Salt Effects on Lamellar Repeat Distance Depending on Head Groups

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Salt effects on lamellar repeat distance depending on head groups of neutrally charged lipids Mafumi Hishida, Yasuhisa Yamamura, and Kazuya Saito Langmuir, Just Accepted Manuscript • DOI: 10.1021/la502576x • Publication Date (Web): 15 Aug 2014 Downloaded from http://pubs.acs.org on August 16, 2014

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Salt Effects on Lamellar Repeat Distance Depending on Head Groups of Neutrally Charged Lipids Mafumi Hishida, Yasuhisa Yamamura, and Kazuya Saito∗ Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan. E-mail: [email protected]

Phone: +81 (29) 853 4239. Fax: +81 (29) 853 6503

Abstract Change in lamellar repeat distances of neutrally charged lipids upon addition of monovalent salts was measured with small-angle X-ray scattering for combinations of two lipids (PC and PE lipids) and six salts. Large dependence on lipid head group is observed in addition to those on added cation and anion. The ion and lipid dependences have little correlation with measured surface potentials of lipid membranes. These results indicate that the lamellar swelling by salt is not explained through balance among interactions considered previously (van der Waals interaction, electrostatic repulsion emerged by ion binding, etc.). It is suggested that effect of water structure, which is affected by not only ions but also lipid itself, should be taken into account for understanding membrane-membrane interactions, as in Hofmeister effect.

∗

To whom correspondence should be addressed

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Introduction Self-assembly of various kinds of soft matter or biomolecules coexisting with electrolytes is often important for their structures and functions. However, a lot remains to be clarified concerning the effect of electrolytes on interactions between them and, consequently, on the mechanisms of their self-assembly. Especially, the effect on neutrally charged soft matter, which may be related to the so-called “Hofmeister effect”, 1 is poorly understood in contrast to that on charged one through the Debye screening of the electrostatic interactions. 2 Although the mechanism of Hofmeister effect is still a matter of dispute, it is, in some cases, assumed that disturbance of water structure (hydrogenbond network) around soft matter by added ions results in some changes in the interactions between them. 3–5 For example, the clouding behavior of non-ionic surfactant, polymer or protein caused by dehydration 6,7 is strongly affected by added ions likely through the change in the dehydration behavior of the surfactant. On neutrally charged lipids which form bilayer membranes, Hofmeister effect has been investigated mainly focusing on membrane properties or the phase transition behavior 8–12 such as from the gel (L′β ) to the liquid-crystalline (Lα ) phases. In these studies, changes in the membrane property and the phase transition behaviors by addition of salts have reported. It has been assumed that disturbance of water structure by added ions result in the changes. It has been also reported that the ion-induced change in the phase transition behavior depends on ion species 11,12 even when valency of added ions are the same (e.g., Na+ and K+ ). The ion dependence likely confirms the mechanism of Hofmeister effect because ion radius seems related to the strength of disturbance of water structure. The order of ion dependence is so-called Hofmeister series. These results show that the properties of membrane (including phase transitions) are determined by not only membranes themselves but also their surroundings. Concerning interactions between lipid membranes, which certainly through water in between, the effect of water structure has, by contrast, been discounted in the recent two decades, although the effect was suggested in 80’s. 13,14 In the recent studies, the membrane-membrane interactions of lipids or surfactants have been discussed while solely considering four following interactions: 15 2 ACS Paragon Plus Environment

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van der Waals interactions, short-range repulsion due to protrusion of molecules or strongly bound water molecule, 16,17 long-range steric repulsion due to membrane fluctuation 18,19 and electrostatic interaction. The electrostatic interaction is negligible between neutrally charged membranes. Petrache et al. 20 have reported the effect of two monovalent salts (KCl and KBr) on the membranemembrane interactions of neutrally charged membranes of phosphatidylcholine lipid (PC lipid). They have measured the membrane-membrane distance (lamellar repeat distance d) as functions of salt concentrations, since the change in the balance of these interactions is sensitively reflected in d. Their analysis considering the above-mentioned four interactions has beautifully explained the salt-concentration dependences of d without considering the concept of Hofmeister effect: Both KCl and KBr weaken van der Waals interaction almost equally. Binding of Br ion to PC head groups strengthens the electrostatic interaction whereas Cl ion, which does not bind, affects little the electrostatic interaction. Thus, d more largely increases with KBr salt. However, in their study, the numbers of investigated salts and lipid are only two and one, respectively. The interpretation suffers from a risk of oversimplification as they themselves claimed. The reason why the concept of Hofmeister effect has been discounted in the recent studies of membrane-membrane interactions is that water structure in between lipid membranes has been assumed to have less effect on the interactions because much shorter range of hydration (1–2 Å 17 ) than typical d has been assumed. The trend for the discount was strengthened by such successful studies on intrinsic membrane properties as, for example, papers, 16,21,22 which explained the shortrange repulsive interaction between membranes in terms of lipid protrusion and overlap of head groups. On the other hand, one of the present authors recently revealed the existence of long-range hydration regions with a length scale of 10 Å at the surfaces of neutrally charged lipid membranes when slightly perturbed water is included as hydration water by utilizing THz spectroscopy. 23 The long-range hydration state has also been suggested in other systems. 24–27 This suggests that the membrane-membrane interactions are possibly affected by the hydration states because the length scale is comparable to those of above mentioned interactions. More recently the present authors 28 have also demonstrated that the long-range hydration states largely differ for lipid bilayers with


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different lipid head groups. The difference certainly comes from a tiny change in the chemical structure of the head group as evidenced by a similar difference in simpler salt solutions. The different hydration states were found to dominate the behavior of phase transition of the lipids, which is related to the membrane-membrane interactions. 28 These results imply that the concept of Hofmeister effect is valuable for the membrane-membrane interaction. If the effect is significant, it is natural to expect that the ion effect on membrane-membrane interactions depend on ion and lipid species because disturbance of water structure should depend on both of them. Investigating dependences of d on various ions and lipids is therefore important not only to verify the Petrache’s model of the effect of ions on neutrally charged lipid membranes but also to see whether the concept of Hofmeister effect should be involved or not in the interactions of lipid membranes, i.e., whether the long-range hydration state affects the membrane-membrane interactions or not. In the present study, we investigate the effect of coexisting ion on interactions between membranes of neutrally charged phospholipid with six salt and two lipid species. Six salts are all monovalent 1:1 salt (cation: Li, Na, K, anion: Cl, Br). For the two lipids, POPC and POPE with different head groups were used, since the long-range hydration states of these lipids have been found to be largely different. 28 Before the experiment, we determined experimental conditions using DLPC and KBr which were used in the study by Petrache et al. 20 The lamellar repeat distances d were measured by small-angle X-ray scattering (SAXS) against the salt concentration, and the electrostatic surface potentials of these membranes in saline solutions are measured. The results indicate that the effect of ions on the interactions between these membranes cannot be interpreted through the model Petrache et al. suggested. 20 This plausibly suggests that effect of water structure on membrane-membrane interactions should be considered as in Hofmeister effect.


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Experiments Materials and methods Powders of two neutrally charged phospholipids (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)) were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, U.S) and another one (1,2-dilauroyl-sn-glycero-3phosphocholine (DLPC)) was from Wako Pure Chemical Industries, Ltd (Osaka, Japan). DLPC was used for determination of experimental condition described later. They are used without further purifications. Main transition temperatures of POPC, POPE and DLPC bilayers dispersed in water are −2 ◦ C, 25 ◦ C and −1 ◦ C, respectively. Six monovalent salts, LiCl (> 99%), LiBr (> 98%), NaCl (> 99.5%), NaBr (> 99.5%), KCl (> 99%), KBr (> 99%), were from Nacalai tesque, Inc. (Kyoto, Japan) and used without further purifications. Small-angle X-ray scattering were mainly performed at BL6A and BL15A, Photon factory, KEK, Japan. Wavelength of X-ray was 1.5 Å, and the distance between the sample and the detector was about 1 m. The distance was calibrated with a standard sample (silver behenate). The detectors were PILATUS 300K (DECTRIS Ltd. (Baden, Switzerland)) at BL6A and CCD C7300 (Hamamatsu Photonics K.K. (Shizuoka, Japan)) at BL15A. Lamellar repeat distances d of the lipid membranes were obtained from the peak position q = 2π/d. To determine lipid membrane thickness a⊥ by Luzzati’s method, 30 small-angle X-ray scattering was performed using a laboratory instrument NANO-viewer with MicroMax007HF (Rigaku Corporation (Tokyo, Japan)) and a detector PILATUS 100K (DECTRIS Ltd. (Baden, Switzerland)). The sample-to-detector length (about 0.7 m) was also calibrated using silver behenate. All scattering measurements were done at 50 ◦ C, where both POPC and POPE exhibit the liquid-crystalline phase. Electrostatic surface potentials (ζ potentials) of lipid membranes were measured with a microscopetype ζ potential meter (ZEECOM ZC-3000, Microtec Co., Ltd. (Chiba, Japan)). Voltage for electrophoresis was kept at 1.56 V cm−1 and numbers of lipid aggregative particles for measurements were 500. Surface potentials of the lipid aggregative particles were measured at a stationary level.


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Salt concentrations for the measurements were kept at 100 mM, since at higher concentration proper voltage could not be impressed because of electric current in solutions, whereas effect of salt became unclear at lower concentration. All measurements were done only at room temperature (18 − 22 ◦ C). At room temperature POPE in all the 100 mM saline solutions were confirmed to be in the gel phase using DSC, consistently with an existing report. 11 Because the ion-dependent order of surface potentials is not changed by temperature, 29 it is expected that the ion-dependent order for the liquid crystalline phase at 50 ◦ C is the same as that at room temperature for the gel phase.

Sample preparation Since systems under study are ternary, it is not trivial to choose appropriate expression of composition. We can imagine, at least, two mechanisms for the salt effect on membrane-membrane interaction: net interaction resulting from direct intermolecular interaction between lipid and ion(s) or via saline solution through long-range hydration. If the former is the case, the molar ratio between lipid and salt would be of the primary importance. On the other hand, the salt concentration in solution governs the behavior if the latter is. Prior to the experiments using POPC and POPE, therefore, preliminary experiments were performed to discriminate two possible choice of key variable (ratio to lipid, or ratio to water). For the experiments, we prepared another system with DLPC and KBr, which is the same lipid and salt as Petrache’s study. 20 24 samples with different concentrations of DLPC and KBr were prepared. Concentrations of DLPC were 11.1, 33.3, 100 and 300 mM to water, and concentrations of KBr were prepared so that [KBr] : [DLPC] (mol:mol) is to be 1:1, 1:3, 1:9, 1:27, 1:81, 1:243. In these concentrations of lipid, the presence of enough bulk water was confirmed. The lamellar repeat distances d of these 24 samples with respect to salt/lipid molar ratio are depicted in Fig. 1 (a), and in (b) it is depicted with respect to salt/water concentration. It is clear that d differs depending on the concentration of lipid to water when that is depicted with respect to salt/lipid molar ratio. On the other hand, d is independent of the lipid concentration if depicted against the salt concentration to water. These results indicate that the salt concentration 6 ACS Paragon Plus Environment

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to water is crucial to determine the lamellar repeat distance in the lipid concentration range of at least 11.1-300 mM. Similar results were observed with NaBr. (a)

(b) [DLPC] = 11 mM [DLPC] = 33 mM [DLPC] = 100 mM [DLPC] = 300 mM

[DLPC] = 11 mM [DLPC] = 33 mM [DLPC] = 100 mM [DLPC] = 300 mM

Figure 1: Lamellar repeat distance d of DLPC in KBr solutions as a function of (a) salt/lipid molar ratio, and (b) salt concentration (against water).

According to the results of DLPC described above, we fixed the lipid concentration to be 150 mM and each saline solution was prepared with respect to the concentration to water for the study with POPC and POPE. It is noticed that procedure of sample preparation of salt/POPC and salt/POPE is crucially important for the study. Each saline solution (0.1, 1, 10, 30, 100, 300, 1000, 3000 mM) was mixed with the lipid powders so as to achieve 150 mM of lipids concentration. After sonication in a ultrasonic bath (US-101 from SND Co., Ltd. (Nagano, Japan)) for 1 h at 50 ◦

C and placing at room temperature, POPE/salt samples were adequately homogenized, and then

clear single peaks were observed in the measurements of SAXS. On the other hand, POPC/salt solutions could not be homogenized so easily. When the POPC/salt solutions were examined by SAXS after such a simple procedure, many peaks due to a phase separation of lamellar phases were observed with poor reproducibility, indicating that the samples were not fully homogenized. For adequate homogenization of POPC/salt solutions, we first sonicated the solution using a probe type sonicator (UH-50 from SMT Co., Ltd. (Tokyo, Japan)), of which frequency is (20 ± 3) kHz and amplitude about 15 µm. After the high-power sonication, the solutions were frozen and melted for over five times using a −25 ◦ C freezer. This freeze/melt procedure seems important to make POPC/salt solutions homogeneous. 7 ACS Paragon Plus Environment

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For determining the lipid membranes thicknesses a⊥ by Luzzati’s method, 30 the lipid/salt samples were prepared so that bulk water did not exist. The salt concentrations were 3000 mM. In those cases, a⊥ were calculated from the prepared volume ratio of lipid and saline solution using the formula, a⊥ = (VL d)/(VL + nw Vw ), where VL , Vw and nw are volume of a lipid molecule, that of a water molecule and number of water molecule per a lipid, respectively. 30 In the condition without bulk water, some samples (with 3000 mM KCl and 3000 mM NaCl) of POPE exhibited a phase transition to hexagonal phase at 50 ◦ C, even though lamellar phases were exhibited at lower temperature (e.g., at 40 ◦ C). In those cases the lamellar repeat distances at 50 ◦ C were obtained by extrapolating the results at lower temperatures, since the distance was found to change linearly with respect to temperature in other samples which did not exhibit the phase transition.

Results Lamellar repeat distances d of POPC and POPE lipids are depicted in Fig. 2. Behaviors of d of POPC shown in Fig. 2(a) are more complicated than those of POPE. For POPC, clear cation dependence is observed: d with Na or K ions increases with increasing salt concentration whereas it decreases with Li ions. Anion dependence is also exhibited: Salts with Br ions changes d more largely than with Cl ions for all cations. For the same anion, small difference is observed between Na and K ions. Although the behaviors with Li ions are entirely different from Na and K ions at a first glance, the difference is only in the high concentration region over 300 mM. As shown in the inset in Fig. 2(a), d increases in low salt concentration region similarly to that with Na or K ions. Over 300 mM, it suddenly decreases. The reported behavior of PC lipid, 20 which swells more largely with KBr than with KCl, is reproduced in the present study. However, the present results clearly shows that the change in d by adding monovalent salts are more complicated than the report. That is, the lamellar repeat distance d is affected not only by anions but also by cations. For POPE lipid, simpler behaviors of d are observed in Fig. 2 (b). Most apparent is anion dependence. With Cl ions, the POPE lamella swells more largely than with Br ions for all cations.


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(a) 85 KBr NaBr LiBr KCl NaCl LiCl

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Figure 2: Lamellar repeat distances d of (a) POPC and (b) POPE lipids dispersed in water with different six salts with respect to the salt concentrations [salt]. Solid lines are linear fit to data for guides. Inset to (a) separately shows the result of POPC with LiBr.


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Note that this anion dependence of POPE repeat distance is opposite to that of POPC observed in Fig. 2 (a). As in POPC, the behavior of d with Li ions differs from those with Na or K ions, though its difference seems slighter than that of POPC. In the case of POPE, the increase in d with LiCl is just milder than other salts with Cl anions. In our system of POPE, additive-induced phase transition 31,32 has not been observed between 0.1–3000 mM, whereas co-existence with another phase were observed in some 10000 mM saline solutions. The lamellar repeat distance d is the sum of water layer spacing dw and membrane thickness a⊥ ; d = dw + a⊥ . To discuss membrane-membrane interactions which relate to dw , changes in a⊥ induced by added salts are evaluated through Luzzati’s method. 30 a⊥ of pure POPC is found to be 35.9 Å and that of pure POPE is 37.3 Å at 50 ◦ C. The obtained a⊥ in 3000 mM solutions are summarized in Table 1. It was found that a⊥ becomes basically smaller as ion radius becomes larger for both lipids. Calculated dw described in Fig. 3 indicate that dependences of dw on salt species are similar to that of d for both lipids. That is, for POPC, it decreases with Li cations, while the increase with Na and K are larger with Br anion. For POPE, it increases with all salts and increases more largely with Cl anions. Table 1: Lipid membrane thicknesses a⊥ in 3000 mM saline solutions at 50 ◦ C. POPC (Å) Cl Br

Li Na K 36.2 34.8 34.8 35.7 32.1 32.1

POPE (Å) Cl Br

Li Na K 40.5 39.8 38.3 38.9 36.5 35.6

The observed behavior of ion-dependent lamellar swelling is more complicated than expected. Therefore, it seems natural to ask 33 whether it is really possible to interpret the behavior through the ion binding and weakening of van der Waals interaction. Thus, to estimate the ion binding, electrostatic surface potential of each lipid membrane is measured as shown in Fig. 4. The frequency is the number of lipid aggregative particles in the designated range of surface potential. The frequency of the potentials are fitted with a Gaussian and the center values are listed in Table 2. In contrast to the SAXS results, the cation dependence is clearer. For POPC with Li ions, the potentials exhibit positive value, while it is negative with Na and K. K ion makes POPC membrane 10 ACS Paragon Plus Environment

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(a)

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NaCl

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LiCl LiBr

NaBr KBr LiCl LiBr

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Figure 3: Water layer thicknesses dw of (a) POPC and (b) POPE in 3000 mM saline solutions. Crosses are the result of pure POPC and POPE.

more negatively charged than Na ion for both cases with Cl and Br anions. Anion dependence is small for Li ions, whereas for both Na and K cations, the surface potentials become more negative with Br anions than with Cl. It is to be noted that the potentials with KBr are reproducibly more negative than KCl for PC lipid as Petrache et al. 20 reported. The surface potentials of POPE lipid also exhibit the dependence on both anion and cation, although the lamellar repeat distances show milder dependence on cation. In the case that cations are Na and K, the dependences on anion are similar to those of POPC, i.e., more negative surface potential with Br anions. However, it is emphasized that d of POPE has opposite anion dependences to POPC. Surface potentials of POPE with salts of Li cations behave differently to each other. With LiCl, the surface potential of POPE is positive, while it is negative with LiBr. However, the order of cation dependence is the same for both anions: POPE is the most negatively charged with K cation, next with Na, and the least negatively (or positively) charged with Li cations. From these results, adsorption affinities of each ion to the lipid head groups can be ordered. The each order of anion and cation is the same for both lipids; Anion: Br > Cl, Cation: Li > Na > K, even though anion affinity is almost the same with Li for POPC. For POPC, Li ion binds more strongly than anions, leading to positive potential. Thus, the mixed order of anion and cation


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LiBr NaBr KBr

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Figure 4: Electric surface potentials (ζ potentials) of (a) POPC and (b) POPE with different six salts at 100 mM. Solid lines are the results of Gaussian fits to the data. Obtained center value of the surface potentials are summarized in Table 2.


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adsorption is estimated as, POPC: Li > Br > Cl > Na > K, and POPE: Br > Li > Cl > Na > K. The order of Br and Li is reversed between two lipids, accordingly. Table 2: Center values of the surface potentials obtained from the Gaussian fitting for each samples at 100 mM of salt concentration. POPC (mV) Cl Br

Li Na K 2.9 -2.7 -4.9 3.1 -6.6 -10.9

POPE (mV) Cl Br

Li 4.3 -5.1

Na K -3.4 -7.2 -6.4 -10.0

Discussion As described in the introduction, lamellar repeat distance d has been considered to be determined by the balance among four interactions: 15 van del Waals interactions, short-range repulsion 16,17 (decay length 1–2 Å 15 ), long-range steric repulsion due to fluctuation of membranes (sometimes called as Helfrich repulsion) 19 and electrostatic interaction. In cases of monovalent 1:1 salts, the Debye screening length for the electrostatic interaction is numerically calculated as √ 1/κ = (3.04/ [salt]) Å, 2 with the concentration [salt] in M. For example, 1/κ = ca. 30 Å at 10 mM, 10 Å at 100 mM, 4.3 Å at 500 mM, and 3.0 Å at 1000 mM. According to the traditional view, the effect of adding salt primarily affects the (direct) electrostatic interaction. The electrostatic interaction can be discussed using the result of surface potentials and Debye screening length. Since the salts in the present study are all monovalent resulting in the same ionic strengths at the same concentration, Debye length remains the same when salt species is changed. Thus, the electrostatic interaction is only dependent on the surface potential. Since the electrostatic interaction depends on absolute value of the surface potential, the order of strength of the electrostatic interaction is as follows; for POPC, KBr > NaBr > KCl > NaCl ≈ LiBr ≈ LiCl, and for POPE, KBr > KCl > NaBr > LiBr > LiCl > NaCl. This order has little correlation with the lamellar swelling. On the other hand, trends of the change in the membrane thickness a⊥ seem to have correlation with the electrostatic potential. Large electrostatic potential


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due to strong ion binding likely make lateral headgroup-headgroup interaction be repulsive and lead smaller membrane thickness. Although the electrostatic potential is screened, the lamella swells more largely in saline solutions with higher concentration for most of samples. This contradiction has been explained 20 in terms of the screening of van der Waals interaction. Namely, in high salt concentration, van der Waals attraction is weakened, resulting in increased lamellar repeat distance. Since the screening of van der Waals interaction is known to have weak ion-dependence, 20 the ion-specific lamellar swelling cannot be explained by the weakening of van der Waals interaction. The Helfrich repulsion due to fluctuation of membranes is also affected little by salt ions 34 because the bending rigidities of membranes are almost the same with and without salt. The short-range repulsion has, in the first place, a small effect on the lamellar repeat distance, 16,17 though the effect of ions on the repulsion has not been clarified in detail yet. Thus, the little correlation between lamellar swelling and the surface potentials indicates the difficulty in explaining the ion-induced swelling in terms of the four interactions that have commonly been assumed. 15 Calculated potential within the theory is shown in Fig. 5. Here, superimposed are the four interactions: 15,20 van del Waals interaction, the short-range interaction, Helfrich repulsion, and the electrostatic interaction, resulting in a functional form, [ ] ( ) 1 2 1 d − a⊥ H1 − + + Psho λsho exp − F(d) = − 12π (d − a⊥ )2 d2 (d + a⊥ )2 λsho 2 (kB T ) + 0.115 + 2ϵϵ0 κζ 2 exp[−κ(d − a⊥ )], Kc (d − a⊥ )2

(1)

where H1 is Hamaker constant, Psho and λsho prefactor and decay length for short-range repulsion, Kc bending rigidity of a bilayer, ζ surface potential of lipid membrane. H1 is about 1.2 kB T for pure POPC, whereas 0.4 kB T for POPC with LiCl. 20 Other parameters are from the literatures. 35,36 The surface potential at 100 mM LiCl is used as a crude estimate for ζ of POPC with LiCl, which does not alter the quantitative trend. As clearly seen in Fig. 5, the equilibrium lamellar repeat distance of POPC with 3000 mM LiCl should be larger than that without salt, in contrast to the experimental


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results. Thus, an explanation previously suggested for the change in the repeat distance by adding salts seems to be applicable to only four systems of POPC with NaCl, NaBr, KCl and KBr, and incorrect or inadequate in general. It is necessary to establish a new model for explanation with wider applicability.

30 20 10 0 -10 -20

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80

Figure 5: Calculated membrane-membrane interaction potential of POPC without added LiCl (dotted line) and with 3000 mM of LiCl (solid line). With LiCl, Hamaker constant decreases down to about one third of that without salt due to screening, and electrostatic potential emerges because of ion binding. The minimum is at 73 Å with LiCl.

The heterogeneous distribution of ions may bring additional effects such as different osmotic pressure. It is reported that the exclusion of salt from lamellar phase to bulk aqueous phase occurs when monovalent salts are added in the lamellar phase of a neutrally charged lipid. 37 Salts with Cl anion are excluded more strongly than those with Br anions. The ion-dependent exclusion causes different osmotic pressure depending on anion species. However, the osmotic pressure should make the lamellar repeat distance smaller, since the activity of ions is higher in the bulk aqueous phase. The Debye length also changes to be larger according to the exclusion of ions. This elongation possibly causes the lamellar swelling. However, the elongation is estimated as 1.3 Å at most even when a half of 1000 mM salt is excluded from the lamellar phase. This slight elongation is woefully inadequate for the explanation of the swelling. Besides, although the longer


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Debye length is expected with Cl anions, the observed lamellar swelling is larger with Br anions for POPC samples. Entropy of ions may also be another cause. Confined ions in between membranes are unfavorable entropically compared to those in bulk. However, this also has the opposing effect to the experimental observation, since entropically ions should be excluded, reducing lamellar repeat distance. Finally, the influence of hydration states of lipid and salt is considered in relation to Hofmeister effect. As described in the introduction, it has widely been assumed that Hofmeister effect is caused by disturbance on water structure around solutes by added ions. 3–5 The disturbance has considered to exhibit ion dependence, and the terms, “water-structure-maker/-breaker” or “kosmotrope/chaotrope,” are used to categorize ions based on the strength of ion effect on solutes. 38 In Hofmesiter effect, anions are known to have stronger effect than cations. The observed strong anion dependences of the lamellar swellings of both lipids is reminiscent of the effect, as reported for salting-in/-out of proteins. 39 However, the effect of water structure or hydration water on the membrane-membrane interaction had been investigated only incompletely. The effect of disturbance of water structure by ions on the membrane-membrane interaction is entirely a mystery, accordingly. Not only ions but also lipid molecules can affect the water structure between membranes. When the long-range hydration region at the surface of lipid membrane 23 is taken into account, it is natural to imagine that disturbance of water structure by lipids also affect the membrane-membrane interactions as in Hofmeister effect. The hydration states of PC and PE head groups have been found to differ largely in the length scale of 10 Å. 28 In the region of this long-range hydration, dynamics of water molecules is slowed down around PC head group whereas it is accelerated for PE head group. Indeed, in the present results, the strong dependence on lipid of the lamellar swelling is observed between PC and PE lipids. It implies that the swelling is affected by the change in the water structure caused not only by salt but also by lipids. Since ion binding and screening of van der Waals interaction have small lipid dependences, it is concluded that the large difference in the hydration states of lipids causes the largely different behavior of the lamellar swelling between


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POPC and POPE, where anion dependences are opposite. These results suggest that change in water structure by lipids affect the effective membrane-membrane interactions. In other words, water structure exerts a strong effect on the interactions even when ions are not added in solution. Correlation between the hydration state of lipids and the membrane-membrane interaction should be investigated more in detail in future.

Conclusion Large difference in the change in the lamellar repeat distances of neutrally charged lipids, POPC and POPE, dispersed in water was identified upon adding six monovalent salts. The order of iondependences differs between two lipids. Considering the existing explanation of lamellar swellings of two samples (PC lipid with KCl and KBr) in terms of ion binding on the lipid membrane and screening of van der Waals interaction, electrostatic surface potentials of lipids for all twelve samples were measured. In contrast to the explanation, the lamellar swellings have small correlation with the surface potentials of the membranes, though the results for PC lipid with KCl and KBr were reproduced. The change in the four interactions ever considered also does not explain the ion-dependent lamellar swellings. Although definite explanation of the ion-dependent lamellar swellings of neutrally charged lipids is not proposed in the present study, opposing anion dependence between POPC and POPE implies that the lamellar swelling is caused as a result of disturbance on water structure around membranes as in Hofmeister effect. Significant dependence of lamellar swellings on not only added salts but also lipid molecules implies that lipid itself affects the water structure. In other words, the hydration states of lipids are highly important for the membrane-membrane interactions.

Acknowledgement We thank Dr. H. Seto (High Energy Accelerator Research Organization, Japan) for fruitful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research from JSPS


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(Grant No. 42740289) for M.H. The SAXS experiments were performed under the approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2011G516, 2011G550, 2013G525 and 2013G530).

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(39) Baldwin, R. L. How Hofmeister ion interactions affect protein stability. Biophys. J. 1996, 71, 2056–2063.


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Salt Effects on Lamellar Repeat Distance Depending on Head Groups

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