Chemistry and Physics of Lipids 188 (2015) 61–67

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Effect of n-alkanes on lipid bilayers depending on headgroups Mafumi Hishida, Asami Endo, Koyomi Nakazawa, Yasuhisa Yamamura, Kazuya Saito ∗ Department of Chemistry, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan

a r t i c l e

i n f o

Article history: Received 4 March 2015 Received in revised form 28 April 2015 Accepted 4 May 2015 Available online 7 May 2015 Keywords: Phospholipid n-Alkane Phase transition Lipid packing Head group dependence

a b s t r a c t Phase behavior and structural properties were examined for phospholipid bilayers having different headgroups (DMPC, DMPS and DMPE) with added n-alkanes to study effect of flexible additives. Change in the temperatures of main transition of the lipid/alkane mixtures against the length of added alkanes depends largely on the headgroup. Theoretical analysis of the change of the temperature of transition indicates that the headgroup dependence is dominantly originated in the strong dependence of total enthalpy on the headgroups. The results of X-ray diffraction show that the enthalpic stabilization due to enhanced packing of acyl chains of the lipid by alkanes in the gel phase causes the headgroup-dependent change in the phase transition behavior. The enhanced packing in the gel phase also leads to easy emergence of the subgel phase with very short relaxation time at room temperature in the DMPE-based bilayers. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Biomembranes in all cells are mainly composed of phospholipid bilayers facing hydrophobic parts of the molecules inward. Small organic molecules such as sterols and isoprenoids are also included in the biomembranes. The small organic molecules, typified by cholesterol, are lipophilic and are incorporated into the hydrophobic part of the biomembranes. Although these additives in the biomembranes have been known to be crucial for vital functions of biomembranes (Luckey, 2008), physico-chemical understanding of the effect of these additives on membrane physical properties remains insufficient. Molecular effects of additives on membrane properties have been studied for each molecule separately. The effect of cholesterol on synthetic lipid bilayers has been investigated very intensively (Vist and Davis, 1990; Ohvo-Rekilä et al., 2002; Veatch and Keller, 2003; Shimokawa et al., 2010; Needham and Nunn, 1990; Ivankin et al., 2010). It is well known that cholesterol changes bilayer structures, phase transition behaviors, lipid partitioning, membrane physical property such as elasticity, and so on. However, the knowledge applies only to cholesterol. No universal understanding of molecular mechanism of additives has been reached, accordingly. Molecules of many kinds of additives in real biomembranes have a rigid core and a flexible chain. This is also the case for cholesterol or other sterols. Recent studies on liquid-crystals have indicated

∗ Corresponding author. Tel.: +81 298534239. E-mail address: [email protected] (K. Saito). http://dx.doi.org/10.1016/j.chemphyslip.2015.05.002 0009-3084/© 2015 Elsevier Ireland Ltd. All rights reserved.

that a core part and a flexible chain have intrinsic “role” for emergence of various liquid-crystalline phases (Horiuchi et al., 2010; Adachi et al., 2012, 2013; Saito et al., 2013; Miyazawa et al., 2013), though most properties of thermotropic liquid crystals have been considered to be explained assuming simply rigid rods (Onsager, 1949; Bolhuis and Frenkel, 1997). This suggests that a core part and a flexible chain of an additive molecule in biomembranes also play different “roles” on its effects. Thus, here we focus on the “role” of the flexible chain, and investigate the effect of n-alkane on the physical properties of lipid bilayers. Effects of n-alkanes in lipid bilayers have been investigated mainly focusing attentions on the change in phase behaviors (McIntosh et al., 1980; Pope et al., 1989, 1984; Lohner, 1991; Aagaard et al., 2006; Pope and Dubro, 1986). Change in the main transition (between ordered gel phase (L␤ ) and disordered liquidcrystalline phase (L␣ )) of phosphatidylcholine (PC) lipids has been extensively investigated. Such studies have indicated that balance of the lengths of added alkanes and acyl chains of lipids strongly affects the transition temperature if the concentrations of additive are the same (McIntosh et al., 1980; Pope et al., 1989). With short alkane the transition temperature is lower than that of the neat bilayers, while the temperature becomes higher as the alkane becomes longer. It is noted that similar trends have been reported for bilayers with n-alcohols (Lohner, 1991), the main part of which is also a flexible chain. This implies that the change in the phase behavior is one of the key points to be clarified for understanding a “role” of flexible molecules in lipid bilayers. However, the molecular mechanism how the flexible chain affects the transition behavior has not fully been clarified yet. That is, it is still unclear how the

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flexible chain changes the bilayer structure, and how the resultant structural change affects the transition behavior. To make clear the mechanism, the structural change and its correlation with phase behaviors would shed a light. Needless to say, the phase transition between the gel and liquidcrystalline phases is the phenomenon related to a whole bilayer, not only to the acyl chains. Not only the balance of alkane and acyl chain lengths but also headgroups of lipid molecules would come into play in the effect of alkanes. The headgroup dependences of phase behavior or other bilayer physical properties have attracted much attention (Todd et al., 1997; Mulukutla and Shipley, 1984). The present authors have recently clarified that the different hydration states depending on headgroups are, at least, one of the origin of the headgroup dependence of membrane properties (Hishida et al., 2014a,b). In this context, the headgroup dependence in the effect of additives certainly deserves investigation. In the present study, to clarify the molecular mechanisms of effect of n-alkanes, as representative flexible molecules, on physical properties of bilayers, the correlation between changes in the phase behaviors and bilayer structures is investigated while paying attention on the headgroup dependences. We chose three lipids with different headgroups but the same acyl chains (DMPC, DMPS and DMPE), and four alkanes with different lengths. Through a statistical-mechanical analysis of the phase transition temperatures combined with the structural change observed by X-ray diffractions, we will show that the origin of the effect of alkanes is enhancement of the molecular packing in the gel phase. The appearance in DMPE-based bilayers of a subgel (Lc ) phase, which is usually not observed for these lipids in the ordinary conditions, is explained by the same structural effect.

(a)

O

O O

O H

O P O O

N

+

O

(b) O

O O

O H

O

(c)

O P O O + Na

O

O H

-

O

+ NH3

O O

O H

O P O O

+

NH3

O Fig. 1. Chemical structures of used phospholipids: (a) DMPC, (b) DMPS, and (c) DMPE.

mixed with 3 h-sonication. Since DMPS has a possibility of some aggregation, fusion and break of the lipid membrane by the sonication (Atkinson et al., 1974), the DMPS/alkane/water mixtures were hydrated at 45 ◦ C without sonication for 3 h, and freeze/fusion cycle of the sample between 45 and −25 ◦ C was applied at least twice.

2. Materials and methods 2.2. Differential scanning calorimetry 2.1. Materials and sample preparations Three phospholipids were used in this study: 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero3-phospho-l-serine (DMPS, sodium salt), and 1,2-dimyristoyl-snglycero-3-phosphoethanolamine (DMPE). Molecular structures of these lipids are depicted in Fig. 1. These lipids have different head groups and the same acyl chains. DMPC and DMPE are neutrally charged (zwitterionic) lipids while DMPS is a negatively charged lipid. Powders of DMPC and DMPE were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and that of DMPS was from Avanti Polar Lipids, Inc. (Alabaster, AL). They were used without further purifications because of their high purity (>99%). The main transition temperature of these lipids are: DMPC, 23 ◦ C; DMPS, 35 ◦ C; DMPE, 49 ◦ C. For n-alkane to add to lipid membranes, octane (C8, >99.5%), decane (C10, >99.5%), dodecane (C12, >99.5%) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and tetradecane (C14, >99%) from Sigma–Aldrich co. inc. (St. Louis, MO). These were also used without further purification. Each alkanes were added onto powders of each lipid, and after that, pure water (MilliQ, 18.2 M cm) was added to the lipid/alkane mixture. The concentration of lipid/alkane mixtures against water were 10 wt%. With this concentration, enough excess water exist and phase behavior of a lipid is independent of concentration (Koynova and Caffrey, 1998). For the measurements of differential scanning calorimetry (DSC), molar ratios of alkanes to lipids were 20 and 30 mol%, while those were only 30 mol% for X-ray diffraction measurements. For DMPC and DMPE, the mixtures after adding water to lipid/alkane were sonicated in a ultrasonic bath (US-101 from SND Co., Ltd. (Nagano, Japan)) at temperatures about 10 ◦ C higher than the main transition temperature of neat bilayers of each lipid. We confirmed that the samples were homogeneously

Measurements of differential scanning calorimetry (DSC) were done with a commercial instrument (Q200, TA Instruments (New Castle, DE)). About 5 ␮L of lipid/alkane/water mixtures were sealed in an aluminum pan. Heating/cooling cycles were done for three times. Rate of temperature change for the first cycle was 5 ◦ C min−1 , while those for the second and third cycles were 2 ◦ C min−1 . After confirming that the second and third cycles showed the same thermal behaviors, the transition temperatures of the lipids were determined from the thermal anomaly due to the main transition in the heating process of the third cycle. Since the thermal anomaly is broadened due to phase separation of the binary mixture during the transition, the temperature of the peak of the anomaly is adopted as the “transition temperature”, which is considered to approximate the temperature where the Gibbs energy of two phases match without phase separation. The temperature cycles were done between ±15 ◦ C of the temperatures of main transition of neat bilayers. DSC measurements were performed within one day after the sample preparation. 2.3. Small-angle and wide-angle X-ray diffraction Small-angle and wide-angle X-ray diffractions (SAXD and WAXD) were performed at BL6A, Photon factory, KEK, Japan. Wave˚ The detectors were PILATUS 300K length of X-ray was 1.5 A. (DECTRIS Ltd. (Baden, Switzerland)) for SAXD and PILATUS 100K (DECTRIS Ltd. (Baden, Switzerland)) for WAXD, respectively. The distances between the sample and the detectors and the tilt angle of the WAXD detector were calibrated with a standard sample (silver behenate). WAXD were performed at temperatures where each bilayers formed by lipid/alkane mixture is in the gel phase (DMPC, 10 ◦ C; DMPS, 30 ◦ C; DMPE, 35 ◦ C), while SAXD were performed

M. Hishida et al. / Chemistry and Physics of Lipids 188 (2015) 61–67

at temperature in the liquid-crystalline phase (DMPC, 40 ◦ C; DMPS, 50 ◦ C; DMPE, 65 ◦ C). For DMPE samples, the mixtures were once heated to about 60 ◦ C and cooled before the WAXD and SAXD measurements except for the measurement of subgel phase. Temperatures of the samples were controlled using a hot stage (FP900, Mettler-Toledo Inc. (Columbus, OH)). X-ray measurements were performed within a few days after the sample preparation. 3. Results 3.1. Phase transition temperatures DSC traces during heating processes of the third cycle are depicted in Fig. 2(a) for DMPC samples with 30 mol% of alkanes. The thermal anomaly corresponds to the main transition of each sample. The main transition temperature becomes higher as the length of the added alkane becomes longer. With octane and decane, the transition temperatures are lower than that of the neat bilayer of DMPC, while that is higher with dodecane and tetradecane. With shorter alkanes, the thermal anomaly broadens. The trends in the

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change in transition temperature for DMPC-based bilayers roughly reproduce the reported one (Pope and Dubro, 1986), though the concentration of alkane and ways for determining the transition temperatures are different. With adding alkanes to DMPC, the pre-transition almost disappeared. Slight heterogeneity of distribution of tetradecane in DMPC likely cause a small nubble around 32 ◦ C, which showed little reproducibility. DSC curves of DMPS and DMPE-based samples are depicted in Fig. S1. For samples with DMPS, the increase in the transition temperature was also observed. Obtained “transition temperatures” are shown in Fig. 2(b) for all samples against the number of carbon atoms in an added alkane molecule. As clearly seen, the change in the transition temperature against the length of the added alkane molecule exhibits a strong dependence on the headgroups. The largest slope is found for DMPC-based samples, while the slope is milder for DMPS-based ones and scarce for DMPE-based ones. The dependence is observed for both 20 and 30 mol% samples. For DMPS-based samples, the transition temperature is higher only with tetradecane than that of neat bilayers of DMPS.

(a)

Endo.

3.2. Change in bilayer structures

pure DMPC

Heat flow

C8 C10 C12 C14

15

20

25

30

35

(b) 50

40

30

20

8 10 12 14 number of carbon atoms in n-alkane Fig. 2. (a) DSC curves for DMPC with 30 mol% of n-alkanes (C8: octane, C10: decane, C12: dodecane, C14: tetradecane). (b) Main transition temperatures of lipid/alkane mixtures against the number of carbon atoms in an added n-alkane molecule (red circles, DMPC; blue triangles, DMPS; green squares, DMPE). Open symbols are the results of 20 mol% of alkanes and closed ones are those of 30 mol%. Solid and dotted lines are the fit results for Eq. (1). Dashed lines are the main transition temperatures of pure lipids. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

The effects of added alkanes on the structure of lipid bilayers were studied using WAXD and SAXD. With WAXD, in-plane correlation between lipid molecules is observed. Since acyl chains of lipid molecules form hexagonal-like lattice within a bilayer in the gel phase, the interstitial distance between the chains s can be determined through the WAXD measurement. In our measurement, due to the measurable q range, the distance s (= s11 = s20 in hexagonal lattice) as defined in the literature (Marsh, 2011) is determined. When alkanes are added to the lipid bilayers, the molecules of added alkanes are expected to align together with acyl √ chains, contributing to the X-ray diffraction. Therefore, 2s/ 3 is identified as the average distance between alkyl chains without discriminating whether it belongs to either of acyl chains or alkane. In Fig. 3(a), typical WAXD profiles are shown for DMPC-based samples with/without alkanes. Addition of alkanes makes the Bragg peak position shift to a higher-q side than that of the neat bilayer of DMPC. With the elongation of the added alkane, the peak moves to a much higher-q side. Further, the longer is the alkane, the sharper the diffraction profile. Similar changes in the WAXD profiles are observed for DMPS-based samples, while changes were weak for DMPE-based ones (see Fig. S2). The interstitial distances of the alkyl chains s within the bilayers are shown in Fig. 3(b) against the number of carbon atoms in an added alkane molecule for all samples. s decreases upon the addition of alkanes for all lipids with notable headgroup dependence as in the DSC results. The slope of s is larger for DMPC-based sample, smaller for DMPS-based one and almost null for DMPE-base one. The decrease of s indicates the increase in molecular density in lipid bilayers for DMPC- and DMPS-based samples. For DMPEbased samples, the packing of acyl chains and alkanes changes little. The sharpening of the Bragg peak as increasing the number of carbon atoms in an added molecule is naturally explained by assuming the enhanced order of lipid acyl chains by alkanes. Assuming that the alkyl chains (lipid acyl chains and alkanes) form a complete hexagonal lattice, the cross-sectional area for a lipid acyl chain √ or an alkane is Ach = 2s2 / 3. The cross-sectional area for a lipid molecule is A = 2Ach . For example, A = 41.0 A˚ 2 for pure DMPC, 41.5 A˚ 2 for pure DMPS, 42.7 A˚ 2 for pure DMPE are obtained, which roughly agree with the reported values (Marsh, 2011; Tristram-Nagle et al., 2002; Petrache et al., 2004; Rappolt and Rapp, 1996). With adding 30 mol% of tetradecane, A decreases about 3% from that of pure DMPC (A becomes 39.9 A˚ 2 ). It is noticed that negative charge on PS headgroup affects little the molecular packing of lipids in a bilayer,

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M. Hishida et al. / Chemistry and Physics of Lipids 188 (2015) 61–67

Intensity / a.u.

(a)

pure DMPC C8 C10 C12 C14 1.2

(b)

1.3

1.4

1.5

1.6

4.25

Interstitial distance, s

pure DMPS pure DMPE pure DMPC

4.20

4.15

8

10

12

14

number of carbon atoms in n-alkane Fig. 3. (a) WAXD profiles of pure DMPC and DMPC with 30 mol% of n-alkanes (C8, octane; C10, decane; C12, dodecane; C14, tetradecane) at 10 ◦ C. Upper dotted line is the peak position of DMPC with octane, while lower one is that of DMPC with tetradecane. (b) Interstitial distances s of the alkyl chains in a bilayer obtained from WAXD (red circles, DMPC at 10 ◦ C; blue triangles, DMPS at 30 ◦ C; green squares, DMPE at 35 ◦ C). Dashed lines are s for pure lipids. Solid lines are the guide for eyes to see the gradient against the number of carbon atoms in an added n-alkane molecule. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

as shown in Fig. 3(b) where the interstitial distances do not show large difference from those of PC and PE lipids. Since a clear Bragg peak is not observed in WAXD in the liquidcrystalline phase because of disordered arrangement of lipid acyl chains, the effect of alkanes on the bilayer structure is examined using the result of SAXD. In the small-angle region, Bragg peaks corresponding to the regular order of stacked lipid bilayers (lamellar structure) are observed (Hishida et al., 2014b, 2008; Petrache et al., 1998). The obtained lamellar repeat distances (d = bilayer thickness + water layer thickness) are depicted in Fig. 4 for DMPC- and

70

60

50

40

8

10

12

14

number of carbon atoms in n-alkane Fig. 4. Lamellar repeat distances d of DMPC at 40 ◦ C (red circle) and DMPE at 65 ◦ C (green squares) with alkanes. Dashed lines are those of pure lipids. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

DMPE-based samples. It is clear that d depends little on the number of carbon atoms in an added alkane molecule. It has been widely accepted that the lamellar repeat distance is determined by the balance of van der Waals attraction and two types of repulsive interaction (short-range repulsion due to strongly bound water, and Helfrich steric repulsion due to fluctuation of bilayers) (Petrache et al., 1998; Hishida et al., 2008). If the effect of added alkanes is significant for physical properties such as a bending rigidity of bilayer, these interactions and its balance would be changed. The observed constancy of d, therefore, indicates rather small effect of added alkanes in the liquid-crystalline phase, though a slight enhancement of molecular packing by added alkanes were reported in the liquid-crystalline phase (Aagaard et al., 2006). The negative charge of the headgroup of DMPS lets the repeat distance d diverge due to the electrostatic repulsion, resulting in failure of a stable formation of any stacked lamellar structures. However, considering the results for DMPC- and DMPE-based samples, we expect that the effect of added alkanes on DMPS-based samples is also small in the liquid-crystalline phase. WAXD and SAXD results indicate that the added alkanes change the physical property of the lipid bilayers mainly in the gel phase. Bilayers in the liquid-crystalline phase are less affected by alkanes. Similar trends of the headgroup dependence (the same orders of the slope) in WAXD and DSC result imply that the change in the bilayer structure in the gel phase causes the slope of the transition temperature against the carbon number.

3.3. Emergence of the subgel Lc phase Only for DMPE-based samples, the first cycle of DSC measurements shows different thermal behavior from the later cycles. The curves of the first and second cycles are shown in Fig. 5(a). Differently from the first cooling and the second heating/cooling processes, a large thermal anomaly is observed around 57 ◦ C only in the first heating process. In the process, the main transition around 49 ◦ C is not observed. According to literatures (Wilkinson and Nagle, 1984; Kodama et al., 1995), the large thermal anomaly around 53–58 ◦ C (for DMPE) in the first heating process is due to the sub-transition from the subgel (Lc ) phase to the liquid-crystalline (L␣ ) phase. In the subgel phase, lipid molecules have crystalline order and most of water between lamellae are pushed away (dehydrated) (Kodama et al., 1995, 2000; Tenchov et al., 1988). Since hydration also occurs simultaneously with the chain melting in the sub-transition, the heat of the sub-transition is larger than that of the main transition, where only the chain melting occurs. The subtransition is observed for the all DMPE/alkane mixtures, but not for pure DMPE in our measurement. The result of pure DMPE is consistent with the report that pure DMPE forms the subgel phase only after annealing at a very low temperature for a long time (e.g., at 2 ◦ C for over 6 days) (Wilkinson and Nagle, 1984). The formation of the subgel phase soon after the sample preparation at room temperature for DMPE/alkane mixture indicates that alkane make the relaxation from the gel phase to the subgel phase speedy. Activation energy would become much lower by the effect of alkanes. The emergence of the subgel phase is also confirmed by WAXD at 35 ◦ C as shown in Fig. 5(b) for all samples of DMPE/alkane mixtures. In the WAXD patterns for the mixtures, many diffraction peaks due to the crystalline order of the subgel phase (Tenchov et al., 1988; Marsh, 2011) are clearly observed, in contrast to only a single peak arising from the gel phase for pure DMPE. The emergence of the subgel phase confirms the homogeneous presence of alkanes into the DMPE bilayers though the transition temperature and interstitial distance s change little.

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M. Hishida et al. / Chemistry and Physics of Lipids 188 (2015) 61–67

Heat flow

(a)

40

45

50

55

60

I / a.u.

(b)

pure DMPE C8 C10 C12 C14

1.2

1.3

1.4

1.5

1.6

Fig. 5. (a) First and second cycles of DSC measurements for DMPE with 30 mol% octane. From the top, first heating, first cooling, second heating and second cooling processes. (b) WAXD profiles of DMPE with/without alkanes (C8, octane; C10, decane; C12, dodecane; C14, tetradecane) at 35 ◦ C. For obtaining the profiles, samples were not heated to the liquid-crystalline phase after the sample preparation, and measurements were performed within a day after the preparation.

4. Discussion 4.1. Analysis for temperature of main transition Transition temperature of a first order transition is related to the enthalpy and entropy changes of the total system as Tm = Htot /Stot . It is natural to consider that the addition of alkanes to lipid bilayers affects both enthalpy and entropy change between the gel and liquid-crystalline phases. Thus, it is also natural to ask which change is main origin of the headgroup dependence, i.e., different slopes of the transition temperatures against the number of carbon atoms in an added alkane molecule? To qualitatively understand the effect of enthalpy and entropy changes by additives, a statistical-mechanical model was proposed (Suezaki et al., 1985). The model is applicable when additives are hydrophobic and linear in their molecular shape. In the model, it is assumed that change in the transition temperature is described through the excess change in enthalpy Hex and entropy Sex emerged by additives. Then, the transition temperatures Tm against length of additives (here, m being the number of carbon atoms in an alkane molecule) is described as follows.

Tm

H + Hex = = Tm,0 S + Sex

1+

1 H



2

x mhA x + m 1+x

1+

x(s1 +ms2 ) S



,

(1)

where Tm,0 , H and S are the transition temperature, the transition enthalpy and entropy of neat bilayers, respectively. Tm,0 is

65

known from our measurements of neat lipids. Since amounts of lipids in sample pans were difficult to be controlled in our measurements due to heterogeneity in the distribution of bilayers in solution, reported values in the literatures (Lewis and McElhaney, 2000; Pope and Dubro, 1986; Mulukutla and Shipley, 1984) were used for H and S. x is the molar ratio of alkane with respect to lipid (x = [alkane]/[lipid]). These known parameters are listed in Table 1. hA = (HAl − HAg )/m, where HAl and HAg are the enthalpy of alkane inside a bilayer in the liquid-crystalline and gel phases, respectively.  represents a change in the alkane–alkane interaction between in the gel and liquid-crystalline phases, where  = g − l . The excess entropy change Sex is divided into mdependent and independent parts as Sex = s1 + ms2 , since, for example, the translational entropy of alkane molecules is mindependent while a linear dependence on m is expected for the conformational entropy. It is noted that s2 as well as  can be assumed to be independent of headgroup of lipids, because both are originated in chain conformation and alkane–alkane interaction, i.e., lipids are unconcerned. Four unknown parameters were obtained through the fitting with Eq. (1) for our data as listed in Table 2. Global fitting for all data of three headgroups and two concentrations have been performed simultaneously, and the fit results are depicted as solid and dotted lines in Fig. 2(b). The fit results show that values of both hA and s1 are DMPC >DMPS >DMPE, which is the same orders as that of slopes of DSC and WAXD results. Compared to 10 J K−1 (mol of CH2 )−1 , which is the conformational entropy change per mole of methylene group during the complete chain melting (Sorai et al., 1980; Sorai and Saito, 2003; Horiuchi et al., 2010), m-dependent part of the excess entropy s2 is smaller enough. This is consistent with the fact that chain melting during the main transition is not complete (Pink et al., 1980). For m = 14 and DMPC, total excess entropy is 44.8 J K−1 mol−1 . The excess entropy of two moles of tetradecane (89.6 J K−1 ) is comparable to the transition entropy of one mole of DMPC (84.9 J K−1 ). This indicates that degree of the chain melting of alkane and lipid acyl chains are almost the same during the main transition. This is reasonable because an alkane molecule locally looks the same as an acyl chain. Compared to hA ,  is much smaller, indicating the alkane–alkane interaction affects little for the transition behavior. Although both hA and s1 are dependent on headgroup, it was found through the fitting that the slope of Tm is mainly dependent on hA , i.e., the enthalpy term of the system. The slope of  = dT /dm) is sensitive over 50 times transition temperature (Tm m to a change in hA than that in s1 as shown in Fig. S3 for the  changes about 60% when h changes 30%, DMPC-based case (Tm A while it changes only 1% with the change of 30% in s1 ). It was also Table 1 Known parameters (Lewis and McElhaney, 2000; Pope and Dubro, 1986; Mulukutla and Shipley, 1984) used for the fitting with Eq. (1).

DMPC DMPS DMPE a

Tm,0 (◦ C)

H (kJ mol−1 )

S (J K−1 mol−1 )

x

22.8 35.7 49.1

25.1 32.6 24.1

84.9 105.3 73.6

0.429 (30 mol%), 0.250 (20 mol%)a

These are common for all lipids.

Table 2 Parameters obtained from the fitting for all DSC data (Fig. 2) by Eq. (1).

DMPC DMPS DMPE a

hA (kJ mol−1 )

s1 (J K−1 mol−1 )

s2 (J K−1 mol−1 )

 (kJ mol−1 )

1.06 0.870 0.749

12.6 6.85 0.31

2.30a

0.02a

These are common for three lipids.

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M. Hishida et al. / Chemistry and Physics of Lipids 188 (2015) 61–67

2

1

0

-1

8 10 12 14 number of carbon atoms in n-alkane

Fig. 6. Change in Gibbs energy of alkanes between in the gel and liquid-crystalline phases against the number of carbon atoms in an added alkane molecule (red circles, DMPC; blue triangles, DMPS; green squares, DMPE). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

found that s1 determines the absolute values of Tm . These indicate that the headgroup-dependent changes in Tm are originated in the change in enthalpy of alkanes between the two phases. Validity of the fit results is confirmed by the calculating Gibbs energy. Change in Tm from that of pure one (Tm,0 ) can be discussed by the Gibbs energy of added alkanes (GA ) at Tm,0 (Suezaki et al., 1985). At Tm,0 , the change in Gibbs energy of lipid itself must be zero. Thus, if alkane is favorable more in the gel phase than in the liquid-crystalline phase at Tm,0 , which correspond to a lower Gibbs energy of alkane (GA ) in the gel phase, Tm becomes higher than Tm,0 . From the model, the difference in Gibbs energy of alkane (GA ) between in the gel (GAg ) and liquid-crystalline (GAl ) phases is described as, GA = GAl − GAg = mhA − Tm,0 (s1 + ms2 ).

(2)

Calculated m-dependence of GA is shown for each headgroup in Fig. 6. For example, GA of DMPC crosses zero at around m = 10, indicating Tm is lower than Tm,0 for m < 10 and higher for m > 10. This certainly agrees with the experimental results. Besides, the gradient of GA , corresponding to that of Tm , is also consistent with the results. These consistencies assure the validity of the fit results. Further, the results of GA qualitatively explain the broadening of thermal anomaly in the DSC results. Large absolute value of GA suggests that the phase separation occurs severely during the phase transition, while no separation occur with zero GA . Broadening of the thermal anomaly in DMPC/alkane mixture is considered to be from the largeness of GA . An explanation in the same scenario is applicable for DMPE-based case where the broadening was rarely observed.

gel phase against the number of carbon atoms in an added alkane molecule is the largest for DMPC and the smallest for DMPE. Thus, the total enthalpy change of the system Htot increases the most largely for DMPC. This is consistent with the order of hA for headgroups. What it comes down to is that enhanced molecular packing in the gel phase by alkanes dominates the change in Tm . Emergence of the subgel phase for DMPE-based sample is also explained within this scenario. In the subgel phase, lipid molecules are highly condensed and have crystalline order (Kodama et al., 1995, 2000). Since the addition of alkane makes lipids be more condensed in the gel phase, it can be said that the structure in the gel phase becomes closer to the subgel phase. Thus, the relaxation time from the gel phase to the subgel phase at room temperature would become shorter than that in the neat system. Indeed, the subgel phase was observed soon after the sample preparation for DMPE/alkane systems. It is impossible to answer why the subgel phase is not observed for DMPC- and DMPS-based samples at present. There is a possibility that the activation energy barrier from the gel to subgel phase is intrinsically lower for pure DMPE than that of pure DMPC and DMPS. In this study, the origin of the change in the phase transition behaviors caused by alkanes is clarified from the view point of structural thermodynamics, though it is still unclear how chemical structures of the headgroups are related to the enthalpy change in the gel phase caused by alkane. Although both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) headgroups are zwitterionic and their chemical structures are similar each other, these lipids are known to have much different properties. For example, there is large difference in hydration states (Hishida et al., 2014a) between them. In addition, only PE lipids exhibit lamellar to inverted-hexagonal phase transition in solution. These large differences between PC and PE lipids seem to come from the different molecular packing in bilayers. Lipids with PC headgroup are known to have gaps in the hydrophobic region due to steric interactions between large PC headgroups (McIntosh, 1980). Alkanes are expected to infill the gap and to attract PC lipid. Actually in the present case, although the chain–chain distance decreases with the addition of alkanes (see Fig. 3(b)), an averaged head-to-head dis√ ˚ (2 + x)/2 × 2s/ 3 = 5.29 A) tance of the lipids with alkanes (sh = (x = 0.3/0.7) is larger than that for DMPC with 30 mol% tetradecane √ ˚ On the other hand, lipids without alkanes (sh = 2s/ 3 = 4.87 A). with PE headgroup have smaller gap than PC lipid since the size of PE headgroup is smaller than that of PC headgroup. Therefore, it is expected that the effect of alkanes to PE lipid is smaller than that to PC lipid. However, this mechanism is not applicable to PS lipid, to which the effect of alkanes is smaller than that to PC lipid in spite of the larger headgroup of PS than PC. Some effects likely originate from the charge on the PS headgroup. Thus, detailed mechanisms of correlation between the chemical structures of headgroups and the enthalpy change caused by alkanes should be further investigated in the future.

4.2. Origin of headgroup dependence

5. Conclusion

The above theoretical analysis clearly showed that the headgroup dependence of Tm change is caused by the fact that the enthalpy change of alkane between in the gel and liquid-crystalline phases depends on headgroup. However, there remains an issue to be solved: Which phases do change dominantly? Results of X-ray diffraction answer clearly. While structural changes of bilayers in the liquid-crystalline phase are little for all lipids, molecular packing of lipid acyl chains and alkanes is enhanced by the addition of alkanes in the gel phase. This means that enthalpy of system is lowered by alkane in the gel phase, while such change is little in the liquid-crystalline phase. Magnitude of the reduction in the

In the present study, the headgroup dependences of the effect of n-alkanes on lipid bilayers are investigated focusing attention on the gel/liquid-crystalline phase transition (main transition) and structural changes of the bilayers. DSC results indicate that the temperature of the main transition increases as the number of carbon atoms in an added alkane molecule increases for all lipids of DMPC, DMPS and DMPE. Besides, it is found that the degree of increase (the slope against the alkane length) has a strong dependence on the headgroup. A theoretical analysis shows that the headgroup dependence is originated in the fact that change in the transition enthalpy caused by added alkanes is strongly dependent on the

M. Hishida et al. / Chemistry and Physics of Lipids 188 (2015) 61–67

headgroup. Further, the small-angle and wide-angle X-ray diffractions indicate that the structural change in bilayers is observed mainly in the gel phase. On the basis of these observations, it is concluded that the enthalpy change in the gel phase is the origin of the headgroup-dependent change in the transition temperatures. Enhanced molecular packing of lipid acyl chains and alkanes (equivalently, the reduction in enthalpy of the total system) in the gel phase also causes the emergence of the subgel phase. This study of the headgroup dependence clearly showed that the thermal properties and structural properties have a strong correlation and that the investigation of the correlation is valuable for understanding the effect of additives universally. From the correlation, the “role” of flexible chain was clarified, i.e., a flexible chain makes the lipid–lipid attraction stronger and lowers the enthalpy of the system, in contrast to a naive intuition that the entropy term seems to be crucial. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research from JSPS (Grant No. 42740289) for M.H. The WAXD and SAXD experiments were performed under the approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2013G525 and 2013G530). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphyslip. 2015.05.002 References Aagaard, T.H., Kristensen, M.N., Westh, P., 2006. Packing properties of 1-alkanols and alkanes in a phospholipid membrane. Biophys. Chem. 119, 61–68. Adachi, T., Yamamura, Y., Hishida, M., Ueda, M., Ito, S., Saito, K., 2012. Comprehensive characterisation of the E phase of 6-octyl-2-phenylazulene. Liq. Cryst. 39, 1340. Adachi, T., Saitoh, H., Yamamura, Y., Hishida, M., Ueda, M., Ito, S., Saito, K., 2013. Universality of molten state of alkyl chain in liquid-crystalline mesophases: smectic E phase of 6-alkyl-2-phenylazulene. Bull. Chem. Soc. Jpn. 86, 1022–1027. Atkinson, D., Hauser, H., Shipley, G.G., Stubbs, J.M., 1974. Structure and morphology of phosphatidylserine dispersions. Biochim. Biophys. Acta 339, 10–29. Bolhuis, P., Frenkel, D., 1997. Tracing the phase boundaries of hard spherocylinders. J. Chem. Phys. 106, 666–687. Hishida, M., Seto, H., Yamada, N.L., Yoshikawa, K., 2008. Hydration process of multistacked phospholipid bilayers to form giant vesicles. Chem. Phys. Lett. 455, 297–302. Hishida, M., Tanaka, K., Yamamura, Y., Saito, K., 2014a. Cooperativity between water and lipids in lamellar to inverted-hexagonal phase transition. J. Phys. Soc. Jpn. 83, 044801. Hishida, M., Yamamura, Y., Saito, K., 2014b. Salt effects on lamellar repeat distance depending on head groups of neutrally charged lipids. Langmuir 30, 10583–10589. Horiuchi, K., Yamamura, Y., Pełka, R., Sumita, M., Yasuzuka, S., Massalska-Arodz, M., Saito, K., 2010. Entropic contribution of flexible terminals to mesophase formation revealed by thermodynamic analysis of 4-alkyl-4 -isothiocyanatobiphenyl (nTCB). J. Phys. Chem. B 114, 4870–4875. Ivankin, A., Kuzmenko, I., Gidalevitz, D., 2010. Cholesterol–phospholipid interactions: new insights from surface X-ray scattering data. Phys. Rev. Lett. 104, 108101. Kodama, M., Inoue, H., Tsuchida, Y., 1995. The behavior of water molecules associated with structural changes in phosphatidylethanolamine assembly as studied by DSC. Thermochim. Acta 266, 373–384. Kodama, M., Kato, H., Aoki, H., 2000. The behavior of water molecules in the most stable subgel phase of dimyristoylphosphatidylethanolamine–water system as studied by differential scanning calorimetry. Thermochim. Acta 352–353, 213–221.

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