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Salt-induced phase separation in the liquid crystalline phase of phosphatidylcholines
Colloids and Surfaces A: Physicochemical and Engineering Aspects 183– 185 (2001) 171– 181 www.elsevier.nl/locate/colsurfa
Salt-induced phase separation in the liquid crystalline phase of phosphatidylcholines Michael Rappolt, Georg Pabst, Heinz Amenitsch, Peter Laggner * Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Steyrergasse 17, A-8010 Graz, Austria
Abstract The effects of alkali chlorides on multilamellar vesicles of various phosphatidylcholines in the lamellar liquid crystalline La-phase were investigated by using small-angle X-ray scattering. At alkali chloride concentrations above 70 mM (LiCl) a phase separation in the liquid crystalline phase of POPC is induced. The splitting of the first and second order diffraction peaks into two major discrete components indicates a separation into different lamellar liquid crystalline (smectic A) phases. Detailed data-analysis applying the modified Caille´ theory proves that the phases mainly differ in the interbilayer water thickness by about two hydration layers. The lipid bilayer profile itself remains essentially the same in all liquid crystalline phases. A comparison of differently prepared samples and rapid mixing experiments in combination with simultaneous time-resolved X-ray diffraction suggest that the phase separation is osmotically driven. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Lecithin; Small-angle X-ray scattering; La-phase; Lithium chloride; Phase separation; Manic depressive disease
1. Introduction In a recent study [1] we have focused on alkaliion induced La-phase separation in the liquid crystalline phosphatidylcholine – water bilayer system by simultaneous small- and wide-angle X-ray scattering studies. Particular attention has been Abbre6iations: DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; EYPC, eggyolk phosphatidylcholine; POPC, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine; SAXS, small-angle X-ray scattering. * Corresponding author. Tel.: +43-316-4120300; fax + 43316-4120390. E-mail address: [email protected] (P. Laggner).
given to the lightest alkali metal, lithium, also because of its pharmacological potential in the treatment of manic-depressive diseases [2,3]. Alkali ions have considerable effects on the structural and conformational behaviour of phospholipid bilayers. Phosphatidylcholine head groups behave as ‘sensors’ of the electrostatic charge at the membrane surface [4] such that, for example, ions induce conformational changes in the polar head group region in the liquid crystalline La-phase [5–7], reorder the packing geometry of saturated acyl chains in the ripple phase, and enhance the appearance of the submain transition [8]. The interaction between Li+ and negatively charged phosphatidylserine leads to dehydration of the phosphatidylserine head
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groups, and to the formation of high-melting ion –lipid complexes [9,10], whereas Na+ and K+ only produce minor changes [11,12]. Further, a fluid –fluid phase separation was demonstrated in liquid crystalline phosphatidylserine–phosphatidylcholine mixtures [13]. Until our first report [1], however, phase separation was only known to occur in phospholipid mixtures, and not in one-component phospholipid bilayer systems. Therefore, the effect of induced phase separation has been investigated under various conditions [1] and may be summarised as follows: The alkali chlorides LiCl, KCl and NaCl may induce up to three different, distinct lamellar lattices in
the liquid crystalline phase, e.g., DSPC, POPC and EYPC, where lithium shows the strongest effect with respect to its concentration. The number and repeat distance of the coexisting lamellar phases depend on the nature and concentration of the alkali chloride, the lipid concentration, and on the degree of acyl chain unsaturation. However, within a given ternary system such as LiCl– POPC –H2O the separation effect stays constant above a certain salt concentration. The present work addresses two open questions, the first concerning the structure of the bilayer phases in the presence of LiCl. This is accomplished by applying a modified Caille´ theory (MCT) [14] analysis to a high quality diffraction pattern of the first five diffraction orders of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in an equimolar LiCl solution. The second question aims at the physical origin of the observed phase splitting. Here, X-ray experiments on differently prepared liposomal dispersions, as well as a rapid mixing of the pure sample with the salt solution provide further insight into the phase separation.
2. Materials and methods
2.1. Sample preparation
Fig. 1. Salt induced phase separation in POPC (20% w/w) dispersions at 2°C. Small-angle X-ray diffraction pattern are displayed in the regime covering the first two orders. Solid lines indicate samples, which have been hydrated from dry films, and dashed lines indicate samples, which have been hydrated from dry powder, respectively. (A) The influence of LiCl and (B) KCl, respectively, each with an lipid-to-salt molar ratio of 1:1 is presented.
1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids, Birmingham AL, and used without further purification. Multilamellar liposomes were prepared in two ways: either by dispersing weighted amounts of dry lipid powder, or by dissolving first the lipids in CHCl3/ CH3OH (2:1, v/v). The solutions were then dried under a stream of N2 and after that kept in a vacuum oven overnight. Both, weighted amounts (20–40% w/w) of dry lipid powder and dry lipid films, respectively, were then hydrated in aqueous solution of LiCl and KCl adjusting a salt-to-lipid molar ratio of 1:1. Quartz-bidistilled, deionized water was used throughout. To ensure complete
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Fig. 2. Schematic drawing of the formation of multilamellar liposomes by a hydration from dry film (A) or dry powder (B). (A) In the initial state (i), the dry film features a layered structure, which exhibits only few defects. Thus, during hydration (ii) only a small number of lithium ions (solid circles) penetrate into the interbilayer regime. The result are vesicles, which incorporate far less lithium ions (iii) compared to the excess aqueous solution. The ion gradient induces an osmotic pressure onto the liposomes. (B) The initial state (i) of the dry powder is characterised by randomly ordered crystallites. The high number of defect lines facilitate the diffusion of lithium ions inbetween the lipid bilayers (ii). The defect lines will heal out during the hydration process. However, the fully hydrated vesicles (iii) will contain more ions in the interbilayer regime than in case (A/iii). Thus, the liposomes will be subjected to a weaker osmotic pressure.
Table 1 Overview of salt-induced phase separation in POPC at 2°C Sample treatment
20% 20% 20% 20% a b
w/w w/w w/w w/w
in in in in
0.33 0.33 0.33 0.33
M M M M
KCla KClb LiCla LiClb
Hydration from dry powder. Hydration from dry film.
Fraction of phase 2
d-spacings (A, ) phase 1/phase 2
Dd= d1−d2 (A, )
30% 70% 20% 80%
67.0/62.9 66.7/61.1 66.7/62.0 66.7/61.5
4.1 5.6 4.7 5.2
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Fig. 3. The first five orders (h= 1,…,5) of Bragg peaks of POPC at 2°C (40% w/w in 0.88 M LiCl: lipid/salt molar ratio 1:1) are presented. MCT-fits to the data are shown as solid lines, different components of phase 1 and phase 2, respectively, are given in dotted lines. Within in the first three orders additional diffuse scattering between the Bragg-peaks of phase 1 and 2 was fitted with a Gaussian distribution. The 4th order is only visible for phase 2, while the 5th is only displayed by phase 1. The results of the MCT fit is given in Table 2.
hydration, the lipid dispersions were incubated for about 4 h at least 10°C above the main transition temperature. During this period the lipid dispersions were vigorously vortexed. Aqueous dispersions of this lipids display narrow, co-operative melting transitions within the limits of published values, thus proving that the lipid purity corresponds to the claimed one of 99%. Thin layer chromatography tests on silica gel-plates 60 (Merck, Darmstadt) before and after the experiments showed no signs of degradation. The solvent used was chloroform/methanol/water (65/25/4).
2.2. X-ray small-angle diffraction Diffraction patterns were recorded on the Austrian SAXS beamline at ELETTRA, Trieste [15,16]
using an one-dimensional position sensitive detector [17] covering the corresponding s-range (s=2 sin(q)/u) of interest from about 1/200 A, − 1 to 1/12 Table 2 MCT [14,23] fit results for POPC 40% w/w in an equimolar LiCl solution at 2°C (see Fig. 3)a Fit parameter
Phase 1
Phase 2
d(A, ) F2/F1 2 F3/F1 2 F4/F1 2 F5/F1 2 p1
64.7 90.1 1.15 90.01 0.271 9 0.01 0 0.125 90.006 0.030 90.004
58.8 90.1 0.667 90.002 0.92 9 0.03 0.63 9 0.05 0 0.070 90.004
(a.u.) (a.u.) (a.u.) (a.u.)
a d, d-spacing; Fi/F1, ratio of the ith form factor to the first diffraction order form factor; p1, Caille´ parameter.
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Fig. 4. Different continuous transforms for POPC in the liquid crystalline phase are given. (A) The transform of phase 1 (POPC 40% w/w 0.88 M LiCl) is identical to fully hydrated POPC (20% w/w) in pure water [30]; a deviation is only seen in the 5th order. (B) The transform of phase 2 is compared to the ones given under different relative humidity (RH) conditions [29].
Fig. 5. Absolute electron densities of POPC at 2°C. Phase 1 and the ‘pure water phase’ [30] are practically identical. In phase 2 the bilayer motif is conserved, but slightly thicker. The interbilayer thickness of phase 2 is clearly reduced and here the density is less than in pure water. The results are summarised in Table 1.
A, − 1. The angular calibration of the detector was performed by silver-behenate (CH3(CH2)20COOAg: d-spacing = 58.38 A, ) [18].
For the X-ray diffraction experiments at thermal equilibrium a sample holder of stainless-steal sealed with 25 mm thick mica windows was used,
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as described previously [19]. Rapid mixing experiments were performed with a stopped flow apparatus (Unisoku Scientific Instruments, Osaka, Japan) in combination with simultaneous time-resolved X-ray diffraction. The device consists of two reservoirs (4 ml each) containing the solutions to be mixed. Each reservoir is connected to a pneumatically driven syringe with a volume of 0.3 ml for each shot. When actuated by an electronic trigger signal both solutions are injected into the X-ray mixing chamber (specified dead time less than 1 ms). The X-ray cell is contained in a ceramic block (1×1 × 1 cm) with an optical path length of 1 mm between two sapphire windows of 0.05 mm thickness each (transmission 0.55 at 8 keV). Fresh lipid dispersions (no salt) of 20% w/w of DMPC and DPPC were mixed with 0.33 M LiCl-solutions reaching a final salt-to-lipid ratio of 1:1. For POPC several diluted samples (6% w/w) of the same stock were investigated to check the reproducibility. Each experiment consisted of 460 time frames in which the rapid mixing was triggered at the beginning of the first frame. Here, the maximum time resolution was 50 ms. The complete reaction was followed over a period of 15 min.
2.3. Data analysis The static diffraction patterns were analysed in terms of the modified Caille´ theory (MCT) [2], which has been demonstrated to be superior to
the paracrystalline theory [20,21] by Nagle and co-workers [22]. The program written in the IDL (Interactive Data Language) is based on the FORTRAN version by Petrache [23] and uses a minimization routine MPFIT [24] that is based on the MINPACK library [25]. The Bragg peaks were fitted within an area of Ds = 9 0.0017 A, − 1 from the respective peak centre position for all recorded orders at once. The model function was chosen to be the sum of two MCT and one Gaussian peak for each order of diffraction. The Gaussian accounts for the diffuse scattering between the Bragg peaks. The raw data of the time-resolved experiments were normalised for the integration time of each time-frame and the background was subtracted. The time course of the ‘fraction of phase 2’ during the rapid-mixing was calculated from the area of the corresponding first-order reflection with respect to the sum of the peak-areas of phase 1 and phase 2 [26].
3. Results and discussion In Fig. 1 the phase separation induced in the La-phase is compared for two differently prepared samples of POPC, i.e., one sample has been hydrated from dry powder and the other from dry film (see Section 2.1). Each sample consists of two distinct lamellar phases coexisting at 2°C. The phase with the greater d-spacing (phase 1) typi-
Table 3 Structural parameters for the POPC dispersions at 2°C. The phase separation results are compared with POPC 20% w/w in pure water, which have been taken from [30]a POPC 20% w/w pure water
A (A, 2) d (A, ) dC (A, ) dB’ (A, ) dW’ (A, ) nW nW’
57 66.2 16.1 50.1 16.1 22 6
POPC 40% w/w 0.88 M LiCl (lipid/salt molar ratio 1:1) Phase 1
Phase 2
57 64.7 15.9 49.8 14.9 21 7
56 58.8 16.3 50.6 8.2 14 6
a A, area per phospholipid moleculed; d, spacing; dC, hydrocarbon chain length; dB’, bilayer thickness (steric definition [30]); dW’, interbilayer water (solution) thickness; nW, number of waters per lipid molecule; nW’, number of bound waters per lipid head group.
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Fig. 6. Contour plot of a series of SAXS-pattern during the rapid mixing of POPC/water dispersion with an 0.33 M LiCl solution at 10°C. For better contrast the 1st order diffraction maxima are white, while the 2nd order diffraction maxima are black. The reaction from fully hydrated bilayer stacks in phase 1 to the partially dehydrated phase 2 was triggered at the beginning of the experiment, and the complete turnover was followed over a period of 15 min. Note: in the 2nd order regions are spurs of another intermediate phase visible. The d-spacings of the phase 1 and phase 2, respectively, are 64.5 and 59.1 A, .
cally shows the same d-spacing as in pure water [1], whereas the phase with the smaller d-spacing (phase 2) may vary, depending on the salt-concentration. For the ternary LiCl– POPC – H2O system, a maximum deviation of about 6 A, was found [1]. The present experiments (Fig. 1) reveal that the multilamellar vesicles, which have been hydrated from dry film, exhibit a greater amount of phase 2, although the chemical compositions are the same. The findings can be explained by assuming that the salt-gradient from the excess solution region to the inside of the each liposome is greater in systems hydrated from dry film. This argument is supported by the notion that the liposome formation from dry film will take place
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with less structural defects, since the dehydrated film features already a layered structure, whereas the powder contains randomly ordered crystallites (Fig. 2). As the bilayers present a considerable diffusion barrier to the lithium ions (Pm : 10 − 12 cm/s − 1 [27]), structural defects display an important alternative route for a faster solute transport across the membrane. Thus, the vesicles, which have been hydrated from dry film will sense a stronger osmotic pressure (see Fig. 2). This may in turn cause the increase of the fraction of phase 2. The results of the different d-spacings and induced fraction of phase 2 are summarised in Table 1. In Fig. 3 the splitting of the diffraction peaks into two discrete components are presented in detail for a 40% w/w POPC dispersion in 0.88 M LiCl. The recorded orders of the phase with the largest d-spacing (d =64.7 A, ) — designated as phase 1 — are h= 1, 2, 3, 5, whereas the first four orders are observed for phase 2, the phase with the smallest d-spacing (d =58.8 A, ). The diffraction pattern has been fitted with a MCT structure factor according to [14,23] (see Section 2.3); the fit results are given in Table 2. The continuous transform of the form factors, given by the sampling theorem [28]
Fig. 7. Sketch of the proposed phase separation in a liposome. Solid lines represent bilayers in phase 1 and phase 2, respectively. Throughout osmosis the water flux (bold arrows) is directed outwards the liposome, while the very slow salt flux (dashed arrow) is directed inwards. Between phase 1 and phase 2 a disordered bilayer region is presented by dashed lines.
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have been calculated from the published results for oriented POPC bilayers at 70, 80 and 100% relative humidity. The transforms systematically expand outward with increasing levels of hydration, thus the motif of phase 2 compares best to samples with relative humidities below 70%, i.e., phase 2 appears to be partially dehydrated. Finally, using the appropriate phase-factors h1 = (−1,− 1,+ 1,/, − 1) for phase 1 and h2 = (−1,− 1,+ 1,−1) for phase 2 and the resulting form factors from the MCT-fits (cf. Fig. 2 and Table 2) the electron density profiles have been calculated with the standard Fourier synthesis (Fig. 5). The density profiles have been set on an absolute scale according to the formalism described by Petrache [23], where the lipid volume, VL = 1223 A, 3, was extracted from volumetric
Fig. 8. Diffraction pattern of (A) DMPC at 26°C and (B) DPPC at 45°C before (dashed line) and to the end of the experiment (solid line) after rapid mixing with 0.33 M LiCl solution. The complete reaction was followed over a period of 15 min. hmax
sin[y(s d − h)] y(s d − h)
F(s)= % hhFh h=0
(1)
is usually taken as a proof that the features of the bilayer structure remain invariant [22,29] during swelling experiments. Fig. 4a shows a plot of the continuous transform of the pure water phase (POPC 20% w/w; results were taken from [30]). The form factors Fh of phase 1 (solid circles) lie on about the same curve. This is — besides nearly identical d-spacing — another indication that the two phases are structurally very similar; small differences may occur from altered lipid concentrations, and the deviation of the 5th order is hardly significant within the counting statistics. The continuous transform of phase 2 is plotted together with the experimental results of Katsaras and co-workers [29] in Fig. 4b. The transforms
Fig. 9. Fraction of phase 2 during the first few seconds after the rapid mixing with 0.33 M LiCl. The half time, t1/2, is defined as the time, when 50% of final fraction of phase 2 are attained. The reaction of (A) the DMPC/water system is about twice as slow as (B) the DPPC/water system.
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measurements on POPC by Hianik et al. [31]. It has been assumed that the lipid volume is not changed upon the phase separation. The electron density of pure water is 0.333 e A, − 3, and 0.311 e A, − 3 for a 0.88 M LiCl solution is, which corresponds to the offset of the profiles. Table 3 lists the results for the structural parameters of phase 1 and 2 together with the POPC results in pure water [30]. Structural parameters have been obtained according to the procedure introduced by McIntosh and Simon [32] and Nagle and co-workers [22]. The parameters of phase 1 differ only marginally and within the error-bars from the parameters of the pure water phase, thus, leading to the notion that phase 1 has the same lamellar structure as the fully hydrated ‘pure water’ phase. On the other hand, comparing the structural parameters of phase 1 with the ones of phase 2 reveals a main difference in the interbilayer water spacing by about 7 A, , which corresponds to about 7 water molecules less per lipid for phase 2 (Table 3). Referring to the interbilayer water thickness this means a decrease of 3.5 A, per lipid which can be explained by a loss of one hydration layer (d 3 A, ) per phospholipid head group. A further difference is seen in the reduced density of the interbilayer region. In fact, the electron densities of hydrated lithium and chloride ions are with values of 0.24–0.27 e A, − 3 and 0.18 e A, − 3, respectively, lower than the density of water. Therefore, the presence of salt in the interbilayer region of phase 2 could explain the relatively lower density. Giving a first summary of the characteristics of phase 2, the two major findings are: (1) with respect to the ‘pure water’ situation the phase is partially dehydrated, which is (2) most likely osmotically induced by a salt gradient from the outside to the inside of the multilamellar vesicles. This encouraged us to carry out rapid mixing experiments. Here, the salt distribution is strictly defined, i.e., the ions are originally outside the vesicles at the time the lipid dispersion is mixed with the salt solution. Thereafter the ions may diffuse (e.g., via defects) into the interbilayer regime. Fig. 6 presents the events after a rapid-mixing of a LiCl-solution with a POPC/H2O as observed
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by time-resolved X-ray diffraction. The lipid-system reacts immediately to the addition of salt by the formation of a thin La-phase, which is completed after 1–2 min. Only a small amount (B 3%) remains in the original phase 1. We note, that the difference in the d-spacing of 5.4 A, between the parent and nascent lattice agrees well with the maximum observed splitting in previous experiments [1] (cf. Table 1). Furthermore, traces of a third intermediate lattice (d=60.1 A, ) are visible in the second-order regime, as indicated by the dashed arrow. Such a third intermediate lattice has also been reported to occur in other lipid systems, e.g., EYPC [1]. However, it must be underlined that this repeat distance is rarely expressed in a sharp Bragg reflection, but in the majority of cases as a diffuse scattering contribution between the reflections of phase 1 and 2. After rapid mixing, the water and ion flux across the bilayers takes place in opposite directions, i.e., water is osmotically driven out of the multilamellar vesicles and ions into the interbilayer water regions. Since the ion transport takes place on a much longer time scale (see above), the reaction of the system will be dominated by a partial dehydration of the liposomes. Therefore, we assume that phase 2 forms shell by shell from outside to inside (see Fig. 7). In this picture the phase with the intermediate lattice could play a geometrically matching role between phase 1 and phase 2 [33]. Similar rapid mixing experiments have been performed with DMPC and DPPC dispersions. The experimental results are summarised in Figs. 8 and 9. Both systems form a partially dehydrated liquid crystalline phase after the mixing (Fig. 8, solid line). The nascent phase 2 does not only have a reduced d-spacing, but also displays sharper Bragg peaks up to the 3rd order. This is a clear sign for an increase in long-range order (reduced disorder of the 2nd kind [34]), as also demonstrated in various osmotically dehydrated phosphatidylcholine systems [32,35]. However, while the DPPC sample completely converts from phase 1 to 2, the DMPC sample displays a broad diffuse scattering around the original diffraction angle of s= 0.015 A, − 1, i.e., the turnover in the DMPC system is only up to 80% complete. Most
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likely, DMPC vesicles are not as stable as DPPC vesicles, so that defects or even rupture might cause highly disordered bilayer stacking. Therefore, a reduced ion-gradient throughout defected DMPC-vesicle and their leakage may also explain the two-fold lower formation rate of the phase 2 (Fig. 9). While the thickness of the hydrophobic region should have only a minor influence on water permeability, the different occurrence of fluctuating lattice defects in DMPC and DPPC systems could be of considerable influence in the osmotically driven water flux [36]. As an outlook for future investigations we would like to point out two main issues. A conspicuous feature in the presented results for the POPC/LiCl/H2O system under relatively high salt conditions is the occurrence of the discrete splitting of about 6 A, , which has been demonstrated to be due to a dehydration corresponding to two water layers. The natural question arising is: do also other physico-chemical conditions exist for phosphatidylcholine systems, which prefer discrete hydration steps (see Fig. 6)? We note, that commonly applied osmotic pressure techniques [37] may induce a continuous variation of interbilayer distances, as demonstrated, e.g., in EYPC and DMPC [32]. The second point concerns the effect of lithium in combination with antidepressants. It is known that the fatty acid composition of brain glycerophospholipids in, for example, peroxisomal disorders is altered [38], and hence, probably also the hydration state of myelin. Thus, with the above results given, lithium should also dehydrate myelin. It could be an interesting question to investigate this effect of lithium with respect to its pharmological potential in the treatment of manic-depressive diseases.
[5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
[26]
[27] [28] [29]
References [30] [1] M. Rappolt, K. Pressl, G. Pabst, P. Laggner, Biochim. Biophys. Acta 1372 (1998) 389. [2] M.T. Diog, M.G. Heyl, D.F. Martin, J. Chem. Educ. 50 (5) (1973) 343. [3] N.J. Birch, Lithium and the Cell. Pharmacology and Biochemistry, Academic Press, London, 1991. [4] J. Seelig, P.M. Macdonald, P.G. Scherrer, Biochemistry 26 (1987) 7535.
[31]
[32] [33] [34]
M.F. Brown, J. Seelig, Nature 269 (1977) 7535. H. Akutsu, J. Seelig, Biochemistry 20 (1981) 7366. H. Akutsu, T. Nagamori, Biochemistry 30 (1991) 4510. K. Pressl, K. Jørgensen, P. Laggner, Biochim. Biophys. Acta 1325 (1997) 1. H.L. Casal, A. Martin, H.H. Mantsch, F. Paltauf, H. Hauser, Biochemistry 26 (1987) 7395. J. Mattai, H. Hauser, R.A. Demel, G.G. Shipley, Biochemistry 28 (1989) 2322. H. Hauser, G.G. Shipley, Biochemistry 23 (1984) 34. H. Hauser, G.G. Shipley, Biochim. Biophys. Acta 813 (1985) 343. A.K. Hinderliter, J. Huang, G.W. Feigenson, Biophys. J. 67 (1994) 1906. R. Zhang, W. Sun, S. Tristram-Nagle, R.L. Headrick, T.C. Irving, R.M. Suter, J.F. Nagle, Biophys. J. 70 (1996) 369. H. Amenitsch, M. Rappolt, M. Kriechbaum, H. Mio, P. Laggner, S. Bernstorff, J. Synchrotron Rad. 5 (1998) 506. S. Bernstorff, H. Amenitsch, P. Laggner, Synchrotron Rad. 5 (1998) 1215. A. Gabriel, Rev. Sci. Instrum. 48 (1977) 1303. T.C. Huang, H. Toraya, T.N. Blanton, Y.J. Wu, Appl. Cryst. 26 (1993) 180. M. Rappolt, G. Rapp, Ber. Bunsenges. Phys. Chem. 100 (1996) 1153. R. Hosemann, S.N. Bagchi, Direct Analysis of Diffraction by Matter, North-Holland, Amsterdam, 1962. A. Guinier, X-ray Diffraction, Freeman, San Francisco, 1963. J.F. Nagle, R. Zhang, S. Tristram-Nagle, W.-J. Sun, H.I. Petrache, S.M. Suter, Biophys. J. 70 (1996) 1419. H.I. Petrache, 1998. Ph.D. thesis, Carnegie Melon University. C. Markwardt, Documentation for MPFIT, internet site: http://cow.physics.wisc.edu/ craigm/idl/idl.html. B.S. Garbow, K.E. Hillstrom, J.J. Moore, Documentation for MINPACK, internet site: http://www.zrz.tuberlin.de/ zrz/software/numerik/minpack. H.P. Klug, L.E. Alexander, X-ray Diffraction Procedures: for Polycrystalline and Amorphous Materials, Wiley, New York, 1974. G. Cevc, in: G. Cevc (Ed.), Phospholipids Handbook, Marcel Dekker, New York, 1993 Chap. 18. C.E. Shannon, Proc. I.R.E.N.Y. 37 (1949) 10. J. Katsaras, K.R. Jeffrey, D.S.-C. Yang, R.M. Epand, Biochemistry 32 (1993) 10700. G. Pabst, M. Rappolt, H. Amenitsch, P. Laggner, Phys. Rev. E, in press. T. Hianik, M. Haburcak, K. Lohner, E. Prenner, F. Paltauf, A. Hermetter, Colloids Surfaces A 139 (1998) 189. T.J. McIntosh, S.A. Simon, Biochemistry 25 (1986) 4948. P. Laggner, M. Kriechbaum, Chem. Phys. Lipids 57 (1991) 121. A.E. Blaurock, Biochim. Biophys. Acta 650 (1982) 167.
M. Rappolt et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 183–185 (2001) 171–181 [35] H.I. Petrache, S. Tristram-Nagle, J.F. Nagle, Chem. Phys. Lipids 95 (1998) 83. [36] M. Jansen, A. Blume, Biophys. J. 68 (1995) 997.
.
181
[37] T.J. McIntosh, S.A. Simon, Annu. Rev. Biophys. Biomol. Struct. 23 (1994) 27. [38] M. Martinez, I. Mougan, Lipids 34 (7) (1999) 733.
Salt-induced phase separation in the liquid crystalline phase of phosphatidylcholines Michael Rappolt, Georg Pabst, Heinz Amenitsch, Peter Laggner * Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Steyrergasse 17, A-8010 Graz, Austria
Abstract The effects of alkali chlorides on multilamellar vesicles of various phosphatidylcholines in the lamellar liquid crystalline La-phase were investigated by using small-angle X-ray scattering. At alkali chloride concentrations above 70 mM (LiCl) a phase separation in the liquid crystalline phase of POPC is induced. The splitting of the first and second order diffraction peaks into two major discrete components indicates a separation into different lamellar liquid crystalline (smectic A) phases. Detailed data-analysis applying the modified Caille´ theory proves that the phases mainly differ in the interbilayer water thickness by about two hydration layers. The lipid bilayer profile itself remains essentially the same in all liquid crystalline phases. A comparison of differently prepared samples and rapid mixing experiments in combination with simultaneous time-resolved X-ray diffraction suggest that the phase separation is osmotically driven. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Lecithin; Small-angle X-ray scattering; La-phase; Lithium chloride; Phase separation; Manic depressive disease
1. Introduction In a recent study [1] we have focused on alkaliion induced La-phase separation in the liquid crystalline phosphatidylcholine – water bilayer system by simultaneous small- and wide-angle X-ray scattering studies. Particular attention has been Abbre6iations: DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; EYPC, eggyolk phosphatidylcholine; POPC, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine; SAXS, small-angle X-ray scattering. * Corresponding author. Tel.: +43-316-4120300; fax + 43316-4120390. E-mail address: [email protected] (P. Laggner).
given to the lightest alkali metal, lithium, also because of its pharmacological potential in the treatment of manic-depressive diseases [2,3]. Alkali ions have considerable effects on the structural and conformational behaviour of phospholipid bilayers. Phosphatidylcholine head groups behave as ‘sensors’ of the electrostatic charge at the membrane surface [4] such that, for example, ions induce conformational changes in the polar head group region in the liquid crystalline La-phase [5–7], reorder the packing geometry of saturated acyl chains in the ripple phase, and enhance the appearance of the submain transition [8]. The interaction between Li+ and negatively charged phosphatidylserine leads to dehydration of the phosphatidylserine head
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groups, and to the formation of high-melting ion –lipid complexes [9,10], whereas Na+ and K+ only produce minor changes [11,12]. Further, a fluid –fluid phase separation was demonstrated in liquid crystalline phosphatidylserine–phosphatidylcholine mixtures [13]. Until our first report [1], however, phase separation was only known to occur in phospholipid mixtures, and not in one-component phospholipid bilayer systems. Therefore, the effect of induced phase separation has been investigated under various conditions [1] and may be summarised as follows: The alkali chlorides LiCl, KCl and NaCl may induce up to three different, distinct lamellar lattices in
the liquid crystalline phase, e.g., DSPC, POPC and EYPC, where lithium shows the strongest effect with respect to its concentration. The number and repeat distance of the coexisting lamellar phases depend on the nature and concentration of the alkali chloride, the lipid concentration, and on the degree of acyl chain unsaturation. However, within a given ternary system such as LiCl– POPC –H2O the separation effect stays constant above a certain salt concentration. The present work addresses two open questions, the first concerning the structure of the bilayer phases in the presence of LiCl. This is accomplished by applying a modified Caille´ theory (MCT) [14] analysis to a high quality diffraction pattern of the first five diffraction orders of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in an equimolar LiCl solution. The second question aims at the physical origin of the observed phase splitting. Here, X-ray experiments on differently prepared liposomal dispersions, as well as a rapid mixing of the pure sample with the salt solution provide further insight into the phase separation.
2. Materials and methods
2.1. Sample preparation
Fig. 1. Salt induced phase separation in POPC (20% w/w) dispersions at 2°C. Small-angle X-ray diffraction pattern are displayed in the regime covering the first two orders. Solid lines indicate samples, which have been hydrated from dry films, and dashed lines indicate samples, which have been hydrated from dry powder, respectively. (A) The influence of LiCl and (B) KCl, respectively, each with an lipid-to-salt molar ratio of 1:1 is presented.
1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids, Birmingham AL, and used without further purification. Multilamellar liposomes were prepared in two ways: either by dispersing weighted amounts of dry lipid powder, or by dissolving first the lipids in CHCl3/ CH3OH (2:1, v/v). The solutions were then dried under a stream of N2 and after that kept in a vacuum oven overnight. Both, weighted amounts (20–40% w/w) of dry lipid powder and dry lipid films, respectively, were then hydrated in aqueous solution of LiCl and KCl adjusting a salt-to-lipid molar ratio of 1:1. Quartz-bidistilled, deionized water was used throughout. To ensure complete
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Fig. 2. Schematic drawing of the formation of multilamellar liposomes by a hydration from dry film (A) or dry powder (B). (A) In the initial state (i), the dry film features a layered structure, which exhibits only few defects. Thus, during hydration (ii) only a small number of lithium ions (solid circles) penetrate into the interbilayer regime. The result are vesicles, which incorporate far less lithium ions (iii) compared to the excess aqueous solution. The ion gradient induces an osmotic pressure onto the liposomes. (B) The initial state (i) of the dry powder is characterised by randomly ordered crystallites. The high number of defect lines facilitate the diffusion of lithium ions inbetween the lipid bilayers (ii). The defect lines will heal out during the hydration process. However, the fully hydrated vesicles (iii) will contain more ions in the interbilayer regime than in case (A/iii). Thus, the liposomes will be subjected to a weaker osmotic pressure.
Table 1 Overview of salt-induced phase separation in POPC at 2°C Sample treatment
20% 20% 20% 20% a b
w/w w/w w/w w/w
in in in in
0.33 0.33 0.33 0.33
M M M M
KCla KClb LiCla LiClb
Hydration from dry powder. Hydration from dry film.
Fraction of phase 2
d-spacings (A, ) phase 1/phase 2
Dd= d1−d2 (A, )
30% 70% 20% 80%
67.0/62.9 66.7/61.1 66.7/62.0 66.7/61.5
4.1 5.6 4.7 5.2
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Fig. 3. The first five orders (h= 1,…,5) of Bragg peaks of POPC at 2°C (40% w/w in 0.88 M LiCl: lipid/salt molar ratio 1:1) are presented. MCT-fits to the data are shown as solid lines, different components of phase 1 and phase 2, respectively, are given in dotted lines. Within in the first three orders additional diffuse scattering between the Bragg-peaks of phase 1 and 2 was fitted with a Gaussian distribution. The 4th order is only visible for phase 2, while the 5th is only displayed by phase 1. The results of the MCT fit is given in Table 2.
hydration, the lipid dispersions were incubated for about 4 h at least 10°C above the main transition temperature. During this period the lipid dispersions were vigorously vortexed. Aqueous dispersions of this lipids display narrow, co-operative melting transitions within the limits of published values, thus proving that the lipid purity corresponds to the claimed one of 99%. Thin layer chromatography tests on silica gel-plates 60 (Merck, Darmstadt) before and after the experiments showed no signs of degradation. The solvent used was chloroform/methanol/water (65/25/4).
2.2. X-ray small-angle diffraction Diffraction patterns were recorded on the Austrian SAXS beamline at ELETTRA, Trieste [15,16]
using an one-dimensional position sensitive detector [17] covering the corresponding s-range (s=2 sin(q)/u) of interest from about 1/200 A, − 1 to 1/12 Table 2 MCT [14,23] fit results for POPC 40% w/w in an equimolar LiCl solution at 2°C (see Fig. 3)a Fit parameter
Phase 1
Phase 2
d(A, ) F2/F1 2 F3/F1 2 F4/F1 2 F5/F1 2 p1
64.7 90.1 1.15 90.01 0.271 9 0.01 0 0.125 90.006 0.030 90.004
58.8 90.1 0.667 90.002 0.92 9 0.03 0.63 9 0.05 0 0.070 90.004
(a.u.) (a.u.) (a.u.) (a.u.)
a d, d-spacing; Fi/F1, ratio of the ith form factor to the first diffraction order form factor; p1, Caille´ parameter.
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Fig. 4. Different continuous transforms for POPC in the liquid crystalline phase are given. (A) The transform of phase 1 (POPC 40% w/w 0.88 M LiCl) is identical to fully hydrated POPC (20% w/w) in pure water [30]; a deviation is only seen in the 5th order. (B) The transform of phase 2 is compared to the ones given under different relative humidity (RH) conditions [29].
Fig. 5. Absolute electron densities of POPC at 2°C. Phase 1 and the ‘pure water phase’ [30] are practically identical. In phase 2 the bilayer motif is conserved, but slightly thicker. The interbilayer thickness of phase 2 is clearly reduced and here the density is less than in pure water. The results are summarised in Table 1.
A, − 1. The angular calibration of the detector was performed by silver-behenate (CH3(CH2)20COOAg: d-spacing = 58.38 A, ) [18].
For the X-ray diffraction experiments at thermal equilibrium a sample holder of stainless-steal sealed with 25 mm thick mica windows was used,
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as described previously [19]. Rapid mixing experiments were performed with a stopped flow apparatus (Unisoku Scientific Instruments, Osaka, Japan) in combination with simultaneous time-resolved X-ray diffraction. The device consists of two reservoirs (4 ml each) containing the solutions to be mixed. Each reservoir is connected to a pneumatically driven syringe with a volume of 0.3 ml for each shot. When actuated by an electronic trigger signal both solutions are injected into the X-ray mixing chamber (specified dead time less than 1 ms). The X-ray cell is contained in a ceramic block (1×1 × 1 cm) with an optical path length of 1 mm between two sapphire windows of 0.05 mm thickness each (transmission 0.55 at 8 keV). Fresh lipid dispersions (no salt) of 20% w/w of DMPC and DPPC were mixed with 0.33 M LiCl-solutions reaching a final salt-to-lipid ratio of 1:1. For POPC several diluted samples (6% w/w) of the same stock were investigated to check the reproducibility. Each experiment consisted of 460 time frames in which the rapid mixing was triggered at the beginning of the first frame. Here, the maximum time resolution was 50 ms. The complete reaction was followed over a period of 15 min.
2.3. Data analysis The static diffraction patterns were analysed in terms of the modified Caille´ theory (MCT) [2], which has been demonstrated to be superior to
the paracrystalline theory [20,21] by Nagle and co-workers [22]. The program written in the IDL (Interactive Data Language) is based on the FORTRAN version by Petrache [23] and uses a minimization routine MPFIT [24] that is based on the MINPACK library [25]. The Bragg peaks were fitted within an area of Ds = 9 0.0017 A, − 1 from the respective peak centre position for all recorded orders at once. The model function was chosen to be the sum of two MCT and one Gaussian peak for each order of diffraction. The Gaussian accounts for the diffuse scattering between the Bragg peaks. The raw data of the time-resolved experiments were normalised for the integration time of each time-frame and the background was subtracted. The time course of the ‘fraction of phase 2’ during the rapid-mixing was calculated from the area of the corresponding first-order reflection with respect to the sum of the peak-areas of phase 1 and phase 2 [26].
3. Results and discussion In Fig. 1 the phase separation induced in the La-phase is compared for two differently prepared samples of POPC, i.e., one sample has been hydrated from dry powder and the other from dry film (see Section 2.1). Each sample consists of two distinct lamellar phases coexisting at 2°C. The phase with the greater d-spacing (phase 1) typi-
Table 3 Structural parameters for the POPC dispersions at 2°C. The phase separation results are compared with POPC 20% w/w in pure water, which have been taken from [30]a POPC 20% w/w pure water
A (A, 2) d (A, ) dC (A, ) dB’ (A, ) dW’ (A, ) nW nW’
57 66.2 16.1 50.1 16.1 22 6
POPC 40% w/w 0.88 M LiCl (lipid/salt molar ratio 1:1) Phase 1
Phase 2
57 64.7 15.9 49.8 14.9 21 7
56 58.8 16.3 50.6 8.2 14 6
a A, area per phospholipid moleculed; d, spacing; dC, hydrocarbon chain length; dB’, bilayer thickness (steric definition [30]); dW’, interbilayer water (solution) thickness; nW, number of waters per lipid molecule; nW’, number of bound waters per lipid head group.
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Fig. 6. Contour plot of a series of SAXS-pattern during the rapid mixing of POPC/water dispersion with an 0.33 M LiCl solution at 10°C. For better contrast the 1st order diffraction maxima are white, while the 2nd order diffraction maxima are black. The reaction from fully hydrated bilayer stacks in phase 1 to the partially dehydrated phase 2 was triggered at the beginning of the experiment, and the complete turnover was followed over a period of 15 min. Note: in the 2nd order regions are spurs of another intermediate phase visible. The d-spacings of the phase 1 and phase 2, respectively, are 64.5 and 59.1 A, .
cally shows the same d-spacing as in pure water [1], whereas the phase with the smaller d-spacing (phase 2) may vary, depending on the salt-concentration. For the ternary LiCl– POPC – H2O system, a maximum deviation of about 6 A, was found [1]. The present experiments (Fig. 1) reveal that the multilamellar vesicles, which have been hydrated from dry film, exhibit a greater amount of phase 2, although the chemical compositions are the same. The findings can be explained by assuming that the salt-gradient from the excess solution region to the inside of the each liposome is greater in systems hydrated from dry film. This argument is supported by the notion that the liposome formation from dry film will take place
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with less structural defects, since the dehydrated film features already a layered structure, whereas the powder contains randomly ordered crystallites (Fig. 2). As the bilayers present a considerable diffusion barrier to the lithium ions (Pm : 10 − 12 cm/s − 1 [27]), structural defects display an important alternative route for a faster solute transport across the membrane. Thus, the vesicles, which have been hydrated from dry film will sense a stronger osmotic pressure (see Fig. 2). This may in turn cause the increase of the fraction of phase 2. The results of the different d-spacings and induced fraction of phase 2 are summarised in Table 1. In Fig. 3 the splitting of the diffraction peaks into two discrete components are presented in detail for a 40% w/w POPC dispersion in 0.88 M LiCl. The recorded orders of the phase with the largest d-spacing (d =64.7 A, ) — designated as phase 1 — are h= 1, 2, 3, 5, whereas the first four orders are observed for phase 2, the phase with the smallest d-spacing (d =58.8 A, ). The diffraction pattern has been fitted with a MCT structure factor according to [14,23] (see Section 2.3); the fit results are given in Table 2. The continuous transform of the form factors, given by the sampling theorem [28]
Fig. 7. Sketch of the proposed phase separation in a liposome. Solid lines represent bilayers in phase 1 and phase 2, respectively. Throughout osmosis the water flux (bold arrows) is directed outwards the liposome, while the very slow salt flux (dashed arrow) is directed inwards. Between phase 1 and phase 2 a disordered bilayer region is presented by dashed lines.
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have been calculated from the published results for oriented POPC bilayers at 70, 80 and 100% relative humidity. The transforms systematically expand outward with increasing levels of hydration, thus the motif of phase 2 compares best to samples with relative humidities below 70%, i.e., phase 2 appears to be partially dehydrated. Finally, using the appropriate phase-factors h1 = (−1,− 1,+ 1,/, − 1) for phase 1 and h2 = (−1,− 1,+ 1,−1) for phase 2 and the resulting form factors from the MCT-fits (cf. Fig. 2 and Table 2) the electron density profiles have been calculated with the standard Fourier synthesis (Fig. 5). The density profiles have been set on an absolute scale according to the formalism described by Petrache [23], where the lipid volume, VL = 1223 A, 3, was extracted from volumetric
Fig. 8. Diffraction pattern of (A) DMPC at 26°C and (B) DPPC at 45°C before (dashed line) and to the end of the experiment (solid line) after rapid mixing with 0.33 M LiCl solution. The complete reaction was followed over a period of 15 min. hmax
sin[y(s d − h)] y(s d − h)
F(s)= % hhFh h=0
(1)
is usually taken as a proof that the features of the bilayer structure remain invariant [22,29] during swelling experiments. Fig. 4a shows a plot of the continuous transform of the pure water phase (POPC 20% w/w; results were taken from [30]). The form factors Fh of phase 1 (solid circles) lie on about the same curve. This is — besides nearly identical d-spacing — another indication that the two phases are structurally very similar; small differences may occur from altered lipid concentrations, and the deviation of the 5th order is hardly significant within the counting statistics. The continuous transform of phase 2 is plotted together with the experimental results of Katsaras and co-workers [29] in Fig. 4b. The transforms
Fig. 9. Fraction of phase 2 during the first few seconds after the rapid mixing with 0.33 M LiCl. The half time, t1/2, is defined as the time, when 50% of final fraction of phase 2 are attained. The reaction of (A) the DMPC/water system is about twice as slow as (B) the DPPC/water system.
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measurements on POPC by Hianik et al. [31]. It has been assumed that the lipid volume is not changed upon the phase separation. The electron density of pure water is 0.333 e A, − 3, and 0.311 e A, − 3 for a 0.88 M LiCl solution is, which corresponds to the offset of the profiles. Table 3 lists the results for the structural parameters of phase 1 and 2 together with the POPC results in pure water [30]. Structural parameters have been obtained according to the procedure introduced by McIntosh and Simon [32] and Nagle and co-workers [22]. The parameters of phase 1 differ only marginally and within the error-bars from the parameters of the pure water phase, thus, leading to the notion that phase 1 has the same lamellar structure as the fully hydrated ‘pure water’ phase. On the other hand, comparing the structural parameters of phase 1 with the ones of phase 2 reveals a main difference in the interbilayer water spacing by about 7 A, , which corresponds to about 7 water molecules less per lipid for phase 2 (Table 3). Referring to the interbilayer water thickness this means a decrease of 3.5 A, per lipid which can be explained by a loss of one hydration layer (d 3 A, ) per phospholipid head group. A further difference is seen in the reduced density of the interbilayer region. In fact, the electron densities of hydrated lithium and chloride ions are with values of 0.24–0.27 e A, − 3 and 0.18 e A, − 3, respectively, lower than the density of water. Therefore, the presence of salt in the interbilayer region of phase 2 could explain the relatively lower density. Giving a first summary of the characteristics of phase 2, the two major findings are: (1) with respect to the ‘pure water’ situation the phase is partially dehydrated, which is (2) most likely osmotically induced by a salt gradient from the outside to the inside of the multilamellar vesicles. This encouraged us to carry out rapid mixing experiments. Here, the salt distribution is strictly defined, i.e., the ions are originally outside the vesicles at the time the lipid dispersion is mixed with the salt solution. Thereafter the ions may diffuse (e.g., via defects) into the interbilayer regime. Fig. 6 presents the events after a rapid-mixing of a LiCl-solution with a POPC/H2O as observed
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by time-resolved X-ray diffraction. The lipid-system reacts immediately to the addition of salt by the formation of a thin La-phase, which is completed after 1–2 min. Only a small amount (B 3%) remains in the original phase 1. We note, that the difference in the d-spacing of 5.4 A, between the parent and nascent lattice agrees well with the maximum observed splitting in previous experiments [1] (cf. Table 1). Furthermore, traces of a third intermediate lattice (d=60.1 A, ) are visible in the second-order regime, as indicated by the dashed arrow. Such a third intermediate lattice has also been reported to occur in other lipid systems, e.g., EYPC [1]. However, it must be underlined that this repeat distance is rarely expressed in a sharp Bragg reflection, but in the majority of cases as a diffuse scattering contribution between the reflections of phase 1 and 2. After rapid mixing, the water and ion flux across the bilayers takes place in opposite directions, i.e., water is osmotically driven out of the multilamellar vesicles and ions into the interbilayer water regions. Since the ion transport takes place on a much longer time scale (see above), the reaction of the system will be dominated by a partial dehydration of the liposomes. Therefore, we assume that phase 2 forms shell by shell from outside to inside (see Fig. 7). In this picture the phase with the intermediate lattice could play a geometrically matching role between phase 1 and phase 2 [33]. Similar rapid mixing experiments have been performed with DMPC and DPPC dispersions. The experimental results are summarised in Figs. 8 and 9. Both systems form a partially dehydrated liquid crystalline phase after the mixing (Fig. 8, solid line). The nascent phase 2 does not only have a reduced d-spacing, but also displays sharper Bragg peaks up to the 3rd order. This is a clear sign for an increase in long-range order (reduced disorder of the 2nd kind [34]), as also demonstrated in various osmotically dehydrated phosphatidylcholine systems [32,35]. However, while the DPPC sample completely converts from phase 1 to 2, the DMPC sample displays a broad diffuse scattering around the original diffraction angle of s= 0.015 A, − 1, i.e., the turnover in the DMPC system is only up to 80% complete. Most
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likely, DMPC vesicles are not as stable as DPPC vesicles, so that defects or even rupture might cause highly disordered bilayer stacking. Therefore, a reduced ion-gradient throughout defected DMPC-vesicle and their leakage may also explain the two-fold lower formation rate of the phase 2 (Fig. 9). While the thickness of the hydrophobic region should have only a minor influence on water permeability, the different occurrence of fluctuating lattice defects in DMPC and DPPC systems could be of considerable influence in the osmotically driven water flux [36]. As an outlook for future investigations we would like to point out two main issues. A conspicuous feature in the presented results for the POPC/LiCl/H2O system under relatively high salt conditions is the occurrence of the discrete splitting of about 6 A, , which has been demonstrated to be due to a dehydration corresponding to two water layers. The natural question arising is: do also other physico-chemical conditions exist for phosphatidylcholine systems, which prefer discrete hydration steps (see Fig. 6)? We note, that commonly applied osmotic pressure techniques [37] may induce a continuous variation of interbilayer distances, as demonstrated, e.g., in EYPC and DMPC [32]. The second point concerns the effect of lithium in combination with antidepressants. It is known that the fatty acid composition of brain glycerophospholipids in, for example, peroxisomal disorders is altered [38], and hence, probably also the hydration state of myelin. Thus, with the above results given, lithium should also dehydrate myelin. It could be an interesting question to investigate this effect of lithium with respect to its pharmological potential in the treatment of manic-depressive diseases.
[5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
[26]
[27] [28] [29]
References [30] [1] M. Rappolt, K. Pressl, G. Pabst, P. Laggner, Biochim. Biophys. Acta 1372 (1998) 389. [2] M.T. Diog, M.G. Heyl, D.F. Martin, J. Chem. Educ. 50 (5) (1973) 343. [3] N.J. Birch, Lithium and the Cell. Pharmacology and Biochemistry, Academic Press, London, 1991. [4] J. Seelig, P.M. Macdonald, P.G. Scherrer, Biochemistry 26 (1987) 7535.
[31]
[32] [33] [34]
M.F. Brown, J. Seelig, Nature 269 (1977) 7535. H. Akutsu, J. Seelig, Biochemistry 20 (1981) 7366. H. Akutsu, T. Nagamori, Biochemistry 30 (1991) 4510. K. Pressl, K. Jørgensen, P. Laggner, Biochim. Biophys. Acta 1325 (1997) 1. H.L. Casal, A. Martin, H.H. Mantsch, F. Paltauf, H. Hauser, Biochemistry 26 (1987) 7395. J. Mattai, H. Hauser, R.A. Demel, G.G. Shipley, Biochemistry 28 (1989) 2322. H. Hauser, G.G. Shipley, Biochemistry 23 (1984) 34. H. Hauser, G.G. Shipley, Biochim. Biophys. Acta 813 (1985) 343. A.K. Hinderliter, J. Huang, G.W. Feigenson, Biophys. J. 67 (1994) 1906. R. Zhang, W. Sun, S. Tristram-Nagle, R.L. Headrick, T.C. Irving, R.M. Suter, J.F. Nagle, Biophys. J. 70 (1996) 369. H. Amenitsch, M. Rappolt, M. Kriechbaum, H. Mio, P. Laggner, S. Bernstorff, J. Synchrotron Rad. 5 (1998) 506. S. Bernstorff, H. Amenitsch, P. Laggner, Synchrotron Rad. 5 (1998) 1215. A. Gabriel, Rev. Sci. Instrum. 48 (1977) 1303. T.C. Huang, H. Toraya, T.N. Blanton, Y.J. Wu, Appl. Cryst. 26 (1993) 180. M. Rappolt, G. Rapp, Ber. Bunsenges. Phys. Chem. 100 (1996) 1153. R. Hosemann, S.N. Bagchi, Direct Analysis of Diffraction by Matter, North-Holland, Amsterdam, 1962. A. Guinier, X-ray Diffraction, Freeman, San Francisco, 1963. J.F. Nagle, R. Zhang, S. Tristram-Nagle, W.-J. Sun, H.I. Petrache, S.M. Suter, Biophys. J. 70 (1996) 1419. H.I. Petrache, 1998. Ph.D. thesis, Carnegie Melon University. C. Markwardt, Documentation for MPFIT, internet site: http://cow.physics.wisc.edu/ craigm/idl/idl.html. B.S. Garbow, K.E. Hillstrom, J.J. Moore, Documentation for MINPACK, internet site: http://www.zrz.tuberlin.de/ zrz/software/numerik/minpack. H.P. Klug, L.E. Alexander, X-ray Diffraction Procedures: for Polycrystalline and Amorphous Materials, Wiley, New York, 1974. G. Cevc, in: G. Cevc (Ed.), Phospholipids Handbook, Marcel Dekker, New York, 1993 Chap. 18. C.E. Shannon, Proc. I.R.E.N.Y. 37 (1949) 10. J. Katsaras, K.R. Jeffrey, D.S.-C. Yang, R.M. Epand, Biochemistry 32 (1993) 10700. G. Pabst, M. Rappolt, H. Amenitsch, P. Laggner, Phys. Rev. E, in press. T. Hianik, M. Haburcak, K. Lohner, E. Prenner, F. Paltauf, A. Hermetter, Colloids Surfaces A 139 (1998) 189. T.J. McIntosh, S.A. Simon, Biochemistry 25 (1986) 4948. P. Laggner, M. Kriechbaum, Chem. Phys. Lipids 57 (1991) 121. A.E. Blaurock, Biochim. Biophys. Acta 650 (1982) 167.
M. Rappolt et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 183–185 (2001) 171–181 [35] H.I. Petrache, S. Tristram-Nagle, J.F. Nagle, Chem. Phys. Lipids 95 (1998) 83. [36] M. Jansen, A. Blume, Biophys. J. 68 (1995) 997.
.
181
[37] T.J. McIntosh, S.A. Simon, Annu. Rev. Biophys. Biomol. Struct. 23 (1994) 27. [38] M. Martinez, I. Mougan, Lipids 34 (7) (1999) 733.