chemical stimuli are also important and are studied in aqueous dispersions of lipids,liposomes,and a lipid-corked capsule memb~-ane.~-l~ For example, permeation through liposomes or lipid-corked capsule membranes containing diacylphosphatidylethanolamineas a n acidic phospholipid can be changed by the addition of Ca2+ ions in the s o l ~ t i o n . ~Ca2+ - ~ ~ions are generally understood to bind to the phosphoethanolamino head groups and change the permeability across the membrane as a result. The binding behavior of Ca2+ions onto the lipid membranes has been studied by using NMR spectra.13 In this paper, we systematically study interactions between Ca2+ions and the monolayer at the air-water + Current address: SupramolecularProject, JRDC, Akikawacho, Kurume, Fukuoka 830, Japan. * Abstract published in Advance ACS Abstracts, June 1, 1994.
(1) Characterization of Langmuir-Blodgett Films. 15. Part 14: Ebara, Y.; Okahata, Y. Langmuir 1993,9,574. (2)Kuba, K; Tanaka, E.; Kumamoto, E.; Minota, S. Pfluegers Arch. . 1989,414,105. (3) Bradshaw, R. A.; Frazier, W. A. Curr. Top. Cell. Regul. 1977,12, 1. (4)Weissmann,G.,Cluiborne,R., Eds. Cell Membranes. Cell Biology and Pathology; Hospital Practice: New York, 1975. (5)Cullis, P.R.;de h i j f f , B. Biochim. Biophys.Acta 1979,539,399. (6)Lawson, D.;Fewtrell, C.: Raff,M. J . Cell Biol. 1978. 79,394. (7)Inesi, G.Annu. Rev. Physiol. 1985,47,573. (8)Katz, B. Nerve, Muscle and Synapse; McGraw-Hik New York, 1966. (9)Vandenvarf, P.; Ullman, E. F. Biochim. Biophys.Acta 1980,596, 302. (10) Liao, M.-J.; Prestegard, G. Biochim. Biophys. Acta 1979,550, 157. (ll)Okahata, Y . ; Lim, H.-J. J . Am. Chem. Soc. 1984,106,4696. Okahata, Y. Acc. Chem. Res. 1986,17,57. (12)Marsh, D.CRCHandbook oflipid Bilayers; CRC Press: Boston, 1990;p 292. (13)Seelig, J.; Macdonald, P. M. ACC.Chem. Res. 1987,20,221.
interface or the transferred bilayers of synthetic 1,3dihexadecylglycero-2-phosphoethanolamine(2ClsPE) by using surface-pressure (JC-A)isotherms, electrochemical reactions, and a quartz-crystal microbalance (QCM) method as a tool. These measurements give the following
information responding to the addition of Ca2+ions: (i) changes of molecular area of the lipid monolayer from JC-Aisotherms, (ii)permeability changes of marker redox ions through LB films on a n electrode, and (iii) binding amounts of Ca2+ions onto the lipid membrane from the frequency changes of the lipid-coated QCM. QCMs are known to provide very sensitive mass measuring devices because their resonance frequency decreases upon the increase of a given mass on the QCM on a nanogram level. I4-I8
Experimental Section (a) Materials. Preparation of 1,3-dihexadecylglycero-2phosphoethanolamine (2C&'E) was reported elsewhere.19~20 Other reagents were used as extra analytical grade without further purification. (b)IC-AIsotherms of the Monolayer and Deposition of Bilayers. The computer-controlled film balance system (FSD20, US1 System, Co., Fukuoka) was used for measurements of n-A isotherms and transfers of bilayers on a substrate by (14)Sauerbrey, G.2.Phys. 1959,155,206. (15) Ebersole, R.; Ward, D. M. J . Am. Chem. Soc. 1988,110,8623. (16)Okahata, Y.;Ebato, H. Anal. Chem. 1991,63, 203. Okahata, Y.; Ebato, H. J . Chem. SOC., Perkin Trans. 2 1991,457. (17)Okahata, Y.; Matsunobu, Y.; Ijiro, K; Mukae, M.; Murakami, A.;Makino, K J . Am. Chem. Soc. 1992,114,8299. (18)Okahata, Y.; Ariga, K.; Tanaka, K. Thin Solid Films 1992,210/ 211, 702. (19)Seki, T.; Okahata, Y. J . Microencapsulation 1986,2,13. (20)Hansen, W. J.;Murai, R.; Wedmid, Y.; Baumann, W. J.Lipids 1982,17,453.
0743-746319412410-2267$04.50100 1994 American Chemical Society
Ebara et al.
2268 Langmuir, Vol. 10, No. 7, 1994 Langmuir-Blodgett (LB) methods.21r2Water for the subphase was purified with the Milli-QII system (Nippon Millipore Co., Tokyo) and poured directly into an LB trough. The specfic resistance of the water was ca. 18MP cm. The maximumsurface area of the water phase on the trough was 475 x 150mm2.The trough surface and a moving barrier were coated with Teflon, and the subphase was temperature-controlled with a thermostat (20 f 0.2 "C). The pH of the subphase was adjusted with HCl and NaOH. The benzendethanol(812 in volume)solution (50150 yL) of lipid molecules (1 mg mL-l) was spread on the subphase. ARer solventevaporation, a monolayer was condensed at a speed of 0.60 cm2 s-1 and n-A isotherms were recorded automatically. A lipid monolayer was transferred onto a SnO2 electrode or a Ag electrode of the QCM plate by a vertical dipping method at a surface pressure of 40 mN m-1 and at a moving speed of the substrateof 60 mm min-1. The substrate,whichhad been soaked in the water phase before spreading the monolayer, was raised and then lowered through the monolayer. A bilayer membrane could be depositedby onedippingcycle: oneside ofthe hydrophilic head group contacts the substrate surface, and the other side faces the aqueous phase. Thebilayer-coveredsubstratewas used in the aqueous solution without raising to the air phase. (c) Electrochemical Measurements. Cyclic voltammetry (CV)was carried out with a potentiostat (Model NPGF2-2501, Nikko Keisoku, Co., Tokyo) equipped with an X-Y recorder (Model RW-11, Rika Denki, Co., Tokyo). A three-electrode configuration was employed a working SnOzelectrodethat was prepared by depositing Sn on a glass plate (1x 2 x 0.2 cm3) and oxidizedin air,a saturated calomelelectrode(SCE)as a reference electrode, and a platinum plate as a counter electrode.23 Lipid bilayers of 2c16PE were deposited on a SnO2 electrode by the LB method as described above. The voltammetric measurements were carried out in an aqueous solution of 1.0 mM &Fe(CN)6 and 0.1 M KC1(pH 7.2 with 0.01 M HEPES buffer) with a sweep rate of 50 mV s-1. The cell (50mL) was temperature-controlled within f O . l "Cby circulatingwater. The oxidationpeak current (i,) was used as a parameter of the barrier effect of LB films for ferrocyanide ions. (a)Quartz-CrystalMicrobalance (QCM) Measurements. The commercially available, 9-MHz, AT-cut QCM (Kyushu Dentsu Co., Tokyo) was used, in which Ag electrodes (area 24 "2) had been depositedon both sides of a quartz plate (diameter 8 mm, area 64 mm2). LB films of 2 c l ~ P E were deposited on one side of a QCM,the other side ofwhich was covered with a rubber case to avoid contact with ionic buffer solutions. A homemade oscillator circuit designed to drive the QCM at its resonant frequency was employed for frequency measurements. The frequency of QCM was followed continuously by an Iwatsu frequencycounter (SC 7201Model)attached to a microcomputer system (NEC, PC 8801 Model).1s-18 The followingequation has been obtained for the AT-cut shear mode QCM:14
AF = (-F,,?A(e#u,)"2)Am (1) where AF is the measured frequency shift (Hz), FOthe parent frequency of the QCM (9 x 106 Hz), Am the mass change (g),A the electrode area (0.238 cm2), e, the density of quartz (2.65 g cm-9, andy, the shear modulus of quartz (2.95 x 10" dyn cm-9. Calibration of the QCM was carried out as follows. When (C17C00-)zCd2+monolayers were transferred with 10layers on one side of the QCM by a vertical dipping method, the frequency decreased 445 f 5 Hz after drying in air. The theoretical mass of 10 dry monolayers of (C17C00-)2Cd2+was calculated to be 575 ng fromthe area per molecule and the electrodearea of the QCM.24 The following calibration equation was obtained from these values, which is closeto the theoretical equation calculated from eq 1 (Am = -1.30 x lO-ghF):16-1* hm = -(im f 0.01) io-~AF (2) When five bilayers of2ClaE were transferred onto a QCM plate, (21) Taneba, S.;Ariga,K.;Tagaki,W.;Okahata,Y.J. Colloidlnterfme Sei. 1989, 131, 561. (22) Okahata,Y.;Kimura,K.;Ariga,K.J.Am.Chem.Soc. 1989,111, 91 no ----. (23)Okahata, Y.; Yokobori, M.; Ebara, Y.; Ebato, H.; Ariga, K. Langmuir 1990, 6, 1148. (24) Okahata, Y.; Ariga, K. J . Chem. Soc., Chem. Commun. 1987, 1535.
A /nm2molec?
Figure 1. Pressure-area (n-A) isotherms of 2C16PE monolayers on the water subphase in the absence (broken lines) and presence (solid lines) of 10mM CaCl2 at 20 "C at pH 2,5.8, and 11.
the frequency of the QCM was decreased 430 5 Hz, which indicates 558 f 5 ng of lipid was transferred onto the substrate accordingto eq 2. This value was consistent with the theoretical mass of five bilayers of 2ClaE (550 ng). The Ca2+ binding experiments were carried out with the 2 c l ~ P E bilayer-deposited QCM in 0.01 M Tris buffer solution containing 0.1 M KCl.
Results and Discussion (a)n-A Isotherms of Monolayers. n-A isotherms of a synthetic phosphoethanolaminelipid, ~ C I ~ Pin E the , presence and absence of Ca2+ions a t pH 2,5.8, and 11are shown in Figure 1. Without Ca2+ions, the molecular area of the lipid increased in the order of pH 5.8 pH 2 pH 11,where the 2C16PE exists as zwitterionic, cationic, and anionic forms, respectively. Thus, intermolecular ionic interaction between phosphate anions and ammonium cations a t pH 5.8 gave the most condensed molecular packing. Although the intermolecular zwitterionic interactions were lost at pH 2, hydrogen bonding between phosphoric acids could compensate the loss of intermolecular ionic interactions and gave the relatively condensed molecular packing in the acidic conditions. On the contrary, repulsion between phosphate anions caused the most expanded molecular structures a t pH 11. The obtained results were consistent with observations for liposome experiments in the l i t e r a t ~ r e . ~ ~ - ~ ' No effects of Ca2+ions were observed in n-A isotherms a t pH 2, due to the repulsion between Ca2+ions and cationic head groups of 2ClaPE monolayers. The addition of Ca2+ ions caused the increase in the molecular area a t pH 5.8, because Ca2+ions might bind to phosphate anions and disturb the intermolecular packing of zwitterionic head groups. On the contrary, the decrease in the molecular area was observed at pH 11by the addition of Ca2+ions. This is due to the decrease of anionic repulsion by binding Ca2+ions to phosphate head groups. When the zwitterionic phosphocholine lipid molecules 2c16Pc or the neutral lipid 2ClsOH was employed as a monolayer, the molecular areas were hardly affected by the addition of Ca2+ions in the subphase at various pH values (results are not shown). (b) Electrochemical Studies of 2ClsPE LB Films. The effects of Ca2+ions and temperature on the molecular packing of 2ClaPE bilayers were studied with electro(25) Harlos, K.; Eibl, H. Biochim. Bwphys. Acta 1980, 601, 113. (26) Jacobson, K.; Papahadjopoulos, D. Biochemistry 1975,14,152. (27) Trauble, H.; Eibl, H. Proc. NatLAcad. Sei. U S A . 1974,71,214.
Langmuir, Vol. 10, No. 7, 1994 2269
Calcium Ion Interactions with Phospholipid Membranes
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II
\j
I
~
0.0
+0.5
+l.OV
(vs.SCE) Figure 2. Cyclic voltammograms of the bare SnO2 electrode and the electrode covered with a bilayer of 2C16PE in 1.0 mM &Fe(CN)e and 0.1 M KC1 aqueous solution at 25 "C (scanning speed 50 mV s-l).
lo4
lo3
10.2
[Ca2'] I M
Figure 4. Effect of Ca2+ion concentrationson the peak currents (idi,")in 1.0 mM &Fe(CN)6 and 0.1 M KCl aqueous solution at 20 and 60 "C. The idiPovalue indicates the ratio of the peak currents for the 2C16PE bilayer-covered and bare electrodes.
Temp. I "C 70 60
50
40
30
20
-4.0
0.5
4 2 -4.5 v
0.2 0.1
bdh
Tc= 51 OC 1
1
1
3.0 T-1
1
3.2
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1
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i 1 0 3 K"
Figure 3. Arrhenius plots of oxidation peak currents (i,) of marker ions (Fe(cN)6*-)on (a) the bare electrode and (b) the electrode covered with a 2C16PE bilayer.
.
10's
10-4
two bilayers-covered
-t--1o - ~
10.2
[Caz'] I M Figure 6. Effect of the number of 2C16PE bilayers on the electrode on the oxidation peak current (idi,")in 1.0 mM &Fe(CN)6 and 0.1 M KC1 aqueous solution at 60 "C.
discontinuously increased in the fluid liquid crystalline state above T,compared with that in the solid state below
Tc*
chemical methods by using cyclic voltammograms of maker ions [Fe(CN)63'4-]in the aqueous phase. Figure 2 shows typical cyclic voltammograms (CVs) of the bare SnO2 electrode and the electrode covered with a bilayer Of 2C16PE in 1.0mM &Fe(CN)6 aqueous solution a t 20 "C. Reversible CV peaks of redox reactions between Fe(CN)e4-and Fe(CNh3- ions were observed for the bare electrode, and they were largely suppressed by the deposition of the 2C16PEbilayer film. The peak potential of the 2C16PE-coveredelectrode is generally shifted to a n overpotential relative to that of the bare electrode.23 The oxidation peak currents (i,) of the bare and the 2C16PE bilayer-covered electrodes were measured a t various temperatures, and the results are shown in Figure 3 as Arrhenius plots. The peak current of the 2C16PEcovered electrode was much reduced in whole temperatures compared with the bare electrode, which indicates that the electrochemical reaction is blocked by a lipid bilayer film. The i, values for the 2Cl6PE-coveredelectrode showed a discontinuity near 51 "C, which was the phase transition temperature (T,)from the solid to liquid crystalline state of 2Cl6PE lipid membranes obtained from differential scanning ~ a l o r i m e t r y . ~Thus, ~ ~ ~ ~ the permeation of marker ions through 2C16PE bilayers
The ratio idipoof oxidation currents for the 2C16PEcovered and the bare electrodes was used as a marker of the barrier effect of the lipid film on the electrode. The influence of Ca2+concentrations on the i&," values was studied both below and above the T,ofthe 2Cl6PE bilayer, and the results are shown in Figure 4. The idi," values increased in the presence of Ca2+ions over 1mM only in the fluid state of the membrane a t 60 "C, but hardly changed in the solid state a t 20 "C. This indicates that Ca2+ions may bind to the head groups and disturb the membrane structures only in the fluid state above the T,. Figure 5 shows the effect of the number of layers of 2C16PE LB films on the oxidation current (idi,") in the fluid state at 60 "C. The large effect of the added Ca2+ ions was observed only for the bilayer-covered electrode, but not for the monolayer and two-bilayer-covered electrodes. The monolayer of 2C16PE could not completely cover the electrode surface and could not reduce the i,,lipo values. Therefore, the small increase of the idi," values (disturbance of the membrane) was obtained in the presence of Ca2+ions. On the other hand, the oxidation currents through two bilayer (four monolayers) were largely suppressed even in the fluid state a t 60 "C. This indicates that the fluid bilayer film is of suitable thickness to change the membrane structures responding to the addition of Ca2+ions. Figure 6 shows reversible changes of the i, values of the 2C16PE bilayer-covered electrode, responding to the alternative addition of Ca2+ions and EDTA. In the fluid
Ebara et al.
2270 Langmuir, Vol. 10,No. 7,1994 20
15
I
20 "C
5 -
7
1
0
5
10
15
"
Number of Bilayers Figure 8. Effect of the number of 2C16PE bilayers on the QCM on frequency changes at 20 and 60 "C in the 0.01 M Tris buffer solution (0.1 M KCl) containing 10 mM Ca2+ions.
5 1
1 5
O/ 20 "C
-1
-6
-5
-4
-3
-2
log [Ca2+]I M Figure 7. Effect of Ca2+ion concentrationon frequencychanges
of the QCM deposited with 15 bilayers of 2C1,jPE in the 0.01 M Tris buffer solution containing 0.1 M KC1 at 20 and 60 "C. 2C16PE bilayer at 60 "C, the z, values increase with the addition of Ca2+ions due to the disturbance of the covered membrane, and decrease reversibly with the addition of EDTA due to the removal of Ca2+ions from the membrane. This behavior was hardly observed a t 20 "C in the solid state of the bilayer film, which was consistent with the results in Figure 4. When the phosphocholine 2ClSPc or the neutral 2C16OH bilayers were deposited on the electrode, the oxidation peak currents were hardly affected by the presence of Ca2+ ions even in their fluid state above their Tc,which indicates Ca2+ions selectively bind to phosphoethanol head groups of bilayers. ( c ) QCM Studies on the Binding Process of Caa+ Ions. A quartz-crystal microbalance (QCM) is known to provide a mass measuring device because the resonance frequency decreases with increasing mass on a n electrode of the QCM on the nanogram level according to eq l.I4-'* A QCM was used here to estimate directly the binding behavior of Ca2+ions onto the 2C16PE bilayers as a mass change. The frequency changes of the QCM deposited with 15 bilayers of 2C16PE responding to the addition of Ca2+ions in the aqueous solution is shown in Figure 7. The negative shift of the frequency indicates the mass increase on the 2C16PE bilayers on the QCM. The large frequency decrease (binding of Ca2+ions) was observed only at 60 "C in the fluid liquid crystalline state of the bilayer above the concentration of 1 x M Ca2+ions, but not in the solid-state bilayers in the whole Ca2+ concentration range. These findings depending on the temperature were consistent with results obtained from electrochemical measurements in Figures 3, 4, and 6. When the zwitterionic 2c16Pc or the neutral 2C16OH LB films were deposited on the QCM and soaked into the aqueous solution of 10 mM Ca2+ ions at 60 "C, the frequency changes due to the binding of Ca2+ions on the membrane were hardly observed.
200
400
600
1
Time / s Figure 9. Reversible frequency changes of the 2 C d E (15 bilayers)-depositedQCM, respondingto the alternative addition of 0.3 mM Ca2+ions and 0.1 mM EDTA at 20 and 60 "C in the 0.01 M Tris buffer solution containing 0.1 M KC1.
Figure 8 shows the effects of the number of layers of 2c16PE bilayers on the frequency decrease (the binding amount of Ca2+ions) in aqueous solution of 10 mM Ca2+ ions. The binding amount of Ca2+ions increased linearly with increasing number of bilayers in the fluid state a t 60 "C, but hardly increased in the solid state of the membrane at 20 "C. Thus, Ca2+ions can deeply penetrate into the fluid LB films above the Te;on the contrary, Ca2+ ions can bind near the surface of the solid membrane a t 20 "C. In the fluid state, about 20 ng (0.5 nmol) of Ca2+ions was calculated to bind to 15 bilayers (825 ng, 1.25 nmol) of 2Cl6PE from Figures 7 and 8, which indicates one Ca2+ ion binds per two or three 2 c l ~ P E molecules. In the solid state, the binding amount of Ca2+ions can be estimated to be less than 10 ng (0.25 m o l ) which means binding only to the surface part of the film. Dissociation constants of Ca2+ ions can be roughly calculated from Figure 7 to be & = 1 x M and more than M in the fluid state and the solid state of 2 C l ~ PE bilayers, respectively. Similar & values could be obtained from the electrochemical experiments in Figures 4 and 5. Ka values of Ca2+ions for phosphatidylglycerol membranes were reported to be lo-' to M by NMR mea~urements.'~J~ These values are a factor of 100larger than our & values. Since the experimental conditions (aqueous dispersion or planar bilayers), chemical structures of the lipid, and measurement tools are largely M e r e n t , we cannot explain this difference a t this moment. Figure 9 shows the reversible frequency changes of the 2cl6PE (30 layers)-deposited QCM, responding to the alternative additions of Ca2+ions and EDTA. In the fluid state of the membrane a t 60 "C, the frequency decreased (the mass increased) due to the binding of Ca2+ions to the
Calcium Ion Interactions with Phospholipid Membranes head groups, and the frequency reverted to the original value (mass decreased) due to the removal of Ca2+ions from the membrane by the addition of EDTA. These changes were hardly observed in the solid state of the membrane a t 20 "C. These reversible changes in the binding behavior of Ca2+ions are very consistent with those in the electrochemical reactions shown in Figure 6.
Conclusion The interaction between the phosphoethanolamine2C16PE monolayers or LB films and Ca2+ions was investigated by three different methods: n-A isotherms, electrochemical reactions, and QCM measurements. It was clarified
Langmuir, Vol. 10,No.7,1994 2271 directly from QCM measurements that Ca2+ions can bind selectively to the fluid state of the 2C16PE bilayers, but hardly to the solid state of the bilayers. It was confirmed from n-A isotherms of the monolayers that the molecular packing of the 2Cl6PE membranes was increased by the addition of Ca2+ ions due to the diminishment of the intermolecular zwitterionic interactions at the neutral pH region. The disturbance of the 2C16PE bilayers caused by the addition of Ca2+ ions was also confirmed from electrochemical reactions on the electrode depending on the fluidity of the bilayers. These effects of Ca2+ions were not observed in the membrane ofthe phosphocholine 2ClsPC and the neutral 2ClsOH membranes.
