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Phase behavior of hydrated lipid bilayer composed of binary mixture of phospholipids with different head groups
Chemistry and Physics of Lipids 100 (1999) 139 – 150 www.elsevier.com/locate/chemphyslip
Phase behavior of hydrated lipid bilayer composed of binary mixture of phospholipids with different head groups Tohru Inoue *, Yoshinori Nibu Department of Chemistry, Faculty of Science, Fukuoka Uni6ersity, Nanakuma, Jonan-ku, Fukuoka 814 -0180, Japan Received 27 January 1999; accepted 17 May 1999
Abstract Phase behavior of hydrated lipid bilayer was investigated for the mixtures of two phospholipid species chosen from phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) with the same acyl chains. The pseudo-binary phase diagrams constructed by a differential scanning calorimetry (DSC) were analyzed based on a thermodynamic model applying the Bragg – Williams approximation for non-ideality of mixing. The interchange energy parameters, r0, derived from this approach were positive for all mixture systems in both gel and liquid–crystalline phase bilayers, and increased in the order PG/PEB PC/PABPC/PEB PG/PA with a few exception. This suggests that the energetical disadvantage for the mixed-pair formation relative to the like-pair formation in the hydrated bilayer increases in this order. In addition, the r0 values increased with the increase in the acyl chain length of the phospholipids. These experimental results were discussed in terms of an intermolecular interaction of the phospholipid species in hydrated bilayer. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phospholipid mixture; Lipid bilayer; Bilayer phase transition; Phase diagram; Non-ideal mixing; Differential scanning calorimetry
1. Introduction Biomembranes are constituted of various kinds of lipids and proteins (Hauser and Poupart, 1992), and the physical state of phospholipids and the lipid composition of local regions of biomembranes is relevant to many biological functions of the membranes, such as enzyme function, ion
* Corresponding author. Tel.: + 81-92-8716631, ext. 6219; fax: + 81-92-8656030. E-mail address: [email protected] (T. Inoue)
transport, antibody stimulated cell lysis, and cell fusion (Bloom et al., 1991). Thus, the phase behavior of hydrated bilayer of phospholipid mixtures is a subject of fundamental importance in relation to membrane biophysics. From this point of view, a number of investigations have so far been reported concerning the phase behavior of hydrated bilayer of binary phospholipid mixtures (Do¨rfler et al., 1988, 1990a; Do¨rfler and Miethe, 1990; Marsh, 1990a; Wiedmann et al., 1993; Polozov et al., 1994; Johann et al., 1996; Rolland et al., 1997; Garidel et al., 1997; Garidel and Blume, 1998).
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The phase diagram becomes a basis to understand the phase behavior of mixture systems. The pseudo-binary phase diagram for hydrated twocomponent phospholipid mixture is governed by the miscibility of each lipid species in the bilayer of both gel and liquid – crystalline states. The miscibility is determined, as a first approximation, by the difference in the pair-interaction energies among the like-pair and mixed-pair formed in the mixture. Thus, the shape of phase boundaries provides information about an intermolecular interaction between phospholipid species in hydrated bilayers of respective phase states. It is expected that the systematic study for the phase behaviors of binary mixtures of various phospholipid species would be useful to elucidate the factors governing the miscibility of different phospholipid species in hydrated bilayers and the phase behavior of phospholipid mixtures. Based on the above consideration, we have been investigating the phase behavior of binary mixture systems of different phospholipid species (Inoue et al., 1992, 1999; Nibu and Inoue, 1995; Inoue and Nibu, 1995a; Nibu et al., 1995). In previous papers, we have reported mixtures of several types of phospholipid species having the same head group but different acyl chains. The present paper is concerned with mixtures composed of several combinations of two phospholipid species with the same acyl chain but different head groups. Two series experiments were made with respect to the acyl chain length, i.e. dipalmitoyl and distearoyl. The phase boundaries for hydrated bilayers of these phospholipid mixtures were determined by a differential scanning calorimetry. The obtained phase diagrams were analyzed based on a thermodynamic model which attributes the non-ideality of mixing or excess free energy of mixing to the difference in the pair-interaction energies among the like-pair and mixedpair. The purpose of the present study is to get information concerning the interaction between different head groups in hydrated lipid bilayer by examining the pseudo-binary phase diagram for various mixtures of phospholipids with different head groups.
2. Experimental
2.1. Materials The phospholipid species used in the present study were dipalmitoylphosphatidic acid (sodium salt; DPPA), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (sodium salt; DPPG), and their distearoyl analogs (DSPA, DSPC, DSPE, and DSPG). These phospholipids of synthetic origin were obtained from Sigma, and were used without further purification. The purities of the lipids were 99% or better than 99%.
2.2. Preparation of lipid mixtures The following four combination of binary lipid mixtures were examined for both dipalmitoyl and distearoyl series; PC/PA, PC/PE, PG/PA, and PG/PE. The lipid mixtures for DSC measurements were prepared by the following procedure. The appropriate amounts of two dry lipids were weighed into a glass cuvette to give a desired composition, to which a small amount of methanol/chloroform (1/4 in volume) mixed solvent was added to dissolve the lipid mixture. Then, the solvent was removed by gentle blowing of dry N2 gas, followed by evaporation under reduced pressure.
2.3. DSC measurements DSC measurements were performed using a Seiko Denshi Model SSC5200 (Tokyo, Japan). The calibration with respect to temperature and caloric was made by the use of gallium and indium. The dry lipid mixture of about 5 mg was weighed in a sealable sample pan made from aluminum, to which 20 ml of water was added, and then the pan was sealed. As a reference material, alumna was used, the weight of which was about 20 mg. The sample was held at 90°C for about 1 h in the oven of the DSC apparatus in order to assure homogeneous mixing of the lipid and water. Then, a cooling/heating cycle was repeated for several times at the rate of 2°C/min in
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the temperature range from 20 or 30 to 90°C. A good reproducibility was obtained for the DSC thermograms recorded by the repeated scan.
3. Results and discussion As examples of DSC thermograms obtained for the present mixture systems, Figs. 1 and 2 show those for DPPC/DPPE and DSPC/DSPE mixtures, respectively, at various compositions. The composition of these mixtures are expressed in terms of the mole fraction of DPPE, xDPPE, and DSPE, xDSPE. The endothermic peaks in these thermograms are ascribed to the gel (L%b)-to-liquid-crystalline (La) phase transition of the hydrated lipid bilayers (Small, 1986). The small
Fig. 2. DSC thermograms obtained for hydrated DSPC/DSPE mixture of various composition. The mole fraction of DSPE in the lipid mixture is indicated in the figure.
Fig. 1. DSC thermograms obtained for hydrated DPPC/DPPE mixture of various composition. The composition expressed in terms of the mole fraction of DPPE in the lipid mixture, xDPPE, is indicated in the figure.
endotherms observed for pure DPPC and DSPC at lower temperature are so-called pre-transition, and are attributed to the phase transition from L %b to P %b (Small, 1986). We are concerned about the behavior of gel-to-liquid-crystalline phase transition with respect to the composition of the lipid mixtures. When PE is added to pure PC species, another endothermic peak appears at higher temperature (black arrows in Figs. 1 and 2) in addition to the endotherms corresponding to those for respective PC species (white arrows). With the increase in PE content in the mixture, this peak moves toward higher temperature concomitantly with the increase in its intensity. On the other hand, the lower temperature peaks reduce their intensity with the increase in PE content in the mixture. They are appreciable up to xPE : 0.5 for both
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DPPC/DPPE and DSPC/DSPE mixtures in the thermograms drawn in more expanded scale, although they are barely seen in Figs. 1 and 2. The peak temperatures in the DSC thermograms such as presented in Figs. 1 and 2 are regarded to define phase boundaries among the gel phase, liquid–crystalline phase, and their coexisting region in the hydrated bilayer of the lipid mixtures, and hence, a pseudo-binary phase diagram can be constructed by plotting these temperatures against the composition. Fig. 3 shows the phase diagrams thus obtained for four dipalmitoyl series mixture systems, i.e. (a) DPPC/DPPA; (b) DPPC/DPPE; (c) DPPG/DPPA; and (d) DPPG/ DPPE. Corresponding phase diagrams for distearoyl series mixtures are shown in Fig. 4(a – d). In these phase diagrams, the composition is expressed by the mole fraction of the component with higher transition temperature of the two lipid species. It can be seen in these figures that the shape of the ‘solidus’ and ‘liquidus’ lines obtained by connecting the peak temperatures of the DSC thermograms is considerably different from that expected for ideal mixing of the two components. Phase behavior of DPPC/DPPE mixture has so far been investigated by many authors using various detection techniques for bilayer phase transition (Shimshick and McConnell, 1973; Lee, 1975; Sklar et al., 1977; Mendelsohn and Koch, 1980; Arnold et al., 1981; Do¨rfler et al., 1990b). The phase diagram obtained in the present work for this mixture system (Fig. 3b) is in fairly good agreement with those constructed from Raman spectroscopy (Mendelsohn and Koch, 1980), 31PNMR (Arnold et al., 1981), and DSC (Do¨rfler et al., 1990b). On the other hand, the phase diagrams obtained by spin label (Shimshick and McConnell, 1973) and fluorescence probe (Lee, 1975; Sklar et al., 1977) techniques are somewhat different from the above ones, particularly, in a solidus line around xDPPE =0.5, although they exhibit a good agreement within themselves. This disagreement between the two groups of detection techniques may be attributed to the existence of probe molecules in the latter cases; it is likely that these probe molecules have affected the phase behavior of the lipid mixture.
Other than DPPC/DPPE system, the phase diagram of DPPC/DPPA mixture at different pH has been recently reported by Blume and co-workers (Garidel et al., 1997). They have determined the onset and end temperatures of the bilayer phase transition of multilamellar liposomes suspended in excess water by simulating the heat-capacity curves obtained by DSC experiments. Comparing the present result for this mixture system (Fig. 3a) with the phase diagram at pH 7.0 reported by Garidel et al., the overall shape of the phase boundaries is similar to each other, although the width of the cigar-like shape is considerably broader in the present case. This difference in the temperature range of the coexisting region of gel and liquid–crystalline phases is attributed to the difference in the determination procedure of the phase boundaries, as described by Johann et al. (1996). It should be mentioned here about possible problems associated with the present experimental condition. In the present experiments, the hydrated phospholipid samples were prepared by adding pure water to dry lipids without using buffer solution; i.e. no pH control was made. This might cause some problems for the case of acidic phospholipids, PA and PG, since the protonation state of their head groups is affected by pH of excess water phase [for PA, pKa(PO4): 3.5 and 9.0, and for PG, pKa(PO4): 3 (Marsh, 1990b)]. In fact, the phase diagrams of DPPC/DPPA mixture obtained by controlling the pH at 7.0 and 4.0, respectively are quite different from each other (Garidel et al., 1997). The present phase diagram for the same mixture system is close to that obtained at pH 7.0 by Garidel et al., as mentioned above. In addition, the phase diagram of hydrated DMPA/DPPA mixture prepared by using pure water (Inoue and Nibu, 1995b) is similar to that obtained for the same mixture at pH 7.0 (Garidel and Blume, 1998). Furthermore, the thermodynamic data for pure DPPA and DPPG determined in the present work, which are listed in Table 1 together with those reported in literature, are almost in agreement with literature values obtained at neutral pH. Judging from these results, it may be safely assumed that the preparation procedure of hydrated lipid samples
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employed in the present study does not cause such a significant effect on the protonation state of the
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lipid head groups as to affect the phase behavior of the hydrated lipid bilayers, or in other words,
Fig. 3. Pseudo-binary phase diagrams of dipalmitoyl series lipid mixtures. The mixture systems are (a) DPPC/DPPA; (b) DPPC/DPPE; (c) DPPG/DPPA; and (d) DPPG/DPPE. Solid lines in these figures are phase boundaries calculated from Eqs. (1) and (2).
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Fig. 4. Pseudo-binary phase diagrams of distearoyl series lipid mixtures. The mixture systems are (a) DSPC/DSPA; (b) DSPC/DSPE; (c) DSPG/DSPA; and (d) DSPG/DSPE. Solid lines in these figures are phase boundaries calculated from Eqs. (1) and (2).
the preparation of hydrated lipid samples with pure water keeps the neutral pH of excess water phase. For PG/PE mixture systems, another problem
would arise due to the preparation of lipid samples with pure water. It has ported that the bilayer phase transition ture of dimyristoylphosphatidylglycerol
hydrated been retempera(DMPG)
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depends on the Na + concentration in the bulk water phase (Salonen et al., 1989; Kodama and Miyata, 1995). In the present experimental condition, the Na + concentration in excess water phase of PG/PE mixtures varies from 300 mM (xPG = 1) to 30 mM (xPG =0.1) in rough estimation. According to Kodama and Miyata, the main phase transition temperature of DMPG bilayer is lowered by approximately 1.5°C as the Na + concentration is decreased from 300 to 30 mM. If the effect of Na + concentration on the bilayer phase transition of DPPG and DSPG is similar to that of DMPG, the temperatures of phase boundaries of DPPG/DPPE and DSPG/ DSPE mixtures obtained in the present study would be slightly different from those expected for the case containing externally added Na + at high concentration. As described above, the phase diagrams presented in Figs. 3 and 4 exhibit a non-ideal nature of mixing of the two lipid species in both gel and liquid–crystalline phases. Several methods have so far been applied to simulate the binary phase diagrams for non-ideal lipid mixtures (Shimshick and McConnell, 1973; van Dijck et al., 1977; Lee, 1977; Mendelsohn and Koch, 1980; Davis and Keough, 1984; Tenchov, 1985; Koynova et al.,
1988; Brumbaugh et al., 1990; Johann et al., 1996). The most frequently used method utilizes the thermodynamic equations derived by considering the excess free energy of mixing. To evaluate the excess free energy of mixing, the Bragg– Williams approximation (Moore, 1972) may be valid for the lipid mixtures such as the present case, because the components in the mixture have a similarity in both molecular structure and molecular volume. Recently, Blume and co-workers developed a new approach which enables one to simulate the heat-capacity curves obtained by DSC measurements and phase diagram of pseudobinary phospholipid mixtures (Johann et al., 1996). In this approach, the composition dependence of the cooperativity of transition was incorporated to simulate the heat-capacity curves and to estimate the temperatures corresponding to the onset and the end of the transition. In addition, the four non-ideality parameters of mixing were used to simulate the phase diagrams taking into account the nonsymmetrical excess free energy with respect to composition of the mixture. We analyzed the phase diagrams presented in Figs. 3 and 4 in terms of the usual two-parameter Bragg– Williams approximation instead of the more refined four-parameter model proposed by Jo-
Table 1 Thermodynamic data for the main phase transition of the pure phospholipids This work
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Literature
References
Tm (°C)
DH (kJ/mol)
Tm (°C)
DH (kJ/mol)
DPPC DPPG
41.2 39.7
30.5 30.5
DPPA
63.2
29.3
DPPE DSPC DSPG DSPA DSPE
64.0 54.7 54.7 74.4 74.7
30.1 38.2 43.5 39.3 37.1
41.49 0.5 34.8 93.2 41.5 37.2 (pH 7) 41.5 36.8 (pH 7) 41 33.0 (pH 7) 39.5 38.1 (pH 7) 40.0 (pH 7, 0.1 M NaCl) 58 (pH 2, 0.1 M NaCl) 65.0 33.0 (pH 6) 67 22.0 (pH 6.5) 64.7 32.4 (pH 7) 65.4 28.8 (pH 4) 64.0 9 2.2 35.4 9 3.3 55.1 9 1.5 42.3 93.7 54.5 43.9
Marsh (1990c) Findlay and Barton (1978) Van Dijck et al. (1978) Jacobson and Papahadjopoulos (1975) Van Dijck (1979) Garidel and Blume (1998) Garidel and Blume (1998) Blume (1983) Jacobson and Papahadjopoulos (1975) Garidel et al. (1997) Garidel et al. (1997) Marsh (1990c) Marsh (1990c) Findlay and Barton (1978)
74.2 9 2.2
Marsh (1990c)
44.3 91.7
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hann et al. in order to facilitate the comparison with previous results reported for various phospholipid mixture systems. According to this treatment, the phase boundaries corresponding to the solidus and liquidus lines are given by solving the following simultaneous equations (Lee, 1977; Tenchov, 1985; Inoue et al., 1992). ln
2 (S) (S) 2 r (L)(1−x (L) x (L) A A ) −r 0 (1 − x A ) + 0 (S) xA RT
(1)
(2)
DHA 1 1 − T TA R (L) 2 (S) (S) 2 r (L) 1− x (L) A 0 (x A ) −r 0 (x A ) + ln 1− x (S) RT A
=−
=−
1 DHB 1 − R T TB
(S) where x (L) A and x A refer to the mole fractions of component A in liquid – crystalline and gel phase bilayers being in equilibrium with each other, TA and TB are the transition temperatures and DHA and DHB are the transition enthalpies for pure species denoted by the subscripts, and r (L) and 0 r (S) are parameters characterizing the non-ideal 0 behavior of mixing in liquid – crystalline and gel phases, respectively. r0 is given by
r0 = z uAB −
uAA +uBB 2
(3)
where z is the first coordination number and uAA, uBB, and uAB are the molar energies of A – A, B – B, and A–B pair interactions. The present experimental phase diagrams were analyzed by applying Eqs. (1) and (2) using a curve fitting procedure. The values of TA, TB, DHA, and DHB for pure lipid species, which are required for the calculation, are listed in Table 1. The non-ideality parameters r (L) and r (S) were 0 0 determined so as to obtain the calculated phase boundaries reproducing the experimental phase boundaries most satisfactorily. This was done by using a least-squares method as has been described elsewhere in some detail (Nibu and Inoue, 1995). The solid lines in Figs. 3 and 4 were drawn according to Eqs. (1) and (2) using the non-ideality parameters thus determined. The agreement between the experimental and computed phase boundaries is satisfactory, although it is rather
Table 2 Non-ideality parameters of mixing obtained for various binary mixtures of phospholipids with different head groups Mixture
(kJ/mol) r (L) 0
r (S) 0 (kJ/mol)
DP series PC/PA PC/PE PG/PA PG/PE
3.3 3.4 4.7 2.8
4.0 4.6 4.9 3.1
DS series PC/PA PC/PE PG/PA PG/PE
3.7 4.1 4.5 4.0
5.9 6.1 6.7 5.6
poor for the case of DSPG/DSPA mixture (Fig. 4c). The values of the non-ideality parameters of mixing obtained for various mixture systems are summarized in Table 2. The non-ideality parameter of mixing for DPPC/DPPE mixture system has been estimated by some authors based on the same thermodynamic approach as the present case. Arnold et al. have reported the r0 values as r (L) 0 = 3.8 kJ/mol and r (S) 0 = 5.4 kJ/mol by analyzing the phase diagram constructed from 31P-NMR (Arnold et al., 1981). According to Mendelsohn and Koch, (S) r (L) 0 = 3.1 kJ/mol and r 0 = 4.4 kJ/mol have been estimated for the mixture of deuterated DPPC and DPPE from the phase diagram obtained by Raman spectroscopy (Mendelsohn and Koch, 1980). The r0 values derived in the present study for DPPC/DPPE mixture (Table 2) are comparable to these reported values. The parameter, r0, is a measure of non-ideality of mixing, and serves to interpret the mixing behavior on the basis of intermolecular interaction energies. The ideal mixing corresponds to r0 = 0, i.e. identical intermolecular interaction energies for both mixed-pair and like-pair. The nonideality of mixing is reflected in the deviation of r0 from 0; the positive r0 means that the A–B pair formation in the mixture is energetically unfavorable compared with A–A and B–B pair formation, while the negative r0 corresponds to the situation that the A–B pair formation is more preferable than the A–A and B–B pair forma-
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tion. Thus, a large positive r0 tends to lead to a phase separation, while a large negative r0 tends to result in a regular arrangement of the two components in the mixture. Some general trends can be drawn from Table 2 regarding the dependence of r0 on various parameters, as described below. The r0 values are positive for all mixture systems in both gel and liquid–crystalline phases. This means that in hydrated bilayers of both phase states, the pair interaction for mixed-pair is repulsive relative to the corresponding like-pairs. Comparing the r0 values obtained for gel and liquid – crystalline bilayers, r (S) is greater than r (L) for all mixture 0 0 systems. This difference in r0 values between the two phase states may be readily understood by considering the solid-like nature of the gel phase and the liquid-like nature of the liquid – crystalline phase. It is usual that the miscibility of different species is extremely reduced in a solid state compared with a liquid state. In gel state bilayer, the lipid molecules with rigid hydrocarbon chain of all-trans conformation are forced to arrange within a definite crystalline-like lattice, whereas in liquid–crystalline state bilayer, the lipid molecules with flexible disordered hydrocarbon chain are loosely packed and can easily move laterally. Thus, the difference in pair-interaction energies between like-pair and mixed-pair may become more significant in gel state bilayer than in liquidcrystalline state bilayer, which results in highly non-ideal behavior of mixing in gel phase. The r0 values for different combination of two lipid species increases in the order PG/PE B PC/ PAB PC/PE B PG/PA commonly to dipalmitoyl (DP) and distearoyl (DS) series mixtures and also to gel and liquid–crystalline phases except for the case of r (L) of DS series, in which the order for 0 PG/PE and PC/PA is reversed in the above sequence. This variation of r0 is seen more clearly in Fig. 5 where the r0 values listed in Table 2 are represented graphically. This result for the order of r0 values for different phospholipid pairs suggests that the energetical disadvantage for the mixed-pair formation relative to corresponding like-pair formation in hydrated bilayer increases in this order. In the present experimental conditions, as described above, the head groups of PG
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and PA bear monovalent negative charge, while PC and PE are zwitterionic, and hence, have no net charge [pKa(PO4): 1 and pKa(NH3+ ) for PE: 10–11 (Marsh, 1990b)]. It might be considered that the largest r0 value for PG/PA mixture is attributed to the fact that the both head groups have a negative charge. However, the situation is more or less similar for both mixed-pair and like-pairs. The head group of PG has two hydroxyl groups, and thus, it is likely that the hydrogen bond formed among neighboring molecules in hydrated bilayer brings about an attractive (energetically favorable) contribution to the intermolecular interaction of PG species. When PA is added to PG, this network of hydrogen bond existing in pure PG bilayer would be disturbed, and hence, the formation of mixed-pair of PG and PA would become highly unfavorable compared with the formation of like-pair between themselves. The hydrogen bond network of hydrated PG bilayer is also disturbed by the addition of PE to PG. In this case, however, the repulsive electrostatic interaction for negatively charged PG/PG pair may be reduced significantly by forming the PG/PE pair because of the zwitterionic nature of PE species. This gain of interac-
Fig. 5. Dependence of the non-ideality parameter of mixing, r0, on the combination of phospholipid species with different head groups. Circles: dipalmitoyl (DP) series lipid mixtures; Squares: distearoyl (DS) series lipid mixtures. Filled and open symbols correspond to the gel and liquid-crystalline phase bilayers, respectively.
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tion energy due to the reduced electrostatic interaction would overcome the disadvantage of the destruction of hydrogen bond network, and hence, the excess energy for PG/PE mixed-pair would become minimal. As for the PC species, no such additional interaction as hydrogen bonding for PG is expected in hydrated bilayer. Therefore, the r0 values for the mixtures associated with PC would fall in between PG/PA and PG/PE mixtures. It can be seen in Table 2 or Fig. 5 that the r0 values obtained for DS series mixtures are larger than those for DP series mixtures in both gel and liquid–crystalline bilayers with an exception of liquid–crystalline bilayer of PG/PA mixture. This means that the effect of difference in head group type on the pair-interaction becomes more pronounced as the lipid acyl chain becomes longer. At a first glance, this trend seems somewhat strange. Because the relative contribution of head group to the overall intermolecular interaction would be reduced as the lipid acyl chain becomes long, the difference in the head group type among the mixed-pair and like-pairs is considered to become less striking with the increase in the acyl chain length. Thus, it may be expected that the r0 values are smaller for DS series mixtures than for DP series mixtures. The actually obtained tendency is, however, opposite. This may be interpreted as follows. The intermolecular separation of phospholipid molecules in hydrated bilayer depends on the lipid acyl chain length. Fig. 6 depicts the plot of S, the average area available to a head group on the hydrated bilayer, as a function of NC, the number of carbon atoms in the lipid acyl chain. These S values were compiled from the data reported for PC species with different acyl chain length (Tardieu et al., 1973; Janiak et al., 1976, 1979; Inoko and Mitsui, 1978; Lis et al., 1982). The values of S, and hence, the intermolecular distance is larger for liquid – crystalline (La) phase bilayer than for gel (L %b) phase bilayer, reflecting the looser packing of the lipid molecules in the former than the latter bilayer state. As for the chain length dependence, the intermolecular separation decreases with the increase in NC in both gel and liquid-crystalline bilayers. This may be interpreted as due to the enhanced cohesive
Fig. 6. Plot of the average area available to a head group on the hydrated bilayer, S, against the number of carbon atoms in the acyl chain, NC, for phosphatidylcholines with two identical saturated acyl chains. Circles and squares correspond to the gel (L %b) and liquid-crystalline (L %a) phase bilayers. The data were taken from literature (Tardieu et al., 1973; Janiak et al., 1976, 1979; Inoko and Mitsui, 1978; Lis et al., 1982).
force caused by the increase in the hydrocarbon chain length. When the separation distance between head groups is shortened, their interaction becomes strong, and hence, the difference in the interaction energy between mixed-pair and likepairs would be intensified. It is likely that the larger r0 values for DS series mixtures than for DP series mixtures is attributed to the shorter separation distance or tighter packing of lipid molecules in hydrated bilayers for DS series mixtures. The above consideration regarding the dependence of r0 on the lipid acyl chain length predicts that the r0 values for dimyristoyl (DM) series mixtures would be smaller that those for DP series mixtures. Actually, this can be recognized in the phase diagram for DMPC/DMPE mixture reported by Chapman et al. (1974) and Polozov et al. (1994). The phase boundaries obtained from DSC experiments for DMPC/DMPE mixture by these authors are closer to a cigar-like shape than those for DPPC/DPPE mixture are. This indicates that the r0 values for DMPC/DMPE mixture is smaller than those for DPPC/DPPE mixture.
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Additional noticeable feature in Table 2 or Fig. 5 is that the difference in r0 between gel and liquid–crystalline bilayers is much larger for DS series mixtures than for DP series mixtures. In the latter case, the difference is not so significant except for PC/PE mixture. A remarkable difference between r (S) and r (L) for DS mixtures is 0 0 ascribed to the extremely large r (S) values, since 0 between the two series mixthe difference in r (L) 0 tures is rather small. Thus, it is regarded that the energetical disadvantage for the mixed-pair formation is enhanced by the increase in acyl chain length to a greater extent in gel phase bilayer than in liquid–crystalline bilayer. This may be explained again, at least in part, based on the chain length dependence of the intermolecular separation of phospholipids in gel and liquid – crystalline phase bilayers. As is seen in Fig. 6, the variation of S with NC is rather parallel for both gel and liquid–crystalline phases. However, the relative decrease, or decrement, of intermolecular separation with the increase in NC becomes larger for gel phase bilayer than for liquid – crystalline bilayer; on going from NC =16 to NC =18, the separation distance decreases by nearly 5.7 and 3.9% for gel and liquid–crystalline bilayers, respectively. Thus, a mixed-pair formation in gel state bilayer would become more unfavorable than that in liquid – crystalline bilayer when the length of the lipid acyl chain is increased.
Acknowledgements This work was supported in part by funds from the Central Research Institute of Fukuoka University.
References Arnold, K., Lo¨sche, A., Gawrisch, K., 1981. 31P-NMR investigations of phase separation in phosphatidylcholine/phosphatidylethanolamine mixtures. Biochim. Biophys. Acta 645, 143 – 148. Bloom, M., Evans, E., Mouritsen, O.G., 1991. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q. Rev. Biophys. 24, 293–397.
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Blume, A., 1983. Apparent molar heat capacities of phospholipids in aqueous dispersion. Effects of chain length and head group structure. Biochemistry 22, 5436 – 5442. Brumbaugh, E.E., Johnson, M.L., Huang, C.-H., 1990. Nonlinear least squares analysis of phase diagrams for nonideal binary mixtures of phospholipids. Chem. Phys. Lipids 52, 69 – 78. Chapman, D., Urbina, J., Keough, K.M., 1974. Biomembrane phase transitions. Studies of lipid – water systems using differential scanning calorimetry. J. Biol. Chem. 249, 2512 – 2521. Davis, P.J., Keough, K.M.W., 1984. Phase diagrams of bilayers of dimyristoyl lecithin plus heteroacid lecithins. Chem. Phys. Lipids 35, 299 – 308. Do¨rfler, H.D., Brezesinski, G., Miethe, P., 1988. Phase diagrams of pseudo-binary phospholipid systems I. Influence of the chain length differences on the miscibility properties of cephaline/cephaline/water systems. Chem. Phys. Lipids 48, 245 – 254. Do¨rfler, H.D., Miethe, P., 1990. Phase diagrams of pseudo-binary phospholipid systems II. Selected calorimetric studies on the influence of branching on the mixing properties of phosphatidylcholines. Chem. Phys. Lipids 54, 61 – 66. Do¨rfler, H.D., Miethe, P., Mo¨ps, A., 1990a. Phase diagrams of pseudo-binary phospholipid systems III. Influence of the head group methylation on the miscibility behavior of N-methylated phosphatidylethanolamine mixtures in aqueous dispersion. Chem. Phys. Lipids 54, 171 – 179. Do¨rfler, H.-D., Miethe, P., Meyer, H.W., 1990b. Phase diagrams of pseudo-binary phospholipid systems IV. Preliminary results about the effects of LiCl and CaCl2 on the phase transitions of amphoteric phospholipids in aqueous dispersions. Chem. Phys. Lipids 54, 181 – 192. Findlay, E.J., Barton, P.G., 1978. Phase behavior of synthetic phosphatidylglycerols and binary mixtures with phosphatidylcholines in the presence and absence of calcium ions. Biochemistry 17, 2400 – 2405. Garidel, P., Johann, C., Blume, A., 1997. Nonideal mixing and phase separation in phosphatidylcholine-phosphatidic acid mixtures as a function of acyl chain length and pH. Biophys. J. 72, 2196 – 2210. Garidel, P., Blume, A., 1998. Miscibility of phospholipids with identical headgroups and acyl chain lengths differing by two methylene units: effect of headgroup structure and headgroup charge. Biochim. Biophys. Acta 1371, 83 – 95. Hauser, H., Poupart, G., 1992. Lipid structure. In: Yeagle, P. (Ed.), The Structure of Biological Membrane. CRC Press, London, pp. 3 – 72. Inoko, Y., Mitsui, T., 1978. Structural parameters of dipalmitoylphosphatidylcholine lamellar phases and bilayer phase transition. J. Phys. Soc. Jpn. 44, 1918 – 1924. Inoue, T., Tasaka, T., Shimozawa, R., 1992. Miscibility of binary phospholipid mixtures under hydrated and unhydrated conditions. I. Phosphatidic acids with different acyl chain length. Chem. Phys. Lipids 63, 203 – 212. Inoue, T., Nibu, Y., 1995a. Miscibility of binary phospholipid mixtures under hydrated and non-hydrated conditions. III.
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Reinvestigation on phosphatidic acids with different acyl chain length. Chem. Phys. Lipids 76, 171–179. Inoue, T., Nibu, Y., 1995b. Miscibility of binary phospholipid mixtures under hydrated and non-hydrated conditions. IV. Phosphatidylglycerols with different acyl chain length. Chem. Phys. Lipids 76, 181–191. Inoue, T., Kitahashi, T., Nibu, Y., 1999. Phase behavior of hydrated bilayer of binary phospholipid mixtures composed of 1,2-distearoylphosphatidylcholine and 1-stearoyl2-oleoylphosphatidylcholine or 1-oleoyl-2-stearoylphosphatidylcholine. Chem. Phys. Lipids 99, 103 – 109. Jacobson, K., Papahadjopoulos, D., 1975. Phase transitions and phase separations in phospholipid membranes induced by changes in temperature, pH, and concentration of bivalent cations. Biochemistry 14, 152–161. Janiak, M.J., Small, D.M., Shipley, G.G., 1976. Nature of the thermal pretransition of synthetic phospholipids: dimyristoyl- and dipalmitoyllecithin. Biochemistry 15, 4575–4580. Janiak, M.J., Small, D.M., Shipley, G.G., 1979. Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin. J. Biol. Chem. 254, 6068–6078. Johann, C., Garidel, P., Mennicke, L., Blume, A., 1996. New approaches to the simulation of heat-capacity curves and phase diagrams of pseudobinary phospholipid mixtures. Biophys. J. 71, 3215 –3228. Kodama, M., Miyata, T., 1995. Effect of Na + concentration on both size and multiplicity of multilamellar vesicles composed of negatively charged phospholipid as revealed by differential scanning calorimetry and electron microscopy. Thermochim. Acta 267, 365–372. Koynova, R.D., Kuttenreich, H.L., Tenchov, B.G., Hinz, H.-J., 1988. Influence of head-group interactions on the miscibility of synthetic, stereochemically pure glycolipids and phospholipids. Biochemistry 27, 4612–4619. Lee, A.G., 1975. Fluorescence studies of chlorophyll a incorporated into lipid mixtures, and the interpretation of ‘phase’ diagrams. Biochim. Biophys. Acta 413, 11–23. Lee, A.G., 1977. Lipid phase transitions and phase diagrams II. Mixtures involving lipids. Biochim. Biophys. Acta 472, 285 – 344. Lis, L.J., McAlister, M., Fuller, N., Rand, R.P., 1982. Interactions between neutral phospholipid bilayer membranes. Biophs. J. 37, 657 – 666. Marsh, D., 1990a. Handbook of Lipid Bilayers. CRC Press, Boston, pp. 229 – 263. Marsh, D., 1990b. Handbook of Lipid Bilayers. CRC Press, Boston, pp. 81 – 85. Marsh, D., 1990c. Handbook of Lipid Bilayers. CRC Press, Boston, pp. 135 – 158. Mendelsohn, R., Koch, C.C., 1980. Deuterated phospholipids as Raman spectroscopic probes of membrane structure.
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Biochim. Biophys. Acta 598, 260 – 271. Moore, W.J., 1972. Physical Chemistry. Prentice-Hall, Englewood Cliffs, New Jersey, pp. 229 – 278. Nibu, Y., Inoue, T., 1995. Miscibility of binary phospholipid mixtures under hydrated and non-hydrated conditions. II. Phosphatidylethanolamines with different acyl chain length. Chem. Phys. Lipids 76, 159 – 169. Nibu, Y., Inoue, T., Motoda, I., 1995. Effect of headgroup type on the miscibility of homologous phospholipids with different acyl chain lengths in hydrated bilayer. Biophys. Chem. 56, 273 – 280. Polozov, I.V., Molotkovsky, J.G., Bergelson, L.D., 1994. Anthrylvinyl-labeled phospholipids as membrane probes: the phosphatidylcholine– phosphatidylethanolamine system. Chem. Phys. Lipids 69, 209 – 218. Rolland, J.-P., Santaella, C., Monasse, B., Vierling, P., 1997. Miscibility of binary mixtures of highly fluorinated doublechain glycerophosphocholines and 1,2-dipalmitoylphosphatidylcholine (DPPC). Chem. Phys. Lipids 85, 135 – 143. Salonen, I.K., Eklund, K.K., Virtanen, J.A., Kinnunen, K.J., 1989. Comparison of the effects of NaCl on the thermotropic behavior of sn-1% and sn-3% stereoisomers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol. Biochim. Biophys. Acta 982, 205 – 215. Shimshick, E.J., McConnell, H.M., 1973. Lateral phase separation in phospholipid membranes. Biochemistry 12, 2351 – 2360. Sklar, L.A., Hudson, B.S., Simoni, R.D., 1977. Conjugated polyene fatty acids as fluorescent probes: synthetic phospholipid membrane studies. Biochemistry 16, 819 – 828. Small, D.M., 1986. The Physical Chemistry of Lipids: From Alkanes to Phospholipids. Plenum Press, New York (Chapter 2). Tardieu, A., Luzzati, V., Reman, F.C., 1973. Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases. J. Mol. Biol. 75, 711 – 733. Tenchov, B.G., 1985. Nonuniform lipid distribution in membranes. Prog. Surface Sci. 20, 273 – 340. van Dijck, P.W.M., Kaper, A.J., Oonk, H.A.J., de Gier, J., 1977. Miscibility properties of binary phosphatidylcholine mixtures. Biochim. Biophys. Acta 470, 58 – 69. Van Dijck, P.W.M., De Kruijff, B., Verkleij, A.J., Van Deenen, L.L.M., 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 – 96. Van Dijck, P.W.M., 1979. Negatively charged phospholipids and their position in the cholesterol affinity sequence. Biochim. Biophys. Acta 555, 89 – 101. Wiedmann, T., Salmon, A., Wong, V., 1993. Phase behavior of mixtures of DPPC and POPG. Biochim. Biophys. Acta 1167, 114 – 120.
Phase behavior of hydrated lipid bilayer composed of binary mixture of phospholipids with different head groups Tohru Inoue *, Yoshinori Nibu Department of Chemistry, Faculty of Science, Fukuoka Uni6ersity, Nanakuma, Jonan-ku, Fukuoka 814 -0180, Japan Received 27 January 1999; accepted 17 May 1999
Abstract Phase behavior of hydrated lipid bilayer was investigated for the mixtures of two phospholipid species chosen from phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) with the same acyl chains. The pseudo-binary phase diagrams constructed by a differential scanning calorimetry (DSC) were analyzed based on a thermodynamic model applying the Bragg – Williams approximation for non-ideality of mixing. The interchange energy parameters, r0, derived from this approach were positive for all mixture systems in both gel and liquid–crystalline phase bilayers, and increased in the order PG/PEB PC/PABPC/PEB PG/PA with a few exception. This suggests that the energetical disadvantage for the mixed-pair formation relative to the like-pair formation in the hydrated bilayer increases in this order. In addition, the r0 values increased with the increase in the acyl chain length of the phospholipids. These experimental results were discussed in terms of an intermolecular interaction of the phospholipid species in hydrated bilayer. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phospholipid mixture; Lipid bilayer; Bilayer phase transition; Phase diagram; Non-ideal mixing; Differential scanning calorimetry
1. Introduction Biomembranes are constituted of various kinds of lipids and proteins (Hauser and Poupart, 1992), and the physical state of phospholipids and the lipid composition of local regions of biomembranes is relevant to many biological functions of the membranes, such as enzyme function, ion
* Corresponding author. Tel.: + 81-92-8716631, ext. 6219; fax: + 81-92-8656030. E-mail address: [email protected] (T. Inoue)
transport, antibody stimulated cell lysis, and cell fusion (Bloom et al., 1991). Thus, the phase behavior of hydrated bilayer of phospholipid mixtures is a subject of fundamental importance in relation to membrane biophysics. From this point of view, a number of investigations have so far been reported concerning the phase behavior of hydrated bilayer of binary phospholipid mixtures (Do¨rfler et al., 1988, 1990a; Do¨rfler and Miethe, 1990; Marsh, 1990a; Wiedmann et al., 1993; Polozov et al., 1994; Johann et al., 1996; Rolland et al., 1997; Garidel et al., 1997; Garidel and Blume, 1998).
0009-3084/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 9 9 ) 0 0 0 6 1 - 4
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The phase diagram becomes a basis to understand the phase behavior of mixture systems. The pseudo-binary phase diagram for hydrated twocomponent phospholipid mixture is governed by the miscibility of each lipid species in the bilayer of both gel and liquid – crystalline states. The miscibility is determined, as a first approximation, by the difference in the pair-interaction energies among the like-pair and mixed-pair formed in the mixture. Thus, the shape of phase boundaries provides information about an intermolecular interaction between phospholipid species in hydrated bilayers of respective phase states. It is expected that the systematic study for the phase behaviors of binary mixtures of various phospholipid species would be useful to elucidate the factors governing the miscibility of different phospholipid species in hydrated bilayers and the phase behavior of phospholipid mixtures. Based on the above consideration, we have been investigating the phase behavior of binary mixture systems of different phospholipid species (Inoue et al., 1992, 1999; Nibu and Inoue, 1995; Inoue and Nibu, 1995a; Nibu et al., 1995). In previous papers, we have reported mixtures of several types of phospholipid species having the same head group but different acyl chains. The present paper is concerned with mixtures composed of several combinations of two phospholipid species with the same acyl chain but different head groups. Two series experiments were made with respect to the acyl chain length, i.e. dipalmitoyl and distearoyl. The phase boundaries for hydrated bilayers of these phospholipid mixtures were determined by a differential scanning calorimetry. The obtained phase diagrams were analyzed based on a thermodynamic model which attributes the non-ideality of mixing or excess free energy of mixing to the difference in the pair-interaction energies among the like-pair and mixedpair. The purpose of the present study is to get information concerning the interaction between different head groups in hydrated lipid bilayer by examining the pseudo-binary phase diagram for various mixtures of phospholipids with different head groups.
2. Experimental
2.1. Materials The phospholipid species used in the present study were dipalmitoylphosphatidic acid (sodium salt; DPPA), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (sodium salt; DPPG), and their distearoyl analogs (DSPA, DSPC, DSPE, and DSPG). These phospholipids of synthetic origin were obtained from Sigma, and were used without further purification. The purities of the lipids were 99% or better than 99%.
2.2. Preparation of lipid mixtures The following four combination of binary lipid mixtures were examined for both dipalmitoyl and distearoyl series; PC/PA, PC/PE, PG/PA, and PG/PE. The lipid mixtures for DSC measurements were prepared by the following procedure. The appropriate amounts of two dry lipids were weighed into a glass cuvette to give a desired composition, to which a small amount of methanol/chloroform (1/4 in volume) mixed solvent was added to dissolve the lipid mixture. Then, the solvent was removed by gentle blowing of dry N2 gas, followed by evaporation under reduced pressure.
2.3. DSC measurements DSC measurements were performed using a Seiko Denshi Model SSC5200 (Tokyo, Japan). The calibration with respect to temperature and caloric was made by the use of gallium and indium. The dry lipid mixture of about 5 mg was weighed in a sealable sample pan made from aluminum, to which 20 ml of water was added, and then the pan was sealed. As a reference material, alumna was used, the weight of which was about 20 mg. The sample was held at 90°C for about 1 h in the oven of the DSC apparatus in order to assure homogeneous mixing of the lipid and water. Then, a cooling/heating cycle was repeated for several times at the rate of 2°C/min in
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the temperature range from 20 or 30 to 90°C. A good reproducibility was obtained for the DSC thermograms recorded by the repeated scan.
3. Results and discussion As examples of DSC thermograms obtained for the present mixture systems, Figs. 1 and 2 show those for DPPC/DPPE and DSPC/DSPE mixtures, respectively, at various compositions. The composition of these mixtures are expressed in terms of the mole fraction of DPPE, xDPPE, and DSPE, xDSPE. The endothermic peaks in these thermograms are ascribed to the gel (L%b)-to-liquid-crystalline (La) phase transition of the hydrated lipid bilayers (Small, 1986). The small
Fig. 2. DSC thermograms obtained for hydrated DSPC/DSPE mixture of various composition. The mole fraction of DSPE in the lipid mixture is indicated in the figure.
Fig. 1. DSC thermograms obtained for hydrated DPPC/DPPE mixture of various composition. The composition expressed in terms of the mole fraction of DPPE in the lipid mixture, xDPPE, is indicated in the figure.
endotherms observed for pure DPPC and DSPC at lower temperature are so-called pre-transition, and are attributed to the phase transition from L %b to P %b (Small, 1986). We are concerned about the behavior of gel-to-liquid-crystalline phase transition with respect to the composition of the lipid mixtures. When PE is added to pure PC species, another endothermic peak appears at higher temperature (black arrows in Figs. 1 and 2) in addition to the endotherms corresponding to those for respective PC species (white arrows). With the increase in PE content in the mixture, this peak moves toward higher temperature concomitantly with the increase in its intensity. On the other hand, the lower temperature peaks reduce their intensity with the increase in PE content in the mixture. They are appreciable up to xPE : 0.5 for both
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DPPC/DPPE and DSPC/DSPE mixtures in the thermograms drawn in more expanded scale, although they are barely seen in Figs. 1 and 2. The peak temperatures in the DSC thermograms such as presented in Figs. 1 and 2 are regarded to define phase boundaries among the gel phase, liquid–crystalline phase, and their coexisting region in the hydrated bilayer of the lipid mixtures, and hence, a pseudo-binary phase diagram can be constructed by plotting these temperatures against the composition. Fig. 3 shows the phase diagrams thus obtained for four dipalmitoyl series mixture systems, i.e. (a) DPPC/DPPA; (b) DPPC/DPPE; (c) DPPG/DPPA; and (d) DPPG/ DPPE. Corresponding phase diagrams for distearoyl series mixtures are shown in Fig. 4(a – d). In these phase diagrams, the composition is expressed by the mole fraction of the component with higher transition temperature of the two lipid species. It can be seen in these figures that the shape of the ‘solidus’ and ‘liquidus’ lines obtained by connecting the peak temperatures of the DSC thermograms is considerably different from that expected for ideal mixing of the two components. Phase behavior of DPPC/DPPE mixture has so far been investigated by many authors using various detection techniques for bilayer phase transition (Shimshick and McConnell, 1973; Lee, 1975; Sklar et al., 1977; Mendelsohn and Koch, 1980; Arnold et al., 1981; Do¨rfler et al., 1990b). The phase diagram obtained in the present work for this mixture system (Fig. 3b) is in fairly good agreement with those constructed from Raman spectroscopy (Mendelsohn and Koch, 1980), 31PNMR (Arnold et al., 1981), and DSC (Do¨rfler et al., 1990b). On the other hand, the phase diagrams obtained by spin label (Shimshick and McConnell, 1973) and fluorescence probe (Lee, 1975; Sklar et al., 1977) techniques are somewhat different from the above ones, particularly, in a solidus line around xDPPE =0.5, although they exhibit a good agreement within themselves. This disagreement between the two groups of detection techniques may be attributed to the existence of probe molecules in the latter cases; it is likely that these probe molecules have affected the phase behavior of the lipid mixture.
Other than DPPC/DPPE system, the phase diagram of DPPC/DPPA mixture at different pH has been recently reported by Blume and co-workers (Garidel et al., 1997). They have determined the onset and end temperatures of the bilayer phase transition of multilamellar liposomes suspended in excess water by simulating the heat-capacity curves obtained by DSC experiments. Comparing the present result for this mixture system (Fig. 3a) with the phase diagram at pH 7.0 reported by Garidel et al., the overall shape of the phase boundaries is similar to each other, although the width of the cigar-like shape is considerably broader in the present case. This difference in the temperature range of the coexisting region of gel and liquid–crystalline phases is attributed to the difference in the determination procedure of the phase boundaries, as described by Johann et al. (1996). It should be mentioned here about possible problems associated with the present experimental condition. In the present experiments, the hydrated phospholipid samples were prepared by adding pure water to dry lipids without using buffer solution; i.e. no pH control was made. This might cause some problems for the case of acidic phospholipids, PA and PG, since the protonation state of their head groups is affected by pH of excess water phase [for PA, pKa(PO4): 3.5 and 9.0, and for PG, pKa(PO4): 3 (Marsh, 1990b)]. In fact, the phase diagrams of DPPC/DPPA mixture obtained by controlling the pH at 7.0 and 4.0, respectively are quite different from each other (Garidel et al., 1997). The present phase diagram for the same mixture system is close to that obtained at pH 7.0 by Garidel et al., as mentioned above. In addition, the phase diagram of hydrated DMPA/DPPA mixture prepared by using pure water (Inoue and Nibu, 1995b) is similar to that obtained for the same mixture at pH 7.0 (Garidel and Blume, 1998). Furthermore, the thermodynamic data for pure DPPA and DPPG determined in the present work, which are listed in Table 1 together with those reported in literature, are almost in agreement with literature values obtained at neutral pH. Judging from these results, it may be safely assumed that the preparation procedure of hydrated lipid samples
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employed in the present study does not cause such a significant effect on the protonation state of the
143
lipid head groups as to affect the phase behavior of the hydrated lipid bilayers, or in other words,
Fig. 3. Pseudo-binary phase diagrams of dipalmitoyl series lipid mixtures. The mixture systems are (a) DPPC/DPPA; (b) DPPC/DPPE; (c) DPPG/DPPA; and (d) DPPG/DPPE. Solid lines in these figures are phase boundaries calculated from Eqs. (1) and (2).
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Fig. 4. Pseudo-binary phase diagrams of distearoyl series lipid mixtures. The mixture systems are (a) DSPC/DSPA; (b) DSPC/DSPE; (c) DSPG/DSPA; and (d) DSPG/DSPE. Solid lines in these figures are phase boundaries calculated from Eqs. (1) and (2).
the preparation of hydrated lipid samples with pure water keeps the neutral pH of excess water phase. For PG/PE mixture systems, another problem
would arise due to the preparation of lipid samples with pure water. It has ported that the bilayer phase transition ture of dimyristoylphosphatidylglycerol
hydrated been retempera(DMPG)
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depends on the Na + concentration in the bulk water phase (Salonen et al., 1989; Kodama and Miyata, 1995). In the present experimental condition, the Na + concentration in excess water phase of PG/PE mixtures varies from 300 mM (xPG = 1) to 30 mM (xPG =0.1) in rough estimation. According to Kodama and Miyata, the main phase transition temperature of DMPG bilayer is lowered by approximately 1.5°C as the Na + concentration is decreased from 300 to 30 mM. If the effect of Na + concentration on the bilayer phase transition of DPPG and DSPG is similar to that of DMPG, the temperatures of phase boundaries of DPPG/DPPE and DSPG/ DSPE mixtures obtained in the present study would be slightly different from those expected for the case containing externally added Na + at high concentration. As described above, the phase diagrams presented in Figs. 3 and 4 exhibit a non-ideal nature of mixing of the two lipid species in both gel and liquid–crystalline phases. Several methods have so far been applied to simulate the binary phase diagrams for non-ideal lipid mixtures (Shimshick and McConnell, 1973; van Dijck et al., 1977; Lee, 1977; Mendelsohn and Koch, 1980; Davis and Keough, 1984; Tenchov, 1985; Koynova et al.,
1988; Brumbaugh et al., 1990; Johann et al., 1996). The most frequently used method utilizes the thermodynamic equations derived by considering the excess free energy of mixing. To evaluate the excess free energy of mixing, the Bragg– Williams approximation (Moore, 1972) may be valid for the lipid mixtures such as the present case, because the components in the mixture have a similarity in both molecular structure and molecular volume. Recently, Blume and co-workers developed a new approach which enables one to simulate the heat-capacity curves obtained by DSC measurements and phase diagram of pseudobinary phospholipid mixtures (Johann et al., 1996). In this approach, the composition dependence of the cooperativity of transition was incorporated to simulate the heat-capacity curves and to estimate the temperatures corresponding to the onset and the end of the transition. In addition, the four non-ideality parameters of mixing were used to simulate the phase diagrams taking into account the nonsymmetrical excess free energy with respect to composition of the mixture. We analyzed the phase diagrams presented in Figs. 3 and 4 in terms of the usual two-parameter Bragg– Williams approximation instead of the more refined four-parameter model proposed by Jo-
Table 1 Thermodynamic data for the main phase transition of the pure phospholipids This work
145
Literature
References
Tm (°C)
DH (kJ/mol)
Tm (°C)
DH (kJ/mol)
DPPC DPPG
41.2 39.7
30.5 30.5
DPPA
63.2
29.3
DPPE DSPC DSPG DSPA DSPE
64.0 54.7 54.7 74.4 74.7
30.1 38.2 43.5 39.3 37.1
41.49 0.5 34.8 93.2 41.5 37.2 (pH 7) 41.5 36.8 (pH 7) 41 33.0 (pH 7) 39.5 38.1 (pH 7) 40.0 (pH 7, 0.1 M NaCl) 58 (pH 2, 0.1 M NaCl) 65.0 33.0 (pH 6) 67 22.0 (pH 6.5) 64.7 32.4 (pH 7) 65.4 28.8 (pH 4) 64.0 9 2.2 35.4 9 3.3 55.1 9 1.5 42.3 93.7 54.5 43.9
Marsh (1990c) Findlay and Barton (1978) Van Dijck et al. (1978) Jacobson and Papahadjopoulos (1975) Van Dijck (1979) Garidel and Blume (1998) Garidel and Blume (1998) Blume (1983) Jacobson and Papahadjopoulos (1975) Garidel et al. (1997) Garidel et al. (1997) Marsh (1990c) Marsh (1990c) Findlay and Barton (1978)
74.2 9 2.2
Marsh (1990c)
44.3 91.7
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146
hann et al. in order to facilitate the comparison with previous results reported for various phospholipid mixture systems. According to this treatment, the phase boundaries corresponding to the solidus and liquidus lines are given by solving the following simultaneous equations (Lee, 1977; Tenchov, 1985; Inoue et al., 1992). ln
2 (S) (S) 2 r (L)(1−x (L) x (L) A A ) −r 0 (1 − x A ) + 0 (S) xA RT
(1)
(2)
DHA 1 1 − T TA R (L) 2 (S) (S) 2 r (L) 1− x (L) A 0 (x A ) −r 0 (x A ) + ln 1− x (S) RT A
=−
=−
1 DHB 1 − R T TB
(S) where x (L) A and x A refer to the mole fractions of component A in liquid – crystalline and gel phase bilayers being in equilibrium with each other, TA and TB are the transition temperatures and DHA and DHB are the transition enthalpies for pure species denoted by the subscripts, and r (L) and 0 r (S) are parameters characterizing the non-ideal 0 behavior of mixing in liquid – crystalline and gel phases, respectively. r0 is given by
r0 = z uAB −
uAA +uBB 2
(3)
where z is the first coordination number and uAA, uBB, and uAB are the molar energies of A – A, B – B, and A–B pair interactions. The present experimental phase diagrams were analyzed by applying Eqs. (1) and (2) using a curve fitting procedure. The values of TA, TB, DHA, and DHB for pure lipid species, which are required for the calculation, are listed in Table 1. The non-ideality parameters r (L) and r (S) were 0 0 determined so as to obtain the calculated phase boundaries reproducing the experimental phase boundaries most satisfactorily. This was done by using a least-squares method as has been described elsewhere in some detail (Nibu and Inoue, 1995). The solid lines in Figs. 3 and 4 were drawn according to Eqs. (1) and (2) using the non-ideality parameters thus determined. The agreement between the experimental and computed phase boundaries is satisfactory, although it is rather
Table 2 Non-ideality parameters of mixing obtained for various binary mixtures of phospholipids with different head groups Mixture
(kJ/mol) r (L) 0
r (S) 0 (kJ/mol)
DP series PC/PA PC/PE PG/PA PG/PE
3.3 3.4 4.7 2.8
4.0 4.6 4.9 3.1
DS series PC/PA PC/PE PG/PA PG/PE
3.7 4.1 4.5 4.0
5.9 6.1 6.7 5.6
poor for the case of DSPG/DSPA mixture (Fig. 4c). The values of the non-ideality parameters of mixing obtained for various mixture systems are summarized in Table 2. The non-ideality parameter of mixing for DPPC/DPPE mixture system has been estimated by some authors based on the same thermodynamic approach as the present case. Arnold et al. have reported the r0 values as r (L) 0 = 3.8 kJ/mol and r (S) 0 = 5.4 kJ/mol by analyzing the phase diagram constructed from 31P-NMR (Arnold et al., 1981). According to Mendelsohn and Koch, (S) r (L) 0 = 3.1 kJ/mol and r 0 = 4.4 kJ/mol have been estimated for the mixture of deuterated DPPC and DPPE from the phase diagram obtained by Raman spectroscopy (Mendelsohn and Koch, 1980). The r0 values derived in the present study for DPPC/DPPE mixture (Table 2) are comparable to these reported values. The parameter, r0, is a measure of non-ideality of mixing, and serves to interpret the mixing behavior on the basis of intermolecular interaction energies. The ideal mixing corresponds to r0 = 0, i.e. identical intermolecular interaction energies for both mixed-pair and like-pair. The nonideality of mixing is reflected in the deviation of r0 from 0; the positive r0 means that the A–B pair formation in the mixture is energetically unfavorable compared with A–A and B–B pair formation, while the negative r0 corresponds to the situation that the A–B pair formation is more preferable than the A–A and B–B pair forma-
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tion. Thus, a large positive r0 tends to lead to a phase separation, while a large negative r0 tends to result in a regular arrangement of the two components in the mixture. Some general trends can be drawn from Table 2 regarding the dependence of r0 on various parameters, as described below. The r0 values are positive for all mixture systems in both gel and liquid–crystalline phases. This means that in hydrated bilayers of both phase states, the pair interaction for mixed-pair is repulsive relative to the corresponding like-pairs. Comparing the r0 values obtained for gel and liquid – crystalline bilayers, r (S) is greater than r (L) for all mixture 0 0 systems. This difference in r0 values between the two phase states may be readily understood by considering the solid-like nature of the gel phase and the liquid-like nature of the liquid – crystalline phase. It is usual that the miscibility of different species is extremely reduced in a solid state compared with a liquid state. In gel state bilayer, the lipid molecules with rigid hydrocarbon chain of all-trans conformation are forced to arrange within a definite crystalline-like lattice, whereas in liquid–crystalline state bilayer, the lipid molecules with flexible disordered hydrocarbon chain are loosely packed and can easily move laterally. Thus, the difference in pair-interaction energies between like-pair and mixed-pair may become more significant in gel state bilayer than in liquidcrystalline state bilayer, which results in highly non-ideal behavior of mixing in gel phase. The r0 values for different combination of two lipid species increases in the order PG/PE B PC/ PAB PC/PE B PG/PA commonly to dipalmitoyl (DP) and distearoyl (DS) series mixtures and also to gel and liquid–crystalline phases except for the case of r (L) of DS series, in which the order for 0 PG/PE and PC/PA is reversed in the above sequence. This variation of r0 is seen more clearly in Fig. 5 where the r0 values listed in Table 2 are represented graphically. This result for the order of r0 values for different phospholipid pairs suggests that the energetical disadvantage for the mixed-pair formation relative to corresponding like-pair formation in hydrated bilayer increases in this order. In the present experimental conditions, as described above, the head groups of PG
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and PA bear monovalent negative charge, while PC and PE are zwitterionic, and hence, have no net charge [pKa(PO4): 1 and pKa(NH3+ ) for PE: 10–11 (Marsh, 1990b)]. It might be considered that the largest r0 value for PG/PA mixture is attributed to the fact that the both head groups have a negative charge. However, the situation is more or less similar for both mixed-pair and like-pairs. The head group of PG has two hydroxyl groups, and thus, it is likely that the hydrogen bond formed among neighboring molecules in hydrated bilayer brings about an attractive (energetically favorable) contribution to the intermolecular interaction of PG species. When PA is added to PG, this network of hydrogen bond existing in pure PG bilayer would be disturbed, and hence, the formation of mixed-pair of PG and PA would become highly unfavorable compared with the formation of like-pair between themselves. The hydrogen bond network of hydrated PG bilayer is also disturbed by the addition of PE to PG. In this case, however, the repulsive electrostatic interaction for negatively charged PG/PG pair may be reduced significantly by forming the PG/PE pair because of the zwitterionic nature of PE species. This gain of interac-
Fig. 5. Dependence of the non-ideality parameter of mixing, r0, on the combination of phospholipid species with different head groups. Circles: dipalmitoyl (DP) series lipid mixtures; Squares: distearoyl (DS) series lipid mixtures. Filled and open symbols correspond to the gel and liquid-crystalline phase bilayers, respectively.
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tion energy due to the reduced electrostatic interaction would overcome the disadvantage of the destruction of hydrogen bond network, and hence, the excess energy for PG/PE mixed-pair would become minimal. As for the PC species, no such additional interaction as hydrogen bonding for PG is expected in hydrated bilayer. Therefore, the r0 values for the mixtures associated with PC would fall in between PG/PA and PG/PE mixtures. It can be seen in Table 2 or Fig. 5 that the r0 values obtained for DS series mixtures are larger than those for DP series mixtures in both gel and liquid–crystalline bilayers with an exception of liquid–crystalline bilayer of PG/PA mixture. This means that the effect of difference in head group type on the pair-interaction becomes more pronounced as the lipid acyl chain becomes longer. At a first glance, this trend seems somewhat strange. Because the relative contribution of head group to the overall intermolecular interaction would be reduced as the lipid acyl chain becomes long, the difference in the head group type among the mixed-pair and like-pairs is considered to become less striking with the increase in the acyl chain length. Thus, it may be expected that the r0 values are smaller for DS series mixtures than for DP series mixtures. The actually obtained tendency is, however, opposite. This may be interpreted as follows. The intermolecular separation of phospholipid molecules in hydrated bilayer depends on the lipid acyl chain length. Fig. 6 depicts the plot of S, the average area available to a head group on the hydrated bilayer, as a function of NC, the number of carbon atoms in the lipid acyl chain. These S values were compiled from the data reported for PC species with different acyl chain length (Tardieu et al., 1973; Janiak et al., 1976, 1979; Inoko and Mitsui, 1978; Lis et al., 1982). The values of S, and hence, the intermolecular distance is larger for liquid – crystalline (La) phase bilayer than for gel (L %b) phase bilayer, reflecting the looser packing of the lipid molecules in the former than the latter bilayer state. As for the chain length dependence, the intermolecular separation decreases with the increase in NC in both gel and liquid-crystalline bilayers. This may be interpreted as due to the enhanced cohesive
Fig. 6. Plot of the average area available to a head group on the hydrated bilayer, S, against the number of carbon atoms in the acyl chain, NC, for phosphatidylcholines with two identical saturated acyl chains. Circles and squares correspond to the gel (L %b) and liquid-crystalline (L %a) phase bilayers. The data were taken from literature (Tardieu et al., 1973; Janiak et al., 1976, 1979; Inoko and Mitsui, 1978; Lis et al., 1982).
force caused by the increase in the hydrocarbon chain length. When the separation distance between head groups is shortened, their interaction becomes strong, and hence, the difference in the interaction energy between mixed-pair and likepairs would be intensified. It is likely that the larger r0 values for DS series mixtures than for DP series mixtures is attributed to the shorter separation distance or tighter packing of lipid molecules in hydrated bilayers for DS series mixtures. The above consideration regarding the dependence of r0 on the lipid acyl chain length predicts that the r0 values for dimyristoyl (DM) series mixtures would be smaller that those for DP series mixtures. Actually, this can be recognized in the phase diagram for DMPC/DMPE mixture reported by Chapman et al. (1974) and Polozov et al. (1994). The phase boundaries obtained from DSC experiments for DMPC/DMPE mixture by these authors are closer to a cigar-like shape than those for DPPC/DPPE mixture are. This indicates that the r0 values for DMPC/DMPE mixture is smaller than those for DPPC/DPPE mixture.
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Additional noticeable feature in Table 2 or Fig. 5 is that the difference in r0 between gel and liquid–crystalline bilayers is much larger for DS series mixtures than for DP series mixtures. In the latter case, the difference is not so significant except for PC/PE mixture. A remarkable difference between r (S) and r (L) for DS mixtures is 0 0 ascribed to the extremely large r (S) values, since 0 between the two series mixthe difference in r (L) 0 tures is rather small. Thus, it is regarded that the energetical disadvantage for the mixed-pair formation is enhanced by the increase in acyl chain length to a greater extent in gel phase bilayer than in liquid–crystalline bilayer. This may be explained again, at least in part, based on the chain length dependence of the intermolecular separation of phospholipids in gel and liquid – crystalline phase bilayers. As is seen in Fig. 6, the variation of S with NC is rather parallel for both gel and liquid–crystalline phases. However, the relative decrease, or decrement, of intermolecular separation with the increase in NC becomes larger for gel phase bilayer than for liquid – crystalline bilayer; on going from NC =16 to NC =18, the separation distance decreases by nearly 5.7 and 3.9% for gel and liquid–crystalline bilayers, respectively. Thus, a mixed-pair formation in gel state bilayer would become more unfavorable than that in liquid – crystalline bilayer when the length of the lipid acyl chain is increased.
Acknowledgements This work was supported in part by funds from the Central Research Institute of Fukuoka University.
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