Langmuir 1994,10, 1415-1420
1415
Thermotropic Behavior of a Phospholipid Bilayer Interacting with Metal Ions Kyung Ok Kwon,? Myung Ja Kim,$ Masahiko Abe,*vtysTadashi Ishinomori,t and Keizo Ogino+l§ Faculty of Science and Technology, Science University of Tokyo, 2641, Yamazaki, Noda, Chiba 278, Japan, Department of Chemistry, Sookmyung Women's University, Chungpa-dong Yongsan-ku, Seoul, Korea, and Institute of Colloid and Interface Science, Science University of Tokyo, 1-3, Kagurazaka, Shinjuku-ku, Tokyo 162, Japan Received August 8, 1993. In Final Form: January 21, 1994@ The metal ion effect on hydrated phospholipid bilayers of dipalmitoylphosphatidylcholine(DPPC) and dipalmitoylphosphatidylglycerol (DPPG), suspended in an aqueous solution placed in a zinc selenide cylindrical internal reflection cell, is studied by means of attenuated total reflection Fourier transform infrared (ATR-FTIR)spectroscopy. The infrared spectra revealed that DPPG liposomes are more unstable than DPPC liposomes with changes in temperature and in the presence of metal ions. In the presence of Ca2+,both DPPC and DPPG liposomes show a conformational change and dehydration as is evident by the ester carbonyl stretching mode. The dehydration ability of metal ions decreases in the order of Ca2+ >> Mg2+> Ba2+in the phosphate ester group of DPPG. The effect of increasing Ca2+ and Mgz+ concentration is cumulative;however, Ba2+does not demonstratea concentration dependence. The infrared spectra and DSC thermogramsindicate that dehydration is the main cause of the transition temperature shift.
Introduction Phospholipids are treated as functional materials as a result of having structures similar to molecular biomembranes.' Their prospective utilities are considered in the area of drug delivery system,2Jfoodchemistry: cosmetics: and so on. Although many studies have been reported,the functional roles of lipids are not understood from the viewpoint of physiology as a result of their complicated behaviors. It is expected that the structure of phospholipid bilayers is changed and exhibits very complex phase behavior in aqueous media by addition of metal ion^.^-'^
* T o whom correspondence should be addressed.
+ Faculty of Science and Technology, Science University of Tokyo. t Department of Chemistry, Sookmyung Women's University. 5 Institute of Colloid and Interface Science, Science University of Tokyo. * Abstract published in Advance ACS Abstracts, April 1, 1994. (1) Gruner, S. M.; Cullis, P. R.; Hope, M. J.; Tilcock, C. P. S. Annu. Rev. Biophys. Biophys. Chem. 1985, 14, 211. (2) Ghosh, P.; Das, P. K.; Bachhawat, B. K. Arch. Biochem. Biophys. 1982,213, 266. (3) Lawman, M. J. P.; Naylor, P. T.;Huang, L.; Courtney,R. J.; Rouse, B. T. J. Immunol. 1981, 126,304. (4) Mellier, A.; Diaf, A. Chem. Phys. Lipids 1988, 46, 51. (5) Rupert, L. A. M.; Engberts, J. B. F. N.; Hoekstra, D. J. Am. Chem. Soc. 1986, 108, 3920. (6) Gabizon, A.; Papahadjopoulos, D. Biochim. Biophys. Acta 1992, I 1 03, 94. (7) Menniti, F. S.;Bird, G. S. J.; Takemura, H.; Thastrup, 0.;Potter, B. V. L.; Putney, J. W. J. Biol. Chem. 1991,266, 13646. (8) Kosk-kosicka, D.; Bzdega, T. Biochemistry 1991,30, 65. (9) Virden, J. W.; Berg, J. C. Langmuir 1992, 8, 1532. (10) Akutsu, H.; Nagamori, T. Biochemistry 1991,30, 4510. (11) Eklund, K. K.; Takkunen, J. E.; Kinnunen, P. K. J. Chem. Phys. Lipids 1991, 57, 59. (12) Choi, S.; Ware, W., Jr.; Lauterbach, S. R.; Phillips, W. M. Biochemistry 1991,30, 8563. (13) Allen, T. M.; Hong, K.; Papahadjopoulos, D. Biochemistry 1990, 29, 2976. (14) Casal, H. L.; Mantach, H. H.; Hauser, H. Biochim. Biophys. Acta 1989,982, 228. (15) Casal, H. L.; Mantsch, H. H.; Hauser, H. Biochemistry 1987,26, 4408. (16) Herbette, L.; Napolitano, C. A,; McDaniel, R. V. Biophys. J. 1984, 46, 617. (17) Lau, A.; McLaughlin,A.;McLaughlin, S. Biochim. Biophys. Acta 1981,645, 279.
0743-7463/94/2410-1415$04.50/0
Interactions of metal ions with lipid membrane have been studied in terms of basic properties of lipid assemblies and possible physiological implications of interest. The fusogenic activity of cations for phosphatidylserine containing cholesterol systems has been reported by Shavnin et a1.18 The phenomena mentioned above are interpreted according to the dehydration ability of the metal ions. Infrared studies (Casal et a1.14J5J9) have shown that differences in Ca2+,Mg2+,and Li+ binding to a series of phosphatidylserine bilayers are dependent on their binding to the phospholipid head groups which is affected by a molecular area, surface charge density, and intermolecular separation (spacing). In a previous paper, we reported that the effect of calcium ion on fusion was dependent on the surface charges of phospholipids prepared by natural extracts, such as phosphatidylserine from ox brain, lecithin from egg yolk, and ghost red cells from human blood.20 In this paper, differential scanning c a l ~ r i m e t r yand ~~-~~ attenuated total reflection (ATR) Fourier transform infrared (FT-IR) spectroscopyapplied to liposomesystems to elucidated the interactions between metal ions and negative phospholipid bilayers. Many papers report studies of deposits or bilayers in a DzO b ~ f f e r ' ~ J 5 J ~ ~ ~ 6 system as a result of interference by the strong infrared absorption of water molecules. In order to overcome this difficulty, the attenuated total reflection technique combined with FT-IR spectroscopy has been applied. ATR(18) Shavnin, S. A.; Lima, M. C. P.; Fedor, J.; Wood, P.; Bentz, J.; Duzgunes Biochim.Biophys. Acta 1988, 405,416. (19) Casal, H. L.; Martin, A.; Mantsch, H. H. Biochemistry 1987,26, 7395. (20) Kwon, K. 0.;Abe, M.; Kim, M. J.; Ogino,K. Jpn. Oil Chem. Soc. 1992, 41, 45. (21) Sunamoto, J.; Gob, M.;Iwamoto, K.; Kondo, H.;Sato, T. Biochim. Biophys. Acta 1990, 1024, 209. (22) Bach, D.; Wachtel, E. Biochim.Biophys. Acta 1989,979, 11. (23) Mannock, D. A.; Lewis, R. N. A. H.; Sen, A.; McElhaney, R. N. Biochemistry 1988, 27, 6852. (24) Hinz, H.-J.; Six, L.; Ruess, K.-P.; Liefliinder, M. Biochemistry 1985, 24, 806. (25) Bach, D. Chem. Phys. Lipids 1984,35, 385. (26) Umemura, J.; Cameron,D. G.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 602, 32.
0 1994 American Chemical Society
Kwon et al.
1416 Langmuir, Vol. 10, No. 5, 1994
FTIR studies of hydrated dimyristoylphosphatidylcholine film cast on germanium or zinc selenide plates has been reported by using of ATR-FTIR;27*28however, few papers regarding liposomes suspended in aqueous solution have been reported. In this experiment, liposomes suspended in HzO buffer solution (100 mM NaC1, TES buffer, pH 7.4) were introduced into a compartment (capacity 2.5 cm3) equipped with a cylindrical internal reflection cell made of zinc selenide. The infrared spectra obtained from a HzO buffer 8 ' 1 1 system are compared with a DzO buffer (NaOD, pD 7.4) system and with spectra obtained by Casal et a1.,14J5J9129 Weers et al.,30 and Mellier et al.31 We discuss the interactions between lipids (dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol)and metal ions and the driving force necessary for creating a similar bilayer environment.
Experimental Section Chemicals. L-a-Dipalmitoylphosphatidylcholineand ~ - a dipalmitoylphosphatidylglycerolwere provided by Nippon Oil & Fats Co., Ltd., Amagasaki, Japan. The purities of phospholipids were confirmed by one-dimensional thin-layer chromatography in chloroform/methanol/water (65:25:4,by volume);0.5
mg of each lipid was chromatographedas a singlespot. Dichloride metal ions of Ca2+,Mgz+,and Ba2+were purchased from Sigma Chemical Co. All other reagents were of analytical grade or of the highest purity available. Fourier Transform Infrared Spectroscopy Measurements. Liposome solutions were prepared by sonication with a
bath type sonicator (BransonB-220)in which phospholipidswere suspended in TES buffer solution (100mM NaCl, 2 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, pH 7.4)for 1 h at 50 "C. The diameter of the multilamellar vesicle measurements ranged from 70 to 250 nm according to dynamic light scattering (Model 4700 type Submicron Particle Analyzer of Malvern Instruments, Ltd., Worcestershire, UK). The attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra were recorded with a JEOL JIR-5300 spectrometer in the 4000-400 cm-l range, operated at a nominal resolution of 4 cm-l with triangular apodization over the entire spectral range. A cylindrical internal reflection (CIR) cell with zinc selenide (TNL-130B Axiom Anal. Inc.) equipped with a temperature controller was used. The spectrum of liquid water at the appropriatetemperature was subtracted from the spectra of the samples to produce the figures. Several of the interferograms were interpolated by a factor of 10 to aid in the comparison of bandshape changes in narrow bands. No smoothing or noise reduction algorithms were applied to any of the spectra.30 All samples were incubated for 30 min at 37 "C after adding metal ion solutions to the liposomes, in which the molecular ratios of metal ion to phospholipid were 0.05,0.1,0.5, 1, 10, and 100.IR measurementswere made at 22 and 70 "C. The final concentration of phospholipid was 6mM. Some sampleswere alsoprepared in a DzO buffer solution (NaOD, pD 7.4). DSC Measurements. Many samples of the phospholipidTES buffer solution, containing 0-40 mM metal ions, were prepared by using a microsyringe to add a certain amount of the dehydrated compounds. The samples were shaken on a Vortex mixer and were cooled to -20 "C after heating to 80 O C to ensure homogeneousmixing (thistemperature treatment was performed 4 times), and then maintained there for about 24 h. A highpressure crucible is used. The DSC measurements were carried out with a DSC 8240 (Rigaku Co.) at a heating rate of 1 K/min ~~~~~
~~
(27) Grainger, D. W.; Sunamoto, J.; Akiyoshi, K.; Goto, M.; Knutson, K. Langmuir 1992,8, 2479. (28) Ter-Minassian-saraga, L.; Okamura, E.; Umemura, J.;Takenaka, T. Biochim.Biophys. Acta 1988, 946, 417. (29) Casal, H. L. J. Phys. Chem. 1989, 93, 4328. (30) Mellier, A,; D i d , A. Chem. Phys. Lipids 1988, 46, 51. (31) Pascher, I.; Sundell, S.; Harlos, K.; Eibl, H. Biochim.Biophys. Acta 1987,896, 77.
I
22'c
1516r
22°C
1 4 1 5 2
0
01
10
' L>
loooL
'
'','I
01
'
"
";
io
loa
Molar ratio (ioniphospholipids)
Figure 1. Infrared spectraof the antisymmetricC-H stretching wavenumbers for DPPC and DPPG at various molar ratios of calcium ion to phospholipid in TES buffer (pH 7.4).
in the range of -40 to 120 "C. The sensitivities employed are indicated in the legends of the figures.
Results and Discussion Acyl Chain Region. Figure 1 depicts the change in the antisymmetric CHZstretching wavenumber for DPPC and DPPG liposome as a function of the Ca2+ ion to phospholipid concentration ratio, at a temperature both above and below the phase transition temperature (70 and 22 "C). In the gel phase, the pure DPPC and DPPG liposomes showed the same wavenumber (2918cm-l), while in the liquid crystalline phase, the band of the DPPG liposome without ions shifted to higher wavenumbers than that of the DPPC liposome (2923 and 2921 cm-', respectively). These results imply that the DPPG liposome exists in a more disordered state than the DPPC liposome in the liquid crystalline phase as a result of the inflation of gauche conformers.3' Furthermore, the addition of Ca2+to the liposome solution caused the frequency shift to disappear in the DPPG liposome. It is considered that the hydrocarbon part of the fatty acyl chain of the DPPG liposome changes to a highly ordered trans form by Ca2+-induced isothermal crystallization.lZ The distance between the layers is shortened from 55 to 50 nm (water content 50%) in the presence of Ca2+as confirmed by small angle X-ray data a t 20 "C (unpublished results). From the results, a higher concentration of Ca2+(molar ratio 10) is needed to the entire gel transition of the fatty acyl chain. Frequency shifts of antisymmetric CHZstretching were not observed by addition of Mg2+and Ba2+. On the other hand, the antisymmetric CHZstretching band for the DPPC liposome was independent of metal ion concentration. The hydrophobic change caused by metal ion binding to the phospholipid head group was prominent in the band of the terminal methyl group. Figure 2 shows the 3000-2800 cm-' region of the infrared spectra of the DPPC and the DPPG liposomes recorded in samples having molar ratio of ref (reference = 01, 0.5, and 10 at 22 "C. The antisymmetric and symmetric stretching bands of the terminal methyl group for DPPC and DPPG liposomes are observed a t the same frequency region, 2956 cm-l. As can be seen in Figure 2, no significant change was shown in the DPPC liposome upon increasing the ion concentration; however, a change was observed in the DPPG liposome system. The structural arrangement of the DPPG liposome bilayer appears to be changed by calcium ion, Le., from a vesicular form having a small curvature to a lamellar, cylindrical, or other form, as a result of the compacted bilayer in which the vibration of the terminal methyl group is restricted. The change in the frequency of the CH2 stretching modes was associated with the melting transitions and the introduction of gauche conformation. Therefore, the CHz
Metal Ion Effect on Phospholipid Bilayers DPPC-Ca*+
22T
I DPPG-Ca*+
Langmuir, Vol. 10, No. 5, 1994 1417 22°C
1738
I
p
c 5: 9
1734
DPPG
3000
2950
2900
2850
3000
2950
2900
2850
28
Wavenumbedcm-1 Figure 2. Infrared spectra of the terminal methyl group for DPPC and DPPG liposomes at antisymmetric (2960,2950cm-l, respectively) and symmetric (2881,2887cm-l) stretching bands as a function of calcium ion to phospholipid molar ratio in TES buffer (pH 7.4) at 22 O C . G
I
"
1
Figure 4. Infrared spectra of the antisymmetric C=O stretching region for DPPC and DPPG liposomes as lipid films with a KBr plate (left side in figure) and liposomes in D20 buffer solution (right side in figure). DPPC
DPPG
/ 0
0.1
1
10
R"
1134
100
Molar ratio (iodphospholipids)
Figure 3. Maximum peak height ratio of 1463 cm-l/1456 cm-l vs molar ratio of the added Ca2+ to DPPG liposome at 70 O C . scissoring mode also represents changes in chain packing arrangements and intermolecular interactions. Figure 3 depicts the peak height ratio of 1463 cm-lI1456 cm-l as a function of the molar ratio of Ca2+to DPPG. The peak height ratio is related to the acyl chain scissoring vibration and to the acyl chain packing conditions in the gel phase of the DPPG liposome. The latter band is associated with gauche conformers.26 Between these bands, the intensity at a lower frequency decreases with increasing calcium ion concentration and these split bands become one peak as a result of lessened interaction coupling of phospholipid hydrocarbon. Thus, the DPPG liposome in the presence of calcium ion, exists as all-trans chains packed in a hexagonal subcell lattice with no interchain coupling at 70 OC.15 Interfacial Region. Figure 4 shows the 1'750-1700cm-l region of antisymmetric carbonyl stretching bands of DPPC and DPPG as solids and as liposomes in D2O buffer solution (NaOD, pD 7.4). These vibrational modes of the ester carbonyl stretching group are related to interactions at the head group and contain information concerning
,750 1700 1750 1700 1750 1700 1750 Wavenumberlcm-1
1700
Figure 5. Infrared spectra of the antisymmetricC=O stretching region for DPPC and DPPG liposomes with Ca2+,Mg2+,and Ba2+ions (the ion to phospholipid ratio is 1)in TES buffer (pH 7.4) at 22 and 70 O C , respectively. membrane structure. The two bands were observed in a DPPG film prepared with a KBr plate (left side in Figure 4, 22 "C). These ester carbonyl stretching bands correspond to the sn-1 chain (1741cm-') and to the sn-2 chain (1730 ~ m - l ) The . ~ ~corresponding bands were observed at 1734cm-l (right side in Figure 4) in the D2O buffer (NaOD, pD 7.4) system. In the case of DPPC, the band for the dry sample was observed at 1738cm-' and was split into several bands in DzO buffer solution. This splitting of the C=O (32) Blume, A.; Hubner, W.; Messner, G. Biochemistry 1988,27,8239.
1418 Langmuir, Vol. 10, No. 5, 1994
Kwon et al.
Wavenum berlcm-I Figure 6. Infrared spectra of the phosphate stretching region for DPPC and DPPG liposomes with various ions, Ca2+,Mg2+,and Bas+ (ion to phospholipid molar ratio is 10) at 70 "C. vibrational bands in D20 is caused by hydrogen bonding of water molecules to both C=O groups. The characteristic hydrogen bonding is observed in the HzO buffer system without metal ions (ref) and with various metal ions (Ca2+, Mg2+,and Ba2+) (Figure 5). Comparing the band with D2O buffer system, sharped and split bands are observed for both the DPPC and the DPPG liposome. With pure samples the conformational differences of the two chains are somewhat averaged in the liquid crystalline phase, and the two C=O stretching bands are of comparable intensity. The same frequency appeared for the pure DPPC and the DPPG liposome in the gel and in the liquid crystalline phase. However, a sharper band was observed for the DPPC liposome compared to the DPPG liposome in the liquid crystalline phase, as opposed to the gel phase where the DPPG liposome appeared narrower. From the bandwidth change with temperature, this suggests that the DPPC liposome is more stable than DPPG liposome with increasing temperature. In the presence of Ca2+, the bands at 1718 and 1701 cm-I for DPPC and the 1716-cm-' band for DPPG disappear. It should be noted that the peak disappeared only with calcium for both DPPC and DPPG in the gel phase. These results lead to the conclusion that the bands are caused from differently hydrated carbonyl bands which are dehydrated by metal ions. Furthermore, the DPPC liposome showed a frequency change in the gel phase and in the liquid crystalline phase separated into three bands at 1743, 1738, and 1730 cm-I which can be assigned to carbonyl groups with different degrees of hydration and conformational change.12 The antisymmetric carbonyl stretching band of DPPG gives a narrow band in the liquid crystalline phase. This is a result of reductions in conformational freedom of fatty acyl chains caused by the presence of the Ca2+. This change may be brought about by changes in the conformation of the glycerol backbone.15 The lack of band splitting may indicate that the conformation of this group is such that no site-symmetrysplitting occurs or that only one rotational isomer of the entire sn-1 chain is found. Thus, the sn-1 chain would be locked in a given position, a situation compatible with the pronounced immobilization of the DPPG molecule brought about by Ca2+ chelation. With Mg2+ and Ba2+, no
significant effect to carbonyl stretching mode was found in either DPPC or DPPG liposomes. Phosphate Group Absorptions. Infrared spectra of the phosphate stretching region of DPPC and DPPG liposomeswith various metal ions at an ion to phospholipid ratio of 10 in the liquid crystalline phase are shown in Figure 6. The antisymmetric and symmetric phosphate stretching absorptions are observed at 1223,1087cm-' for DPPC and at 1215, 1091 cm-' for DPPG, respectively. The antisymmetric phosphate stretching band for DPPC is observed at significantly higher frequency than for DPPG, suggesting that the phosphate group for DPPC is restricted by other nearby molecules such as water and/or the choline group in the neighborhood of the phospholipid molecule. The symmetric (1067 cm-l) stretching mode of the C-0-C band overlapping the strong PO*- band is observed at the same frequency for DPPC and DPPG liposomes. In the presence of Ca2+,the antisymmetric phosphate stretching band of DPPC was shifted to higher frequency. This frequency shift is due to loss of bound water from the DPPC phosphate group. In the case of the DPPG liposome,the metal ion effect was significant. With Ca2+,the C-0-C band frequency of the DPPG liposome was shifted to lower frequency. This shift is due to intramolecular nonequivalence of the two ester groups. Furthermore, the bandwidth of the antisymmetric phosphate stretching peak narrowed and the wavenumber of the symmetric one was observed at a higher side, reflecting the immobilization of the phosphate group as a result of the calcium ion binding. The symmetric phosphate stretching band was split into four bands in the presence of Mg2+, 1120,1108,1103,1095, and 1090 cm-l. The split is attributed to arise from the antiplanar-antiplanar conformation of the torsional angles of the two P-0 ester bonds.I5 These splittings may reflect intra- and intermolecular nonequivalence of the ester groups. In the presence of Ba2+, the phosphate band of DPPG was somewhat sharpened. The effects of Ca2+and Mg2+on DPPG liposome were cumulative while the effect of Ba2+ was not. Figure 7 shows infrared spectra of the symmetric phosphate stretching region with various metal ions and metal ion concentrations for DPPG liposome. The DPPG liposome was revealed to be almost independent of Ba2+
Metal Ion Effect on Phospholipid Bilayers
Langmuir, Vol. 10, NO.5, 1994 1419
I
I IO0 IO50
I I O 0 1050
1242
1221
I
I
I
1270
1240
1210
1
1180
I
1150
Wavenumber/cm-l Figure 9. Infrared spectra of the asymmetric phosphate stretching band for DPPC (solid line) and DPPG (dotted line) (calcium ion to phospholipid ratio is 10) at 22 OC.
I IO0 1050
Wavenumberlcm-I
Figure 7. Infrared spectra of symmetric phosphate stretching band for DPPG in the presence of Ca2+,Mg2+,and Ba2+ (ion to phospholipid ratios are 0.1, 0.5, 1, 10, and 100) at 70 OC. 1120
r"
11085
I
I
/ \Molar ratio (ion/phospholipids)
Figure 8. Changes of wavenumbers of the symmetric -POrstretching band (open circles) and the symmetric -C-0-CStretchingband (open squares)for DPPG liposome as a function of calcium ion to phospholipid molar ratios, 0.05,0.1,0.5,1,and 10, at 70 "C.
concentration. This indicates that there is no strong interaction or chelate formation between Ba2+ and the phosphate group of DPPG molecules. Being at high Mg2+ concentration, the phosphate group remained in a somewhat hydrated state. The symmetric phosphate stretching band was sharpened from complete dehydration by Ca2+ ion. From these results, the ability of metal ions to dehydrate the phosphate group decreases in the order of Ca2+ >> Mg2+ > Ba2+. Figure 8 depicts the frequency change of symmetric phosphate stretching for DPPG as a function of the Ca2+ ion to phospholipid ratio. The frequency change of -PO4was greater than that of -C-0-C-. Furthermore the change of -PO4- began early, i.e., at low concentrations of Ca2+(molar ratio 0.5). It is considered that metal ions affected the -Pod- group more than the -C-0-C- group. These metal ion effects on phospholipid head groups were observed more profoundly in the gel phase than in the liquid crystalline phase. I t is noted that the antisymmetric phosphate stretching band of the DDPGCa2+was shifted to a higher frequency and the peak feature overlapped with that of the DPPC-Ca2+ in the gel phase (Figure 9). However, the 1242-cm-1 band of the dehydrated -Podantisymmetric stretching band disappeared in the liquid crystalline phase. This band was observed at 1223 cm-l
0
0.1
1
10
100
Molar ratio (ionlphospholipids) Figure 10. Plots of the full widths at 0.75 maximum peak height vs molar ratio of various metal ions to DPPC at 22 OC. Squares represent Ca2+, triangles Mg2+, and circles Ba2+.
for DPPC and at 1215 cm-' for DPPG. The pronounced band shift of DPPG was observed. It was concluded that the DPPC liposome is highly ordered in the H2O buffer where the bilayer is stable to temperature and metal ions, whereas the DPPG liposome is unstable to temperature and metal ions. It is generally proposed that the choline group appearing in the infrared spectrum at 960-980 nm interacts with the phosphate group of an adjacent DPPC molecule.26 The changes in choline group bandwidth are depicted as a function of molar ratio of metal ion to phospholipid in Figure 10. It is considered that the binding between the choline and phosphate group is disturbed by Mg2+, resulting in the broadening bandwidth of the choline group. On the contrary, the binding seems to be rigid in the presence of Ca2+and Ba2+. The curves in Figure 10 show saturation at a metal ion to phospholipid ratio of 0.5. This may be an indication that the metal ions only interact with the head group of the phospholipids molecules in the outerlayer of the DPPC liposome. The DSC thermogram changes of DPPC with increasing metal ion concentration are shown in Figure 11. The effects of Ca2+ and Ba2+ ions are observed at the pretransition region. In DPPC with Mg2+,the thermogram change was observed in the lower side of the phase transition temperature, suggesting that the DPPC phospholipid bilayer is disturbed by Mg2+. This complex thermogram caused by Mg2+ion corresponds to the results of the infrared spectrum between the choline and the phosphate groups as shown in Figure 10.
Kwon et al.
1420 Langmuir, Vol. 10, No. 5, 1994
Ba2+
M g 2 + O/mM
-10 0
10 20 30 40 50
-10 0
O/mM
20
20
40
40
10 20 30 40
SO
-10
0
10 20 30 40 SO
t 1°C Figure 11. Thermograms of DPPC as a function of temperature and various metal ion concentrations: sensitivity, 0.01 mcals-1; scan rate, 1K m-l. Ca2+ O/mM
I
;
20
~
I
I
I
I
, I
I
.
'
20
20
V
40
Ba2+ O/mM
Mg2+ O/mM
V A40
V
40
V
V
I , / 1
I
I
I
I
Figure 12. Thermograms of DPPG as a function of temperature and various metal ion concentrations: sensitivity, 0.01 mcal s-l; scan rate, 1 K m-l.
The thermograms of the DPPG liposome depicted in Figure 12 reveal the change in the phase transition temperature with increasing metal ion concentration. The phase transition temperature of DPPG was gradually shifted to 90 "C with Ca2+,to 68 "C with Mg2+,and to 47 "C with Ba2+. It is noted that the transition temperature of the hydrated DPPG remains at 50 "C in high Mg2+ion concentration solution. This thermogram corresponds to the result of the infrared spectrum of symmetric -Podstretching band in which the -POcremains in a somewhat hydrated state with Mg2+. These results suggest that the dehydration ability of the metal ion is a contributing factor to shift the phase transition temperature.
Conclusions The phospholipid bilayers of DPPC and DPPG suspended in H2O buffer (NaC1,TES buffer, pH 7.4) solution were studied by ATR-FTIR. It was found that one of the hydrated carbonyl asymmetric bands, 1718cm-l for DPPC
and 1716 cm-I for DPPG, disappeared when deprived of hydrated water in the presence of Ca2+. This leads to the conclusion that both carbonyl groups of hydrated phospholipids take part in hydrogen bonding and are dehydrated by metal ions. From the results of infrared spectra and the DSC thermogram, the DPPG liposome is completely dehydrated and conformationally changed in the presence of Ca2+. The infrared spectra revealed that DPPG liposomeis more unstable than the DPPC liposome with temperature changes and changes in metal ions. The effects of increasing the concentrations of Ca2+and Mg2+ are cumulative, while Ba2+does not show a concentration dependence on the DPPG liposome. In particular, the band of the choline group of the DPPC liposome was disturbed by Mg2+ which was also evidenced by a thermogram change at the lower side of the phase transition temperature. It was found that the dehydration ability of metal ions is a contributing factor to shifting the phase transition temperature.
