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Effect of the head group of phospholipids on the acyl-chain packing and structure of their assemblies as revealed by microcalorimetry and electron microscopy
ii!lLOIDS A SURFACES
Colloids and Surfaces A: Physicochemical and Engineering Aspects 109 (1996) 283-289
Effect of the head group of phospholipids on the acyl-chain packing and structure of their assemblies as revealed by microcalorimetry and electron microscopy Michiko Kodama *, Takahiro Miyata Department of Biochemistry,
Faculty of Science, Okayama University of Science, 1-l Ridai-cho, Received 23 August 1995; accepted
8 September
Okayama 700, Japan
1995
Abstract By comparing three phospholipids with different head groups, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the effect of the head group on the acyl-chain packing and structure of the lipid assembly was investigated by high sensitivity differential scanning calorimetry and negative stain electron microscopy. The surface curvatures of the three assemblies were revealed to increase in the order PE < PC < PG, that is, in the order of an increase in the areas of the head groups relative to the area of the acyl chains. In accordance with this difference, the acyl-chain packing in the gel phase also differed greatly among the three assemblies, which was reflected in thermotropic behavior such as a very broad peak, a high temperature for the gel to liquid crystal transition and
lack of a pretransition. Keywords:
DMPC;
DMPE;
DMPG;
Electron
microscopy;
1. Introduction
Phospholipids are major constituents of fundamental bilayer structure in biomembranes and a great variety of phospholipids are present in biomembranes [ 1,2]. The phospholipids are generally classified according to their polar head groups consisting of a phosphate acidic group and other groups attached to its common group, such as phosphatidyl-choline (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). However, a specific composition of the lipid head groups is observed in different types of biomembranes [ 11. This suggests specific lateral interaction and packing of each lipid caused by different * Corresponding author. Tel: (+81) 86 252 3161; Fax: (+81) 86 255 7700. 0927-7757/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI
0927-7757(95)03461-7
Microcalorimetry
types of head groups in the bilayer structure. Thus, the head group is shown to be a predominant factor in determining the specificity of the bilayer structure [2,3]. From this viewpoint, the present paper will discuss how the packing of the lipid acyl chain and the structure of the lipid assembly are changed by alteration in the types of head groups such as PC, PE and PG. Both PC and PE, major phospholipid components of most biomembranes, are known to be present at zwitterions at neutral pH, indicating electrostatic attractions operating between the adjacent head groups at the bilayer surface. However, the geometrical sizes of the two head groups differ significantly [ 31. PG, a widely occurring phospholipid in chloroplast and mitochondrial membranes and bacteria, is negatively charged at neutral pH [4]. As for the charged PG,
284
M. Kodama, T MiyataJColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
a contribution from electrostatic repulsion operating between the adjacent head groups should be considered [ 5-73. Additional attention should be paid to PE which interacts intermolecularly by forming hydrogen bonds [S]. In this study, thermotropic properties of the lipid assemblies were investigated by differential scanning calorimetry and their structural information was obtained by negative stain electron microscopy.
2.3. High sensitivity diferential
2. Experimental
2.4. Electron Microscopy
2.1. Materials 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE) and 1,2-dimyristoylsn-glycero-3-[phospho-rat-( l-glycerol)] (DMPG, sodium salt) were purchased from Sigma and used without further purification. Each lipid yielded a single spot in thin-layer chromatography on a silica gel plate (E. Merck) using chloroform/ methanol/7 M ammonia (230 : 90 : 15, v/v/v) [ 93. Lipid concentrations were estimated by a modified Bartlett phosphate assay [lo]. 2.2. Preparation
scanning
calorimetry
All calorimetric experiments were performed with a Microcal MC2 differential scanning calorimeter interfaced to an IBM PC microcomputer system using an A/D converter board (Data Translation DT-2801) for automatic data collection and analysis. The lipid concentrations in the calorimetric experiments were in the range l-2 mM with a calorimetric cell volume of 1.2 ml. A heating scanning rate of 45 “C h- ’ was used.
Preparations of the lipid assemblies were examined by negative stain electron microscopy [ 13-151. The preparations were prepared at temperatures of 5-10°C as follows: a drop of dispersion (lipid concentration: ~2 mmol ml-‘) was placed on a copper grid covered with a carboncoated collodion film, allowed to remain for 5 min, and then drained. A 2% solution of sodium phosphotungstate (pH z 7) was added and after 10 min the excess solution was drained. The preparation was examined immediately in a JEOL JEM-2000EX electron microscope operated at 200 kV. All operations were performed at z 20’ C.
of lipid assemblies
3. Results and discussion Dispersions of lipid assemblies composed of each of DMPC, DMPE and DMPG were prepared according to a Bangham method for the preparation of a vesicle as follows [ 11,121. The lipid films were first prepared by removing chloroform from lipid stock solutions on a rotary evaporator, and then under high vacuum (lop4 Pa) to achieve complete removal of traces of the solvent. The dried lipid films were then suspended in distilled water, and gently vortexed at the required temperatures above the gel to liquid crystal transition, unless otherwise specified. In order to investigate the effect of Na cation, additional DMPG assemblies were prepared by suspending the dried lipid films in aqueous solutions of NaCl at different concentrations up to 1 M in the same way as mentioned above.
3.1. EfSect of the areas occupied by the head groups on the bilayer surface curvature
Some phospholipids exhibit phase transitions depending on whether the lipid assemblies are prepared at temperatures above or below the gel to liquid crystal transition temperature. The present discussion is limited to the lipid assemblies prepared at the liquid crystal temperatures. Thermotropic behaviors of the three dispersions of DMPC, DMPE and DMPG prepared in the absence of salt are shown in Figs. la, lb and lc respectively, where excess heat capacity (AC,) per mole of lipid is plotted against temperature (t). Negative stain electron micrographs of the three lipid assemblies are shown in Figs. 2a, 2b and 2c
M. Kodama, T. MiyataJColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
-
-1
I
1 kcal K-‘mot-’
a
-b
d 10
20
30
40
50
60
t 1°C Fig. 1. Thermotropic behaviors of the lipid assemblies (a) DMPC, (b) DMPE and (c) DMPG prepared at temperatures above the gel to liquid crystal transition and in the absence salt. The behavior of the DMPC sonicated vesicle is shown part (d). Apparent excess heat capacity (AC,) is plotted as function of temperature (t).
of of in a
respectively. The thermotropic behaviors of the three lipids differ greatly, as shown in Fig. 1, which is in accordance with the quite different structures of these lipid assemblies shown in Fig. 2. A comparison of these thermotropic behaviors produces the following results: (1) DMPG shows a noticeably broad peak due to the transition of the gel to liquid crystal phases, in contrast to the highly cooperative transitions of DMPC and DMPE; (2) DMPE shows a fairly high transition temperature of the gel to liquid crystal phases, compared to those of DMPC and DMPG; and (3) no peak other than the transition peak of the gel to liquid crystal is observed for DMPE, indicating the lack of a pretransition, in contrast with DMPC and DMPG. In contrast, the micrographs of Fig. 2 give the following profiles of these lipid assemblies: ( 1) DMPC is present as large multilamellar vesicles (> 500 nm diameter), (2) DMPG, in contrast, is present as unilamellar vesicles of a nearly constant, small size ( z approximately 100 nm diameter); and
285
(3) DMPE shows a planar bilayer surface different from the curved surfaces of DMPC and DMPG. On the basis of the microscopic structural information, we first discuss lateral packings of the three head groups. Fig. 3 schematically illustrates lipid-packing adaptations to differences in the cross-sectional areas occupied by lipid head groups under the same acyl-chain conditions. In Fig. 3, a decrease in the areas of the head groups relative to the acyl chains in the order (A)>(B)>(C) is shown to cause a decrease in the surface curvatures of lipid bilayers from highly curved to planar surfaces, simultaneous with an increase in the size of the lipid assemblies. By reference to information obtained from X-ray crystallographic study [3,16], the geometrical sizes of the three head groups are revealed to increase in the order PE (R 38 A’) < PG( ~44 A’) < PC( x 50 A*). The size of the negatively charged PG, which swells fully in excess water in the absence of salt [ 171, should be evaluated by the effective area, that is, the geometrical size plus a space due to electrostatic repulsion operating between the neighboring head groups at the bilayer surface [S,lS]. Considering the repulsion space, the area occupied by the PG head group is assumed to be the largest amongst the three head groups. This accounts well for the microscopic result shown in Fig. 2 where the surface curvatures of the three assemblies increase in the order DMPE
286
M. Kodama. T. MiyatalColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
Fig. 2. Negative stain electron micrographs of the lipid assemblies of (a) DMPC, (b) DMPE and (c) DMPG characterized by the thermotropic behaviors of Figs. la, lb and lc respectively. The micrograph of the DMPG assembly prepared in the presence of Na+ at 100 mM concentration is shown in part (d).
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0 a
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Fig. 3. Schematic illustration of lipid-packing adaptations to differences in cross-sectional areas occupied by the polar head groups under the same acyl-chain conditions. The areas of the head groups (a) relative to the acyl chains (b=constant) increase in the order (C) c(B) <(A).
face curvature as shown in Fig. 3. Typical thermotropic behavior and a micrograph of the DMPG vesicles prepared in the presence of Na cation are presented in Fig. 5 and Fig. 2d respectively.
3.2. Effect of the head groups on the acyl-chain packing of the gel phase The generally accepted concept for the lipid phase transition of the gel to liquid crystal is a conformational change in the acyl chain from the solid-like to liquid-like states. Accordingly, it is suggested that the packing between the acyl chains of neighboring lipids is close and loose in the gel and liquid crystal phases respectively. In such a situation, the acyl-chain packing of the gel phase could be influenced by the head groups more greatly than that of the liquid crystal phase. From this viewpoint, the different transition behaviors of the three lipids shown in Figs. la, lb, and lc are considered to result from different packing modes of the acyl chains in the gel phase. Therefore, we next discuss the effect of the head groups on the acyl-chain packing of the gel phase, by reference
281
M. Kodama, T. MiyatalColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
d 0
20
40
60
80 100 120
[Na+]/mM Fig. 4. Effect of Na+ concentration on the approximate mean diameter (A) and the number of bilayers (B) of the DMPG vesicle.
to the transition behaviors shown in Fig. 1. Considering a uniform size of DMPG unilamellar vesicles (see Fig. 2c) [ 191, the marked broadening of the transition peak of DMPG shown in Fig. lc is presumed to be related to the highly curved surfaces in both outer and inner membranes of the lipid bilayer. That is, the acyl chains of the outer membrane are caused to adopt a loose packing corresponding to the highly convex curved surface. Similarly, the acyl chains of the inner membrane are forced to pack much more loosely in a reversed micellar form according to the highly concave surface curvature. Such loose packings of the acyl chains of the outer and inner membranes in the gel phase cause their heterogeneous motions and lateral interactions, resulting in a low cooperativity of the phase transition and the marked broadening of the transition peak for the DMPG vesicle.
I
10
I
I
20
,
I
30
I
40
t /“c Fig. 5. Effect of Na+ concentration on the thermotropic behavior of the DMPG vesicle. Apparent excess heat capacity (AC,) is plotted as a function of temperature (t). Na+ concentration (mM): (a) 50; (b) 100; (c) 250; (d) 1000.
However, as shown in Fig. 5, the transition peaks of DMPG in the presence of Na cation become sharper with an increase in Na concentration, indicating an increase in the cooperativity of the transition [ 191. This suggests a change of the acylchain packing into a less loose aggregation, in accordance with a less loose packing of the PG head groups caused by the screening effect of the Na cation mentioned above. Once again, focusing on the DMPG vesicle prepared in the absence of salt, it is noted that the vesicle, though heterogeneous in the acyl chain motions, exhibits a high stability at the gel temperature; that is, its structure is maintained during a period of annealing of over 1 month at this temperature and no change is observed in the transition behavior [ 201. This suggests that the loose packing
288
M. Kodama. T Miyatalcolloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
of the DMPG lipids in the vesicle is just a way of minimizing their potential energy and is related to the electrostatic repulsion force of the head groups and the van der Waals attractive force of the acyl chains. In this way, by sonication, a small size (z 50 nm) unilamellar vesicle of DMPC is formed, which also exhibits a very broad transition peak shown in Fig. Id [21]. However, in contrast to the DMPG vesicle, the DMPC sonicated vesicle shows a low stability, so that by annealing at the gel temperature the vesicle transforms to the large size, multilamellar vesicles [21]. Considering the electrostatic attractive force operating between the PC head groups, the DMPC sonicated vesicle is revealed to be in an enthalpically unstabilized state, in which the PC head groups are very loosely packed beyond their geometrical size. Therefore, the PC head groups move to a state of less loose packing and a less curved surface, approaching the large size, multilamellar vesicle and simultaneously accompanied by a cooperative movement of the acyl chains. This is evidenced by a large difference between the transition enthalpies of the sonicated (4.6 kcal mol- ‘) and large multilamellar vesicles (6.7 kcal mol-‘) [21]. However, similar to the DMPC sonicated vesicle, the DMPG vesicles prepared in the presence of Na cation characterized by the thermotropic behaviors shown in Fig. 5 greatly change their structures when annealed at the gel temperature [22]. Focusing on the pretransition observed for the DMPC and DMPG vesicles shown in Figs. la, lc and Id, the acyl chains of these lipids are revealed to adopt a tilted form at the gel temperature [4,6,23], so as to revise their unfavorable, loose packing caused by the areas of their head groups being larger than that of the acyl chains (37-40 A’) [3]. Furthermore, as shown in Fig. 5, the pretransition of DMPG is found to persist, even in the presence of a Na’ concentration of 1 M. This result indicates that even at such a high Na+ concentration, the packing of PG head groups is not close enough to permit the acyl-chain packing perpendicular to the bilayer surface [6,18]. Thus, the larger area (~48 A’) [ 181 of the PG head group than that predicted at the high Naf concentration suggests a non-specific screening effect of Na cation on the charged surface based on a diffuse
double layer [ 71, in contrast with Ca cation which shows a specific binding to the charged head groups [24]. The lack of a pretransition for the DMPE assembly shown in Fig. lb indicates the perpendicular form of the acyl chains to the bilayer surface [3,25], which is related to a close packing of the PE head groups as evidenced by the planar bilayer surface shown by the micrograph of Fig. 1b. In fact, the area of the PE head group is nearly the same as that of the acyl chains [ 31, in contrast with PC and PG. Additionally, the close packing of PE is presumed to be associated with the intermolecular hydrogen bonds formed between amino and phosphate groups of the adjacent head groups in the intrabilayer [S], which is reflected in the fairly high transition temperature shown in Fig. lb. In connection with the lipid intermolecular hydrogen bonding, when DMPE is suspended at temperatures below the gel to liquid crystal transition, the lipid assembly thus obtained shows a crystalline state composed of closely stacked bilayers and transforms into the liquid crystal phase at a temperature higher than that of the assembly prepared above the gel to liquid crystal transition [25,26]. A similar temperaturedependent phenomenon is observed for the DMPG vesicles prepared in the presence of Na cation above 50 mM [22]. On this basis, it is suggested that when prepared below the gel to liquid crystal transition, these lipids form intermolecular hydrogen bonds between the head groups opposite each other in the interbilayer [ 22,261. Accordingly, in the case of DMPG, we could consider a specific binding of the Na cation to the charged head group, by which its negative charge is sealed to a degree high enough to form intermolecular hydrogen bonding. The details will be discussed in a subsequent paper.
References [l] [2]
R. Harrison and edn., Blackie and G.G. Shipley, in Research, Vol. 4,
G.G. Lunt, Biological Membranes, 2nd Son Ltd., London, 1980, p. 68. D.M. Small (Ed.), Handbook of Lipid Plenum Press, New York, 1986, p. 97.
M. Koduma. T. MiyatalColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289 [3]
[4] [S] [6] [7] [8] [9] [lo] [ 111 [ 121 [ 131 [ 141 [15]
H. Hauser, I. Pascher, R.H. Pearson and S. Sundell, Biochim. Biophys. Acta, 650 (1981) 21. D.A. Wilkinson, D.A. Tirrell, A.B. Turek and T.J. McIntosh, Biochim. Biophys. Acta, 905 (1987) 447. R.M. Epand and SW. Hui, FEBS Lett., 209 (1986) 257. F. Jahnig, K. Harlos, H. Vogel and H. Eibl, Biochemistry, 18 (1979) 1459. H. Trauble, M. Teubner, P. Woolley and H. Eibl, Biophys. Chem., 4 (1976) 319. J.M. Boggs, Biochim. Biophys. Acta, 906 (1987) 353. H. Hauser, Biochem. Biophys. Res. Commun., 45 (1971) 1049. G.V. Marinetti, J. Lipid Res., 3 (1962) 1. A.D. Bangham, M.W. Hill and N.G. Miller, Methods Membr. Biol., 1 (1974) 1. F. Jr. Szoka and D. Papahadjopoulos, Ann. Rev. Biophys. Bioeng., 9 (1980) 467. C. Huang, Biochemistry, 8 (1969) 344. S.M. Johnson, A.D. Bangham, M.W. Hill and E.D. Korn, Biochim. Biophys. Acta, 233 (1971) 820. S.E. Schullery, CF. Schmidt, P. Felgner, T.W. Tillack and T.E. Thompson, Biochemistry, 19 (1980) 3919.
289
[ 163 I. Pascher, S. Sundell, K. Harlos and H. Eibl, Biochim. Biophys. Acta, 896 (1987) 77. [ 171 H. Hauser, Biochim. Biophys. Acta, 772 (1984) 37. [18] A. Watts, K. Harlos and D. Marsh, Biochim. Biophys. Acta, 645 (1981) 91. [19] M. Kodama and T. Miyata, Thermochim. Acta, 267 (1995) 365. [20] M. Kodama and T. Miyata, Biochim. Biophys. Acta, to be submitted. [21] M. Kodama, T. Miyata and Y. Takaichi, Biochim. Biophys. Acta, 1169 (1993) 90. [22] M. Kodama, T. Miyata and T. Yokoyama, Biochim. Biophys. Acta, 1168 (1993) 243. [23] T.J. McIntosh, Biophys. J., 29 (1980) 237. [24] J.M. Boggs and G. Rangaraj, Biochemistry, 22 (1983) 5425. [25] D.A. Wilkinson and J.F. Nagle, Biochemistry, 23 (1984) 1538. [26] M. Kodama, H. Inoue and Y. Tsuchida, Thermochim. Acta, 266 (1995) 373.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 109 (1996) 283-289
Effect of the head group of phospholipids on the acyl-chain packing and structure of their assemblies as revealed by microcalorimetry and electron microscopy Michiko Kodama *, Takahiro Miyata Department of Biochemistry,
Faculty of Science, Okayama University of Science, 1-l Ridai-cho, Received 23 August 1995; accepted
8 September
Okayama 700, Japan
1995
Abstract By comparing three phospholipids with different head groups, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the effect of the head group on the acyl-chain packing and structure of the lipid assembly was investigated by high sensitivity differential scanning calorimetry and negative stain electron microscopy. The surface curvatures of the three assemblies were revealed to increase in the order PE < PC < PG, that is, in the order of an increase in the areas of the head groups relative to the area of the acyl chains. In accordance with this difference, the acyl-chain packing in the gel phase also differed greatly among the three assemblies, which was reflected in thermotropic behavior such as a very broad peak, a high temperature for the gel to liquid crystal transition and
lack of a pretransition. Keywords:
DMPC;
DMPE;
DMPG;
Electron
microscopy;
1. Introduction
Phospholipids are major constituents of fundamental bilayer structure in biomembranes and a great variety of phospholipids are present in biomembranes [ 1,2]. The phospholipids are generally classified according to their polar head groups consisting of a phosphate acidic group and other groups attached to its common group, such as phosphatidyl-choline (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). However, a specific composition of the lipid head groups is observed in different types of biomembranes [ 11. This suggests specific lateral interaction and packing of each lipid caused by different * Corresponding author. Tel: (+81) 86 252 3161; Fax: (+81) 86 255 7700. 0927-7757/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI
0927-7757(95)03461-7
Microcalorimetry
types of head groups in the bilayer structure. Thus, the head group is shown to be a predominant factor in determining the specificity of the bilayer structure [2,3]. From this viewpoint, the present paper will discuss how the packing of the lipid acyl chain and the structure of the lipid assembly are changed by alteration in the types of head groups such as PC, PE and PG. Both PC and PE, major phospholipid components of most biomembranes, are known to be present at zwitterions at neutral pH, indicating electrostatic attractions operating between the adjacent head groups at the bilayer surface. However, the geometrical sizes of the two head groups differ significantly [ 31. PG, a widely occurring phospholipid in chloroplast and mitochondrial membranes and bacteria, is negatively charged at neutral pH [4]. As for the charged PG,
284
M. Kodama, T MiyataJColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
a contribution from electrostatic repulsion operating between the adjacent head groups should be considered [ 5-73. Additional attention should be paid to PE which interacts intermolecularly by forming hydrogen bonds [S]. In this study, thermotropic properties of the lipid assemblies were investigated by differential scanning calorimetry and their structural information was obtained by negative stain electron microscopy.
2.3. High sensitivity diferential
2. Experimental
2.4. Electron Microscopy
2.1. Materials 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE) and 1,2-dimyristoylsn-glycero-3-[phospho-rat-( l-glycerol)] (DMPG, sodium salt) were purchased from Sigma and used without further purification. Each lipid yielded a single spot in thin-layer chromatography on a silica gel plate (E. Merck) using chloroform/ methanol/7 M ammonia (230 : 90 : 15, v/v/v) [ 93. Lipid concentrations were estimated by a modified Bartlett phosphate assay [lo]. 2.2. Preparation
scanning
calorimetry
All calorimetric experiments were performed with a Microcal MC2 differential scanning calorimeter interfaced to an IBM PC microcomputer system using an A/D converter board (Data Translation DT-2801) for automatic data collection and analysis. The lipid concentrations in the calorimetric experiments were in the range l-2 mM with a calorimetric cell volume of 1.2 ml. A heating scanning rate of 45 “C h- ’ was used.
Preparations of the lipid assemblies were examined by negative stain electron microscopy [ 13-151. The preparations were prepared at temperatures of 5-10°C as follows: a drop of dispersion (lipid concentration: ~2 mmol ml-‘) was placed on a copper grid covered with a carboncoated collodion film, allowed to remain for 5 min, and then drained. A 2% solution of sodium phosphotungstate (pH z 7) was added and after 10 min the excess solution was drained. The preparation was examined immediately in a JEOL JEM-2000EX electron microscope operated at 200 kV. All operations were performed at z 20’ C.
of lipid assemblies
3. Results and discussion Dispersions of lipid assemblies composed of each of DMPC, DMPE and DMPG were prepared according to a Bangham method for the preparation of a vesicle as follows [ 11,121. The lipid films were first prepared by removing chloroform from lipid stock solutions on a rotary evaporator, and then under high vacuum (lop4 Pa) to achieve complete removal of traces of the solvent. The dried lipid films were then suspended in distilled water, and gently vortexed at the required temperatures above the gel to liquid crystal transition, unless otherwise specified. In order to investigate the effect of Na cation, additional DMPG assemblies were prepared by suspending the dried lipid films in aqueous solutions of NaCl at different concentrations up to 1 M in the same way as mentioned above.
3.1. EfSect of the areas occupied by the head groups on the bilayer surface curvature
Some phospholipids exhibit phase transitions depending on whether the lipid assemblies are prepared at temperatures above or below the gel to liquid crystal transition temperature. The present discussion is limited to the lipid assemblies prepared at the liquid crystal temperatures. Thermotropic behaviors of the three dispersions of DMPC, DMPE and DMPG prepared in the absence of salt are shown in Figs. la, lb and lc respectively, where excess heat capacity (AC,) per mole of lipid is plotted against temperature (t). Negative stain electron micrographs of the three lipid assemblies are shown in Figs. 2a, 2b and 2c
M. Kodama, T. MiyataJColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
-
-1
I
1 kcal K-‘mot-’
a
-b
d 10
20
30
40
50
60
t 1°C Fig. 1. Thermotropic behaviors of the lipid assemblies (a) DMPC, (b) DMPE and (c) DMPG prepared at temperatures above the gel to liquid crystal transition and in the absence salt. The behavior of the DMPC sonicated vesicle is shown part (d). Apparent excess heat capacity (AC,) is plotted as function of temperature (t).
of of in a
respectively. The thermotropic behaviors of the three lipids differ greatly, as shown in Fig. 1, which is in accordance with the quite different structures of these lipid assemblies shown in Fig. 2. A comparison of these thermotropic behaviors produces the following results: (1) DMPG shows a noticeably broad peak due to the transition of the gel to liquid crystal phases, in contrast to the highly cooperative transitions of DMPC and DMPE; (2) DMPE shows a fairly high transition temperature of the gel to liquid crystal phases, compared to those of DMPC and DMPG; and (3) no peak other than the transition peak of the gel to liquid crystal is observed for DMPE, indicating the lack of a pretransition, in contrast with DMPC and DMPG. In contrast, the micrographs of Fig. 2 give the following profiles of these lipid assemblies: ( 1) DMPC is present as large multilamellar vesicles (> 500 nm diameter), (2) DMPG, in contrast, is present as unilamellar vesicles of a nearly constant, small size ( z approximately 100 nm diameter); and
285
(3) DMPE shows a planar bilayer surface different from the curved surfaces of DMPC and DMPG. On the basis of the microscopic structural information, we first discuss lateral packings of the three head groups. Fig. 3 schematically illustrates lipid-packing adaptations to differences in the cross-sectional areas occupied by lipid head groups under the same acyl-chain conditions. In Fig. 3, a decrease in the areas of the head groups relative to the acyl chains in the order (A)>(B)>(C) is shown to cause a decrease in the surface curvatures of lipid bilayers from highly curved to planar surfaces, simultaneous with an increase in the size of the lipid assemblies. By reference to information obtained from X-ray crystallographic study [3,16], the geometrical sizes of the three head groups are revealed to increase in the order PE (R 38 A’) < PG( ~44 A’) < PC( x 50 A*). The size of the negatively charged PG, which swells fully in excess water in the absence of salt [ 171, should be evaluated by the effective area, that is, the geometrical size plus a space due to electrostatic repulsion operating between the neighboring head groups at the bilayer surface [S,lS]. Considering the repulsion space, the area occupied by the PG head group is assumed to be the largest amongst the three head groups. This accounts well for the microscopic result shown in Fig. 2 where the surface curvatures of the three assemblies increase in the order DMPE
286
M. Kodama. T. MiyatalColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
Fig. 2. Negative stain electron micrographs of the lipid assemblies of (a) DMPC, (b) DMPE and (c) DMPG characterized by the thermotropic behaviors of Figs. la, lb and lc respectively. The micrograph of the DMPG assembly prepared in the presence of Na+ at 100 mM concentration is shown in part (d).
W
0 a
I
Fig. 3. Schematic illustration of lipid-packing adaptations to differences in cross-sectional areas occupied by the polar head groups under the same acyl-chain conditions. The areas of the head groups (a) relative to the acyl chains (b=constant) increase in the order (C) c(B) <(A).
face curvature as shown in Fig. 3. Typical thermotropic behavior and a micrograph of the DMPG vesicles prepared in the presence of Na cation are presented in Fig. 5 and Fig. 2d respectively.
3.2. Effect of the head groups on the acyl-chain packing of the gel phase The generally accepted concept for the lipid phase transition of the gel to liquid crystal is a conformational change in the acyl chain from the solid-like to liquid-like states. Accordingly, it is suggested that the packing between the acyl chains of neighboring lipids is close and loose in the gel and liquid crystal phases respectively. In such a situation, the acyl-chain packing of the gel phase could be influenced by the head groups more greatly than that of the liquid crystal phase. From this viewpoint, the different transition behaviors of the three lipids shown in Figs. la, lb, and lc are considered to result from different packing modes of the acyl chains in the gel phase. Therefore, we next discuss the effect of the head groups on the acyl-chain packing of the gel phase, by reference
281
M. Kodama, T. MiyatalColloids Surfaces A: Physicochem. Eng. Aspects 109 (1996) 283-289
d 0
20
40
60
80 100 120
[Na+]/mM Fig. 4. Effect of Na+ concentration on the approximate mean diameter (A) and the number of bilayers (B) of the DMPG vesicle.
to the transition behaviors shown in Fig. 1. Considering a uniform size of DMPG unilamellar vesicles (see Fig. 2c) [ 191, the marked broadening of the transition peak of DMPG shown in Fig. lc is presumed to be related to the highly curved surfaces in both outer and inner membranes of the lipid bilayer. That is, the acyl chains of the outer membrane are caused to adopt a loose packing corresponding to the highly convex curved surface. Similarly, the acyl chains of the inner membrane are forced to pack much more loosely in a reversed micellar form according to the highly concave surface curvature. Such loose packings of the acyl chains of the outer and inner membranes in the gel phase cause their heterogeneous motions and lateral interactions, resulting in a low cooperativity of the phase transition and the marked broadening of the transition peak for the DMPG vesicle.
I
10
I
I
20
,
I
30
I
40
t /“c Fig. 5. Effect of Na+ concentration on the thermotropic behavior of the DMPG vesicle. Apparent excess heat capacity (AC,) is plotted as a function of temperature (t). Na+ concentration (mM): (a) 50; (b) 100; (c) 250; (d) 1000.
However, as shown in Fig. 5, the transition peaks of DMPG in the presence of Na cation become sharper with an increase in Na concentration, indicating an increase in the cooperativity of the transition [ 191. This suggests a change of the acylchain packing into a less loose aggregation, in accordance with a less loose packing of the PG head groups caused by the screening effect of the Na cation mentioned above. Once again, focusing on the DMPG vesicle prepared in the absence of salt, it is noted that the vesicle, though heterogeneous in the acyl chain motions, exhibits a high stability at the gel temperature; that is, its structure is maintained during a period of annealing of over 1 month at this temperature and no change is observed in the transition behavior [ 201. This suggests that the loose packing
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of the DMPG lipids in the vesicle is just a way of minimizing their potential energy and is related to the electrostatic repulsion force of the head groups and the van der Waals attractive force of the acyl chains. In this way, by sonication, a small size (z 50 nm) unilamellar vesicle of DMPC is formed, which also exhibits a very broad transition peak shown in Fig. Id [21]. However, in contrast to the DMPG vesicle, the DMPC sonicated vesicle shows a low stability, so that by annealing at the gel temperature the vesicle transforms to the large size, multilamellar vesicles [21]. Considering the electrostatic attractive force operating between the PC head groups, the DMPC sonicated vesicle is revealed to be in an enthalpically unstabilized state, in which the PC head groups are very loosely packed beyond their geometrical size. Therefore, the PC head groups move to a state of less loose packing and a less curved surface, approaching the large size, multilamellar vesicle and simultaneously accompanied by a cooperative movement of the acyl chains. This is evidenced by a large difference between the transition enthalpies of the sonicated (4.6 kcal mol- ‘) and large multilamellar vesicles (6.7 kcal mol-‘) [21]. However, similar to the DMPC sonicated vesicle, the DMPG vesicles prepared in the presence of Na cation characterized by the thermotropic behaviors shown in Fig. 5 greatly change their structures when annealed at the gel temperature [22]. Focusing on the pretransition observed for the DMPC and DMPG vesicles shown in Figs. la, lc and Id, the acyl chains of these lipids are revealed to adopt a tilted form at the gel temperature [4,6,23], so as to revise their unfavorable, loose packing caused by the areas of their head groups being larger than that of the acyl chains (37-40 A’) [3]. Furthermore, as shown in Fig. 5, the pretransition of DMPG is found to persist, even in the presence of a Na’ concentration of 1 M. This result indicates that even at such a high Na+ concentration, the packing of PG head groups is not close enough to permit the acyl-chain packing perpendicular to the bilayer surface [6,18]. Thus, the larger area (~48 A’) [ 181 of the PG head group than that predicted at the high Naf concentration suggests a non-specific screening effect of Na cation on the charged surface based on a diffuse
double layer [ 71, in contrast with Ca cation which shows a specific binding to the charged head groups [24]. The lack of a pretransition for the DMPE assembly shown in Fig. lb indicates the perpendicular form of the acyl chains to the bilayer surface [3,25], which is related to a close packing of the PE head groups as evidenced by the planar bilayer surface shown by the micrograph of Fig. 1b. In fact, the area of the PE head group is nearly the same as that of the acyl chains [ 31, in contrast with PC and PG. Additionally, the close packing of PE is presumed to be associated with the intermolecular hydrogen bonds formed between amino and phosphate groups of the adjacent head groups in the intrabilayer [S], which is reflected in the fairly high transition temperature shown in Fig. lb. In connection with the lipid intermolecular hydrogen bonding, when DMPE is suspended at temperatures below the gel to liquid crystal transition, the lipid assembly thus obtained shows a crystalline state composed of closely stacked bilayers and transforms into the liquid crystal phase at a temperature higher than that of the assembly prepared above the gel to liquid crystal transition [25,26]. A similar temperaturedependent phenomenon is observed for the DMPG vesicles prepared in the presence of Na cation above 50 mM [22]. On this basis, it is suggested that when prepared below the gel to liquid crystal transition, these lipids form intermolecular hydrogen bonds between the head groups opposite each other in the interbilayer [ 22,261. Accordingly, in the case of DMPG, we could consider a specific binding of the Na cation to the charged head group, by which its negative charge is sealed to a degree high enough to form intermolecular hydrogen bonding. The details will be discussed in a subsequent paper.
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