- Email: [email protected]
Ethanol and methanol induced changes in phospholipid monolayer
Applied Surface Science 253 (2006) 2425–2431 www.elsevier.com/locate/apsusc
Ethanol and methanol induced changes in phospholipid monolayer M. Weis a, M. Kopa´ni b,*, J. Jakubovsky´ b, L’. Danihel b a
Slovak University of Technology, Faculty of Electrical Engineering and Information Technology, Department of Physics, Bratislava, Slovakia b Comenius University, School of Medicine, Institute of Pathology, Sasinkova 4, 811 08 Bratislava, Slovakia Received 21 April 2006; received in revised form 28 April 2006; accepted 28 April 2006 Available online 30 June 2006
Abstract The main components of cell membranes are phospholipids and proteins. The aim of our study was to examine structural changes of dipalmitoyl-phosphatidylcholine (DPPC) monolayer as a simple model system of a cell membrane in different environments. Pure water, ethanol and methanol solutions were used as subphases of Langmuir films as a membrane models. For detection of changes in charge states of the molecules as well as relation with structural and conformational changes, a contactless method Maxwell’s displacement currents (MDC) was used. Behaviour of DPPC molecules on two different subphases is substantialy different. In DPPC monolayer on the subphase of methanol–water, a gradual absorption (incorporation, penetration) of methanol molecules into the layer can appear. In DPPC monolayer on the subphase of ethanol– water adsorption of ethanol molecules on the layer can be observed. The membrane permeability might change. At both subphases (ethanol–water and methanol–water) the elasticity modulus of the monolayer decreases leading to the loss of membrane elasticity. # 2006 Elsevier B.V. All rights reserved. PACS: 68.08.p Liquid–solid interfaces; 68.18.g Langmuir–Blodgett films on liquids Keywords: Maxwell displacement current; Conformation; Membrane; Ethanol; Methanol; Permeability
1. Introduction Cell membrane is an important component of all cells. It separates intracellular from extracelluar spaces. It forms a selective permeabile border providing dynamic balance between a cell and an outer space. It contains enzymes, receptors, transport and signaling systems and antigens. The main components of cell membranes are phospholipids and proteins. They are incorporated into the membranes or proteins and can be electrostatically bounded to the surface. In addition to phospholipids and proteins, membranes contain water and different chemical compounds. Most important are cholestorol and glycolipids. Cholesterol provides firmness and fluidity of the membrane. Membranes seem to be altered by the presence of ethanol [1–4]. The mechanism by which ethanol affects function of a membrane at the molecular level is still not known. It is likely that ethanol acts in a hydrophobic environment. Alcohols cause the inhibition of molecule transport through the cell membrane.
* Corresponding author. Tel.: +421 2 59357273; fax: +421 2 59357592. E-mail address: [email protected] (M. Kopa´ni). 0169-4332/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2006.04.053
Hutchinson et al. [5] compared the relative potencies of the inhibition caused by the different alcohols. They found that potencies of the inhibition increased with the alcohol chain length. Alcohols can induce a disordering of the acyl chains of the fluid phospholipids, as well as the formation of an interdigitated gel phase [6–8]. From NMR studies it can be concluded that ethanol binding to lipid membrane near lipid– water interface [9–12]. The exact nature of the interactions among ethanol, membrane proteins, and the lipid framework remains obscure. It is known that ethanol fluidizes the bulk lipid of membranes and may alter cell function. The changes of cell membrane involve direct effect of ethanol on proteins, other membrane acting drugs, temperature effects, effects of ethanol on aged membranes and inconsistent effects of chronic ethanol consumption on lipid content [13]. Interaction between ethanol and cell membrane results in a disorder or ‘‘fluidize’’ membranes, or act as a local anesthetic. In some kinds of membranes low concentrations of drugs have an ordering effect [14,15]. Also influence of the alcohol (with various chain lengths) on disorder and rigidity of surfactants was investigated [16]. In the simplest picture, the molecular entropy varies with the area per molecule A as S = kBT ln A (kB and T denotes the
2426
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Boltzmann constant and temperature, respectively). Therefore, incorporation of cosurfactant into the phospholipid leads to disorder due to increasing of area per molecule. Luzzati et al. [17] investigated the structure of toad sciatic nerves exposed to either low temperature or influence of tetracaine. Their results suggest that membranes were thickened and stiffened. These phenomena are typical for lipid-containing systems with disordered chains. Some evidences indicate that interaction between ethanol and membranes have a membrane-fluidizing effect. The chronic response to this effect is not the change of the membrane bulk lipid composition [18]. Properties of the membrane model system may be investigated by examining the dipole moment projection of the monolayer during compression and analysis of the elasticity. Dipole moment projection analysis by the Maxwell displacement current (MDC) [19] is very sensitive method for evaluation of molecular orientation (so-called order parameter) as well as electric state of the molecule. In this way is possible to investigate phospholipid phase transition and influence of alcohol in the subphase. In contrast with surface pressure–area isotherm analysis is MDC measurement is extremely sensitive also in the low surface pressure–area, where these methods are useless. The aim of our study was to examine structural changes of dipalmitoyl-phosphatidylcholine (DPPC) monolayer as a simple model system of a cell membrane in different environments and to compare effects of ethanol and methanol on this system. 2. Material and methods 2.1. Chemicals and preparation of monolayers The material used in this study as model phospholipid was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC) purchased from Sigma–Aldrich. Lipid was dissolved in chloroform at the stock concentration 0.5 mg/ml and spread on the subphase using microsyringe (Hamilton, USA). Pure water (bidistilled deionized water, 15 MV cm) and 20% ethanol and methanol solutions were used as subphases. For alcohol solutions ethyl alcohol and methyl alcohol were used (spectrophotometric grade purity) from Sigma–Aldrich. Subphase was thermostated to the temperature 17 8C. Monolayer
was allowed to equilibrate and solvent to evaporate for 15 min. This time was sufficient for chloroform to evaporate and monolayer to stabilize. 2.2. Experimental methods Various experimental techniques have been designed for the observation of structure properties and order parameter in organic monolayers situated onto the air–water interface [20]. However, the orientational parameter measurement in the time domain is possible only with some of them. For detection of changes in charge states of the molecules as well as relation with structural and conformational changes, a contactless method was developed based on analysis of Maxwell’s displacement currents. This method was originally introduced by Iwamoto and Majima [21,22] and improved by other authors [23–26]. Proposal of MDC experiment application for biological membrane phantom measurement was presented for the first time in our previous work [19]. The basic components of the Maxwell’s displacement current—experimental setup attached to the computer-controlled model Langmuir trough (model 611, Nima Technology, UK) are schematically shown in Fig. 1. The top electrode was suspended in air, parallel to the interface, without a direct mechanical or electrical contact with a floating monolayer on the water surface. The air gap between the top electrode and the water surface was regulated to a certain spacing (approximately 1 mm) by measuring the capacitance of the electrode system. The displacement current was detected with a Keithley 617 electrometer (Keithley Instruments, Cleveland, Ohio, USA). The sensitivity of measuring the current was 0.1 fA, the background noise was suppressed by a multiple electrical shielding of electrode as well as whole measuring system to 2 fA. Langmuir through was situated in a laminar-flow box on an antivibrating stand to avoid any mechanical stress. The total working area of the trough was 600 cm2 and the compression rate was 50 cm2/min, which correspond to ˚ 2/s per one molecule. The area of top electrode was 0.17 A AE = 20 cm2. Due to dynamic processes in the monolayer associated with the change in charge distribution caused by its compression, the induced charge in the top electrode varies with time and this generates a current flowing through the outer circuit via the electrometer.
Fig. 1. Schematic view of the experimental setup for displacement current measurement (left). Rod-like polar molecules execute precessional motion on the air– water interface (right) with maximal tilt angle QA (A and m stand for the area per molecule and the dipole moment of molecule, respectively). Electrical shielding of the top electrode is not drawn.
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
The level of the displacement current detected is very low and therefore the problem of background was carefully considered. The noise signal, known as the Johnson–Nyquist thermal noise, is at the level of 1–3 fA the value being at least one order of magnitude less than the signal of the monolayer. The MDC technique is sensitive only to dynamic charge processes, which in this experimental setup are caused by lateral compression of the monolayer. Therefore any timeindependent charge (mainly structured water layer and additional substances in subphase) distributed near/at the interface has no effect. In comparison with conventional electrical measurements of surface potential (by the Kelvin method) it provides big advantage in time-depended signals. The surface pressure–area isotherms were measured by the Willhelmy plate method with accuracy 0.05 mN/m. Consequently various mechanic and thermodynamic properties as elastic modulus of Langmuir films or thermodynamic properties (Gibbs free energy, entropy and enthalpy) can be evaluated. Membrane curvation due to thermal fluctuations is essential for shape and/or conformations of membranes as well as for cracks and defects generation [27]. The elastic modulus characterizes the elasticity of the monolayer and is in analogy with bulk materials defined as @p jEj ¼ A (1) @A T where p is the surface pressure, A the area per molecule and T is the temperature. The elastic modulus expresses the elasticity of the Langmuir film under influence of the compression force. The stability of the mixed monolayer can be determined by evaluation of excess Gibbs free energy of mixture following the Goodrich method [28] by integration of surface pressure–area isotherm up to selected surface pressure p Z p DG ¼ ðA12 x1 A1 x2 A2 Þ dp (2) 0
where A12 is the molecular area in the mixed monolayer, A1 and A2 the molecular areas in the pure component monolayer and x1 and x2 are the molar ratios of pure components in the mixture (x2 = 1 x1). In our case surfactant is DPPC and cosurfactant is adsorbed alcohol, x2 is time-depending parameter.
2427
where m is the dipole moment of one molecule (mz is projection of m to the normal), N the number of molecules under the top electrode and G is the geometrical factor depending only on the distance between the top electrode and the top plane of the monolayer and on the shape and area of the upper electrode. The hcos Qi stands for the statistical mean value cos Q where Q is the angle between the vector of dipole moment and the normal. Detailed analysis of dipole moment projection of simple fatty acid was described in Ref. [29]. As we show in our previous studies [23] the current flowing in the outer circuit can be expressed as a time change of the induced charge I¼
@Qi @hcos Qi @N þ mhcos QiG ¼ mNG @t @t @t
(4)
By integrating the displacement current with respect to time, the induced charge Q can be obtained and in this way we also evaluated the vertical component of the molecular dipole moment. Thus, the dipole moment projection to the normal mz should be calculated as Z 1 I dt (5) mz ¼ mhcos Qi ¼ G N 3. Results 3.1. MDC measurements During the measurement of the displacement current of the DPPC monolayer situated onto the pure water in relation to area ˚2 per molecule we can notice a sharp maximum at 110 A (Fig. 2). Relation between displacement current of the monolayer DPPC on the subphase methanol–water and area per molecule is depicted in Fig. 3. Our results indicate maximum at about ˚ 2. In addition, we can observe time-shift of a maximum of 90 A the monolayer displacement current to higher values of area per molecule.
2.3. Theoretical background of MDC The analysis of MDC experiment is based on the assumption that each molecule behaves like a weak dipole moment with a negative pole bound to the water surface. Individual molecules have random directions within a certain solid angle and execute a random precessional motion with a maximal possible tilt QA from the vertical axis. Generally, we consider the molecule as a rod-like rigid body without a possibility of bending. If we consider the organic film as a system of electric dipole moments then it is possible to calculate the induces charge on the upper electrode with the method of images. Qi ¼ hmz iNG ¼ mhcos QiNG
(3)
Fig. 2. Record of the displacement current–area isotherm measurement of DPPC monolayer onto the pure water subphase.
2428
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Fig. 3. Records of the displacement current–area isotherm measurements of DPPC monolayer on subphase methanol–water.
Relation between displacement current of the monolayer DPPC on the subphase ethanol–water and area per molecule is ˚ 2. In depicted in Fig. 4. We can notice maximum at about 50 A addition, we can observe a mild time-shift of the monolayer displacement current maximum to hihger values of area per molecule. It is notable, that in all gaseous phase of the monolayer negative displacement current is observed. 3.2. Dipole moment projection analysis By analysis of records of the Maxwell diplacement current projection, we can calculate dependence of the dipole moment on the area per molecule of the monolayer. On the surface of pure water at DPPC monolayer we can see a rapid change of the dipole moment projection at value around ˚ 2 (Fig. 5). 110 A At DPPC monolayer, on the surface of subphase methanol– water, no significant change of the dipole moment projection of DPPC molecule is observed related to the change of the dipole moment projection of DPPC molecule on water, even though the effect of methanol–water subphase is obvious. Rapid growth of the dipole moment projection is observed at around ˚ 2 per molecule (Fig. 6). area 90 A
Fig. 4. Records of the displacement current–area isotherm measurements of DPPC monolayer on subphase ethanol–water.
Fig. 5. The dipole moment projection to the normal calculated from MDC measurement of monolayer on pure water. Rapid growth of dipole moment ˚ 2 indicates ordering of molecules during the gas–liquid projection at 110 A phase transition.
Measurement of the dipole moment projection of DPPC monolayer on the surface of subphase ethanol–water in dependence of area per molecule indicates the rapid phase ˚ 2 (Fig. 7). transition from liquid phase to solid phase at 50 A Moreover, our recordings show a time shift to lower values of the dipole moment projection of DPPC molecule on the surface of subphase ethanol–water. We can observe that dipole moment projection reaches negative values of the polar head, even though the change of the tail dipole moment can occur. 3.3. Surface pressure–area isotherms Fig. 8 depicts relationship between surface pressure and area per molecule of the DPPC layer on the surface of subphase methanol–water. We can observe the time shift of p–A isotherm to higher values of the surface pressure.
Fig. 6. The dipole moment projections to the normal calculated from MDC measurements on subphase methanol–water. Rapid growth of dipole moment ˚ 2 indicates ordering of molecules during the gas–liquid phase projection at 90 A transition.
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Fig. 7. The dipole moment projection to the normal calculated from MDC measurement on subphase ethanol–water. Rapid growth of dipole moment ˚ 2 indicates ordering of molecules during the liquid–solid projection at 50 A phase transition.
Fig. 8. p–A Isotherms of DPPC monolayer on subphase methanol–water.
2429
Fig. 10. Isothermal elastic modulus curves of DPPC monolayer on subphase: (top) ethanol–water, (bottom) methanol–water. Elastic modulus of DPPC situated onto the pure water subphase is presented for a comparison (grey curve).
At subphase ethanol–water, a significant change in the dipole moment projection of DPPC molecules forming the monolayer appeared (Fig. 9). Monolayer exhibits very sharp phase transition, phase transition from gaseous to liquid phase ˚ 2 (observable only for first compression) and at area about 80 A ˚ 2. from liquid phase to solid phase at 50 A Similarly, as at DPPC monolayer on the subphase methanol– water we can observe a time shift of p–A isotherm to higher values. However, this shift is not as obvious as it was at DPPC monolayer on the subphase methanol–water (Fig. 10). From the graph representing the relation between elasticity modules and area per molecule we found out that at DPPC monolayer on the surface of both subphases (ethanol–water and methanol–water) elasticity modulus of the monolayer decreases. Our results suggest a gradual increase of membrane rigidity (decrease of elasticity). Time dependence of Gibbs energy and maximum of dipole moment projection is shown in Fig. 11. The dipole moment projection maximum represents the phase transition area; therefore its change is directly proportional to adsorbed alcohol. In both cases is observable continuous adsorption. The Gibbs free energy characterizes the stability of the phospholipid–alcohol mixture. Methanol molecules destabilize the monolayer in contrast with ethanol, which incorporation into the monolayer carry to lower free energy. 4. Discussion 4.1. Dipole moment and electric properties
Fig. 9. p–A Isotherms of DPPC monolayer on subphase ethanol–water.
4.1.1. Water Results of experiments performed on pure water [20] ˚ 2 area per molecule, suggest that at the value around 100–110 A a phase transition occurs from gaseous phase to liquid phase. From results of the measurement of the surface potential by Kelvin probe [30] the value of the dipole moment of DPPC molecule was determined to be 820 mD.
2430
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Fig. 11. Time dependence of Gibbs free energy of DPPC monolayer onto the ethanol–water (up) and methanol–water (down) subphase for various values of surface pressure (left view). Time dependence of dipole moment maximum position DPPC onto ethanol–water and methanol-water subphase (right view).
Our recordings show the value around 815–825 mD, which is in according with values obtained by independent measurements. 4.1.2. Methanol By the analysis of the measurement of the Maxwell diplacement current in relation with area per molecule, we calculated the dependence of the dipole moment projection on the area per molecule of the DPPC monolayer on the subphase methanol–water [19]. At DPPC monolayer on the surface of subphase methanol–water no significant changes of the dipole moment projection of the DPPC molecule appear when compared to the dipole moment projection of the DPPC molecule on water, even though the effect of subphase methanol–water is obvious. We can observe the time shift of the area per DPPC molecule to higher values. We assume that DPPC molecules move away from each other leading to incorporation (penetration) of methanol molecules into the air–liquid interface [16,31–34]. During incorporation (penetration) into DPPC molecules layer, no significant influence on to DPPC is observed, as we do not observe any changes of the dipole moment projection. Incorporation (penetration) of the methanol molecules into the layer is manifested by the shift of p–A isotherm to higher area values. From the shape of p–A isotherm we can assume a smooth transition from liquid expanded to liquid condensed phase [20]. DPPC monolayer on subphase methanol–water has no distinct electrical properties in comparison to the DPPC monolayer on water. We suppose that incorporation (penetration) of methanol molecules into DPPC layer causes no changes in electrical properties of this layer when compared to electrical properties of DPPC on water [35]. Consequently, we assume no electrical interactions between ions of the polar head and methane ions. Due to moving molecules away from each other, increased membrane permeability can occur [36,37]. 4.1.3. Ethanol Measurement of the dipole moment projections of the DPPC monolayer on the surface of subphase ethanol–water in relation
to area per molecule show two order transitions. As we observe no time shift of the dipole moment projection, we assume that the distance between molecules does not increase. Unlike DPPC monolayer on the subphase methanol–water, adsorption of ethanol to this layer might occur. Yamamoto et al. [38] investigated interaction with DPPC and dihexadecyl phosphate with ethanol. They found different behaviour of ethanol–water solution depending on its concentration. Based on their results, it can be concluded that at low concentration of ethanol, hydrates adsorb on the monolayer–water interface and saturate on the interface. The increase of ethanol concentration causes multilayer formation of hydrates and/or penetration of hydrates into the monolayer core. From NMR results Barry and Gawrisch [6] showed ethanol binding in the lipid–water interface. The interaction of ethanol in the lipid–water interface changed order parameter. The amount of cholesterol influenced the phase transition to the liquid-ordered phase and ethanol binding decreased with increasing amounts of cholesterol. Moreover, measurements show the time shift of the dipole moment projection of the DPPC molecule to lower values. Values of the dipole moment projection reach negative values. Major part of the dipole moment of DPPC molecule consists of the phosphatocholin (PC) group in the polar head. Interaction of DPPC–ethanol influenced bonds in this group. Due to the adsorption of ethane molecules to monolayer consisting of DPPC molecules, reversion of the dipole moment projection of the DPPC molecule is observed. We suppose that either change of bond orientation or disruption of a bond occured [39] resulting in significant change of electric properties of PC group. The change of electric charge on the membrane surface can appears which will influence the diffusion of ions. The change of electric charge on the membrane surface (in natural state the membrane surface is weakly negatively charged due to the PC group) will change the ion diffusion through the membrane (reduction of the negative charge might occur resulting in easier transition of negative ions). Measurements of MDC in dependence of area per molecule on the subphase ethanol–water suggest, that no time-dependent
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
significant changes occur as it was in case of DPPC monolayer on the subphase methanol–water. We suppose that electric properties of this monolayer have not changed significantly. 4.2. Membrane rigidity From graph representing the relation between elasticity modulus and area per molecule of DPPC monolayer on the surface of both subphases (ethanol–water and methanol–water) we found that elasticity modulus of the monolayer decreases. Based on our results, we assume a gradual ‘‘solidification’’ of the membrane and loss of its elasticity [40,41]. Increase of rigidity is proportional to alcohol length, what is in agreement with other studies [16]. Inequality of influence of alcohol molecules on phospholipid monolayer is caused by the different alcohol chain length. Bending rigidity depends on chain length and adsorption rate in complex form. The measurements of the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols revealed that the order in decreasing mechanical properties was butanol > propropanol > ethanol > methanol [42]. Goldstein [43] desribed that animals after chronical treatment of ethanol showed stiffer membranes. This stiffer effect of ethanol may be reduced by cholesterol or saturated fatty acids. Goldstein and Chin [44] examined the influence of ethanol on a cell membrane. The mice were treated with ethanol for 8 days. It was found that ethanol disorders mouse cell membranes, making the lipid matrix more fluid. The consequent disruption of the function of integral membrane proteins may be the cause of ethanol’s central actions. The tolerance to the disordering effect of ethanol was accompanied by an increased proportion of cholesterol in the membranes. 5. Conclusion Behaviour of DPPC molecules on two different subphases is substantialy different. In DPPC monolayer on the subphase of methanol–water, a gradual absorption (incorporation, penetration) of methanol molecules into the layer can appear leading to ‘‘dilution’’ of the layer and thus to the change of monolayer permeability. In DPPC monolayer on the subphase of ethanol– water adsorption of ethanol molecules to the layer can be observed leading to the change of electric properties of the layer surface. Consequently, the membrane permeability might change. At both subphases (ethanol–water and methanol–water) the elasticity modulus of the monolayer decreases leading to the loss of membrane elasticity.
References [1] [2] [3] [4] [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] [30] [31] [32] [33] [34] [35]
[36] [37] [38] [39] [40]
Acknowledgement This work was supported by grant of Science and Technology Assistance Agency nos. APVT-20-003104 and APVT-51-013904.
2431
[41] [42] [43] [44]
G. Mandel, W. Clark, J. Immunol. 120 (1978) 1637. E.S. Rowe, T.A. Cutrera, Biochemistry 29 (1990) 10398. P. Nambi, E.S. Rowe, T.J. McIntosh, Biochemistry 27 (1988) 9175. C.L. Kowalczyk, J.J. Stachecki, J.F. Schultz, R.E. Leach, D.R. Armant, Alcohol Clin. Exp. Res. 20 (1996) 566. B.T. Hutchinson, P.A. Hyslop, C.E. Kuhn, R.D. Sauerheber, Biochem. Pharmacol. 34 (1985) 1079. J.A. Barry, K. Gawrisch, Biochemistry 34 (1995) 8852. H. Komatsu, E.S. Rowe, Biochemistry 30 (1991) 2463. J. Mou, J. Yang, C. Huang, Z. Shao, Biochemistry 33 (1994) 9981. J. Zhang, H. Cao, B. Jing, S.L. Regen, J. Am. Chem. Soc. 128 (2006) 265. J.A. Barry, K. Gawrisch, Biochemistry 33 (1994) 8082. L.L. Hote, K. Gawrisch, Biochemistry 36 (1997) 4669. S.E. Feller, C.A. Brown, D.T. Nizza, K. Gawrisch, Biophys. J. 82 (2002) 1396. W.G. Wood, F. Schroeder, Life Sci. 43 (1988) 467. P. Celec, P. Jani, L. Smrekova, A. Mrlian, M. Kudela, J. Hodosy, P. Boor, V. Kristova, J. Jakubovsky, D. Jezova, L. Halcak, P. Bozek, J. Slamova, O. Ulicna, D. Hojsik, I. Jurkovicova, Life Sci. 74 (2003) 419. D.B. Goldstein, Ann. Rev. Pharmacol. Toxicol. 24 (1984) 43. L. Foret, A. Wu¨rger, Phys. Rev. Lett. 86 (2001) 5930. V. Luzzati, L. Mateu, G. Marquez, M. Borgo, J. Mol. Biol. 286 (1999) 1389. G.Y. Sun, A.Y. Sun, Alcohol Clin. Exp. Res. 9 (1985) 164. M. Weis, M. Kopani, P. Michalka, Cs. Biro, P. Celec, L. Danisovic, J. Jakubovsky, J. Biochem. Biophys. Methods 65 (2005) 81. V.M. Kaganer, H. Mo¨hwald, P. Dutta, Rev. Modern Phys. 71 (1999) 779. M. Iwamoto, Y. Majima, Thin Solid Films 178 (1989) 67. M. Iwamoto, Y. Majima, J. Chem. Phys. 94 (1991) 5135. D. Barancˇok, J. Cira´k, P. Tomcˇ´ık, J. Vajda, Phys. Stat. Sol. 169 (1998) 267. A.V. Zakharov, M. Iwamoto, Phys. Rev. E66 (2002) 0616051. K. Sulaiman, W.H.A. Majid, M.R. Muhamad, Appl. Surf. Sci. 252 (2006) 2875. J. Cira´k, D. Barancˇok, P. Tomcˇ´ık, J. Vajda, Mater. Sci. Eng. C 8–9 (1999) 13. R. Lipowsky, Nature 349 (1991) 475. F.C. Goodrich, in: J.H. Schulman (Ed.), Proceedings of the Second International Congress of Surface Activity, vol. 15, Butterworth, London, 1957, p. 85. J. Vajda, M. Weis, D. Barancˇok, J. Cira´k, P. Tomcˇ´ık, Appl. Surf. Sci. 229 (2004) 183. V. Vogel, D. Mo¨bius, J. Colloid Interface Sci. 126 (1988) 408. H. Nakahara, S. Nakamura, T. Hiranita, H. Kawasaki, S. Lee, G. Sugihara, O. Shibata, Langmuir 22 (2006) 1182. A. Takahara, S. Ge, K. Kojio, T. Kajiyama, J. Biomater. Sci. Polym. Ed. 11 (2000) 111. I. Ohtsuka, S. Yokoyama, Chem. Pharm. Bull. (Tokyo) 53 (2005) 42. W. Caetano, M. Ferreira, O.N. Oliveira Jr., R. Itri, Colloids Surf. B Biointerfaces 38 (2004) 21. R.O. McCracken, A.W. Carr, W.H. Stillwell, K.B. Lipkowitz, R. Boisvenue, G.O. O’Doherty, D.I. Wickiser, Biochem. Pharmacol. 45 (1993) 1873. T. Katsu, Biochem. Int. 23 (1991) 413. B.A. Prior, J. Fennema, O. Pate, Appl. Microbiol. 29 (1975) 685. Y. Yamamoto, K. Taga, T. Yoshida, H. Kamaya, I. Ueda, J. Colloid Interface Sci. 298 (2006) 529. J.G. Petrov, E.E. Polymeropoulos, H. Mo¨hwald, J. Phys. Chem. 100 (1996) 9860. L.M. Chi, W.G. Wu, K.L. Sung, S. Chien, Biochim. Biophys. Acta 1027 (1990) 163. B.G. Vertessy, T.L. Steck, Biophys. J. 55 (1989) 255. H.V. Ly, M.L. Longo, Biophys J. 87 (2004) 1013. D.B. Goldstein, Ann. Emerg. Med. 15 (1986) 1013. D.B. Goldstein, J.H. Chin, Fed. Proc. 40 (1981) 2073.
Ethanol and methanol induced changes in phospholipid monolayer M. Weis a, M. Kopa´ni b,*, J. Jakubovsky´ b, L’. Danihel b a
Slovak University of Technology, Faculty of Electrical Engineering and Information Technology, Department of Physics, Bratislava, Slovakia b Comenius University, School of Medicine, Institute of Pathology, Sasinkova 4, 811 08 Bratislava, Slovakia Received 21 April 2006; received in revised form 28 April 2006; accepted 28 April 2006 Available online 30 June 2006
Abstract The main components of cell membranes are phospholipids and proteins. The aim of our study was to examine structural changes of dipalmitoyl-phosphatidylcholine (DPPC) monolayer as a simple model system of a cell membrane in different environments. Pure water, ethanol and methanol solutions were used as subphases of Langmuir films as a membrane models. For detection of changes in charge states of the molecules as well as relation with structural and conformational changes, a contactless method Maxwell’s displacement currents (MDC) was used. Behaviour of DPPC molecules on two different subphases is substantialy different. In DPPC monolayer on the subphase of methanol–water, a gradual absorption (incorporation, penetration) of methanol molecules into the layer can appear. In DPPC monolayer on the subphase of ethanol– water adsorption of ethanol molecules on the layer can be observed. The membrane permeability might change. At both subphases (ethanol–water and methanol–water) the elasticity modulus of the monolayer decreases leading to the loss of membrane elasticity. # 2006 Elsevier B.V. All rights reserved. PACS: 68.08.p Liquid–solid interfaces; 68.18.g Langmuir–Blodgett films on liquids Keywords: Maxwell displacement current; Conformation; Membrane; Ethanol; Methanol; Permeability
1. Introduction Cell membrane is an important component of all cells. It separates intracellular from extracelluar spaces. It forms a selective permeabile border providing dynamic balance between a cell and an outer space. It contains enzymes, receptors, transport and signaling systems and antigens. The main components of cell membranes are phospholipids and proteins. They are incorporated into the membranes or proteins and can be electrostatically bounded to the surface. In addition to phospholipids and proteins, membranes contain water and different chemical compounds. Most important are cholestorol and glycolipids. Cholesterol provides firmness and fluidity of the membrane. Membranes seem to be altered by the presence of ethanol [1–4]. The mechanism by which ethanol affects function of a membrane at the molecular level is still not known. It is likely that ethanol acts in a hydrophobic environment. Alcohols cause the inhibition of molecule transport through the cell membrane.
* Corresponding author. Tel.: +421 2 59357273; fax: +421 2 59357592. E-mail address: [email protected] (M. Kopa´ni). 0169-4332/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2006.04.053
Hutchinson et al. [5] compared the relative potencies of the inhibition caused by the different alcohols. They found that potencies of the inhibition increased with the alcohol chain length. Alcohols can induce a disordering of the acyl chains of the fluid phospholipids, as well as the formation of an interdigitated gel phase [6–8]. From NMR studies it can be concluded that ethanol binding to lipid membrane near lipid– water interface [9–12]. The exact nature of the interactions among ethanol, membrane proteins, and the lipid framework remains obscure. It is known that ethanol fluidizes the bulk lipid of membranes and may alter cell function. The changes of cell membrane involve direct effect of ethanol on proteins, other membrane acting drugs, temperature effects, effects of ethanol on aged membranes and inconsistent effects of chronic ethanol consumption on lipid content [13]. Interaction between ethanol and cell membrane results in a disorder or ‘‘fluidize’’ membranes, or act as a local anesthetic. In some kinds of membranes low concentrations of drugs have an ordering effect [14,15]. Also influence of the alcohol (with various chain lengths) on disorder and rigidity of surfactants was investigated [16]. In the simplest picture, the molecular entropy varies with the area per molecule A as S = kBT ln A (kB and T denotes the
2426
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Boltzmann constant and temperature, respectively). Therefore, incorporation of cosurfactant into the phospholipid leads to disorder due to increasing of area per molecule. Luzzati et al. [17] investigated the structure of toad sciatic nerves exposed to either low temperature or influence of tetracaine. Their results suggest that membranes were thickened and stiffened. These phenomena are typical for lipid-containing systems with disordered chains. Some evidences indicate that interaction between ethanol and membranes have a membrane-fluidizing effect. The chronic response to this effect is not the change of the membrane bulk lipid composition [18]. Properties of the membrane model system may be investigated by examining the dipole moment projection of the monolayer during compression and analysis of the elasticity. Dipole moment projection analysis by the Maxwell displacement current (MDC) [19] is very sensitive method for evaluation of molecular orientation (so-called order parameter) as well as electric state of the molecule. In this way is possible to investigate phospholipid phase transition and influence of alcohol in the subphase. In contrast with surface pressure–area isotherm analysis is MDC measurement is extremely sensitive also in the low surface pressure–area, where these methods are useless. The aim of our study was to examine structural changes of dipalmitoyl-phosphatidylcholine (DPPC) monolayer as a simple model system of a cell membrane in different environments and to compare effects of ethanol and methanol on this system. 2. Material and methods 2.1. Chemicals and preparation of monolayers The material used in this study as model phospholipid was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC) purchased from Sigma–Aldrich. Lipid was dissolved in chloroform at the stock concentration 0.5 mg/ml and spread on the subphase using microsyringe (Hamilton, USA). Pure water (bidistilled deionized water, 15 MV cm) and 20% ethanol and methanol solutions were used as subphases. For alcohol solutions ethyl alcohol and methyl alcohol were used (spectrophotometric grade purity) from Sigma–Aldrich. Subphase was thermostated to the temperature 17 8C. Monolayer
was allowed to equilibrate and solvent to evaporate for 15 min. This time was sufficient for chloroform to evaporate and monolayer to stabilize. 2.2. Experimental methods Various experimental techniques have been designed for the observation of structure properties and order parameter in organic monolayers situated onto the air–water interface [20]. However, the orientational parameter measurement in the time domain is possible only with some of them. For detection of changes in charge states of the molecules as well as relation with structural and conformational changes, a contactless method was developed based on analysis of Maxwell’s displacement currents. This method was originally introduced by Iwamoto and Majima [21,22] and improved by other authors [23–26]. Proposal of MDC experiment application for biological membrane phantom measurement was presented for the first time in our previous work [19]. The basic components of the Maxwell’s displacement current—experimental setup attached to the computer-controlled model Langmuir trough (model 611, Nima Technology, UK) are schematically shown in Fig. 1. The top electrode was suspended in air, parallel to the interface, without a direct mechanical or electrical contact with a floating monolayer on the water surface. The air gap between the top electrode and the water surface was regulated to a certain spacing (approximately 1 mm) by measuring the capacitance of the electrode system. The displacement current was detected with a Keithley 617 electrometer (Keithley Instruments, Cleveland, Ohio, USA). The sensitivity of measuring the current was 0.1 fA, the background noise was suppressed by a multiple electrical shielding of electrode as well as whole measuring system to 2 fA. Langmuir through was situated in a laminar-flow box on an antivibrating stand to avoid any mechanical stress. The total working area of the trough was 600 cm2 and the compression rate was 50 cm2/min, which correspond to ˚ 2/s per one molecule. The area of top electrode was 0.17 A AE = 20 cm2. Due to dynamic processes in the monolayer associated with the change in charge distribution caused by its compression, the induced charge in the top electrode varies with time and this generates a current flowing through the outer circuit via the electrometer.
Fig. 1. Schematic view of the experimental setup for displacement current measurement (left). Rod-like polar molecules execute precessional motion on the air– water interface (right) with maximal tilt angle QA (A and m stand for the area per molecule and the dipole moment of molecule, respectively). Electrical shielding of the top electrode is not drawn.
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
The level of the displacement current detected is very low and therefore the problem of background was carefully considered. The noise signal, known as the Johnson–Nyquist thermal noise, is at the level of 1–3 fA the value being at least one order of magnitude less than the signal of the monolayer. The MDC technique is sensitive only to dynamic charge processes, which in this experimental setup are caused by lateral compression of the monolayer. Therefore any timeindependent charge (mainly structured water layer and additional substances in subphase) distributed near/at the interface has no effect. In comparison with conventional electrical measurements of surface potential (by the Kelvin method) it provides big advantage in time-depended signals. The surface pressure–area isotherms were measured by the Willhelmy plate method with accuracy 0.05 mN/m. Consequently various mechanic and thermodynamic properties as elastic modulus of Langmuir films or thermodynamic properties (Gibbs free energy, entropy and enthalpy) can be evaluated. Membrane curvation due to thermal fluctuations is essential for shape and/or conformations of membranes as well as for cracks and defects generation [27]. The elastic modulus characterizes the elasticity of the monolayer and is in analogy with bulk materials defined as @p jEj ¼ A (1) @A T where p is the surface pressure, A the area per molecule and T is the temperature. The elastic modulus expresses the elasticity of the Langmuir film under influence of the compression force. The stability of the mixed monolayer can be determined by evaluation of excess Gibbs free energy of mixture following the Goodrich method [28] by integration of surface pressure–area isotherm up to selected surface pressure p Z p DG ¼ ðA12 x1 A1 x2 A2 Þ dp (2) 0
where A12 is the molecular area in the mixed monolayer, A1 and A2 the molecular areas in the pure component monolayer and x1 and x2 are the molar ratios of pure components in the mixture (x2 = 1 x1). In our case surfactant is DPPC and cosurfactant is adsorbed alcohol, x2 is time-depending parameter.
2427
where m is the dipole moment of one molecule (mz is projection of m to the normal), N the number of molecules under the top electrode and G is the geometrical factor depending only on the distance between the top electrode and the top plane of the monolayer and on the shape and area of the upper electrode. The hcos Qi stands for the statistical mean value cos Q where Q is the angle between the vector of dipole moment and the normal. Detailed analysis of dipole moment projection of simple fatty acid was described in Ref. [29]. As we show in our previous studies [23] the current flowing in the outer circuit can be expressed as a time change of the induced charge I¼
@Qi @hcos Qi @N þ mhcos QiG ¼ mNG @t @t @t
(4)
By integrating the displacement current with respect to time, the induced charge Q can be obtained and in this way we also evaluated the vertical component of the molecular dipole moment. Thus, the dipole moment projection to the normal mz should be calculated as Z 1 I dt (5) mz ¼ mhcos Qi ¼ G N 3. Results 3.1. MDC measurements During the measurement of the displacement current of the DPPC monolayer situated onto the pure water in relation to area ˚2 per molecule we can notice a sharp maximum at 110 A (Fig. 2). Relation between displacement current of the monolayer DPPC on the subphase methanol–water and area per molecule is depicted in Fig. 3. Our results indicate maximum at about ˚ 2. In addition, we can observe time-shift of a maximum of 90 A the monolayer displacement current to higher values of area per molecule.
2.3. Theoretical background of MDC The analysis of MDC experiment is based on the assumption that each molecule behaves like a weak dipole moment with a negative pole bound to the water surface. Individual molecules have random directions within a certain solid angle and execute a random precessional motion with a maximal possible tilt QA from the vertical axis. Generally, we consider the molecule as a rod-like rigid body without a possibility of bending. If we consider the organic film as a system of electric dipole moments then it is possible to calculate the induces charge on the upper electrode with the method of images. Qi ¼ hmz iNG ¼ mhcos QiNG
(3)
Fig. 2. Record of the displacement current–area isotherm measurement of DPPC monolayer onto the pure water subphase.
2428
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Fig. 3. Records of the displacement current–area isotherm measurements of DPPC monolayer on subphase methanol–water.
Relation between displacement current of the monolayer DPPC on the subphase ethanol–water and area per molecule is ˚ 2. In depicted in Fig. 4. We can notice maximum at about 50 A addition, we can observe a mild time-shift of the monolayer displacement current maximum to hihger values of area per molecule. It is notable, that in all gaseous phase of the monolayer negative displacement current is observed. 3.2. Dipole moment projection analysis By analysis of records of the Maxwell diplacement current projection, we can calculate dependence of the dipole moment on the area per molecule of the monolayer. On the surface of pure water at DPPC monolayer we can see a rapid change of the dipole moment projection at value around ˚ 2 (Fig. 5). 110 A At DPPC monolayer, on the surface of subphase methanol– water, no significant change of the dipole moment projection of DPPC molecule is observed related to the change of the dipole moment projection of DPPC molecule on water, even though the effect of methanol–water subphase is obvious. Rapid growth of the dipole moment projection is observed at around ˚ 2 per molecule (Fig. 6). area 90 A
Fig. 4. Records of the displacement current–area isotherm measurements of DPPC monolayer on subphase ethanol–water.
Fig. 5. The dipole moment projection to the normal calculated from MDC measurement of monolayer on pure water. Rapid growth of dipole moment ˚ 2 indicates ordering of molecules during the gas–liquid projection at 110 A phase transition.
Measurement of the dipole moment projection of DPPC monolayer on the surface of subphase ethanol–water in dependence of area per molecule indicates the rapid phase ˚ 2 (Fig. 7). transition from liquid phase to solid phase at 50 A Moreover, our recordings show a time shift to lower values of the dipole moment projection of DPPC molecule on the surface of subphase ethanol–water. We can observe that dipole moment projection reaches negative values of the polar head, even though the change of the tail dipole moment can occur. 3.3. Surface pressure–area isotherms Fig. 8 depicts relationship between surface pressure and area per molecule of the DPPC layer on the surface of subphase methanol–water. We can observe the time shift of p–A isotherm to higher values of the surface pressure.
Fig. 6. The dipole moment projections to the normal calculated from MDC measurements on subphase methanol–water. Rapid growth of dipole moment ˚ 2 indicates ordering of molecules during the gas–liquid phase projection at 90 A transition.
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Fig. 7. The dipole moment projection to the normal calculated from MDC measurement on subphase ethanol–water. Rapid growth of dipole moment ˚ 2 indicates ordering of molecules during the liquid–solid projection at 50 A phase transition.
Fig. 8. p–A Isotherms of DPPC monolayer on subphase methanol–water.
2429
Fig. 10. Isothermal elastic modulus curves of DPPC monolayer on subphase: (top) ethanol–water, (bottom) methanol–water. Elastic modulus of DPPC situated onto the pure water subphase is presented for a comparison (grey curve).
At subphase ethanol–water, a significant change in the dipole moment projection of DPPC molecules forming the monolayer appeared (Fig. 9). Monolayer exhibits very sharp phase transition, phase transition from gaseous to liquid phase ˚ 2 (observable only for first compression) and at area about 80 A ˚ 2. from liquid phase to solid phase at 50 A Similarly, as at DPPC monolayer on the subphase methanol– water we can observe a time shift of p–A isotherm to higher values. However, this shift is not as obvious as it was at DPPC monolayer on the subphase methanol–water (Fig. 10). From the graph representing the relation between elasticity modules and area per molecule we found out that at DPPC monolayer on the surface of both subphases (ethanol–water and methanol–water) elasticity modulus of the monolayer decreases. Our results suggest a gradual increase of membrane rigidity (decrease of elasticity). Time dependence of Gibbs energy and maximum of dipole moment projection is shown in Fig. 11. The dipole moment projection maximum represents the phase transition area; therefore its change is directly proportional to adsorbed alcohol. In both cases is observable continuous adsorption. The Gibbs free energy characterizes the stability of the phospholipid–alcohol mixture. Methanol molecules destabilize the monolayer in contrast with ethanol, which incorporation into the monolayer carry to lower free energy. 4. Discussion 4.1. Dipole moment and electric properties
Fig. 9. p–A Isotherms of DPPC monolayer on subphase ethanol–water.
4.1.1. Water Results of experiments performed on pure water [20] ˚ 2 area per molecule, suggest that at the value around 100–110 A a phase transition occurs from gaseous phase to liquid phase. From results of the measurement of the surface potential by Kelvin probe [30] the value of the dipole moment of DPPC molecule was determined to be 820 mD.
2430
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
Fig. 11. Time dependence of Gibbs free energy of DPPC monolayer onto the ethanol–water (up) and methanol–water (down) subphase for various values of surface pressure (left view). Time dependence of dipole moment maximum position DPPC onto ethanol–water and methanol-water subphase (right view).
Our recordings show the value around 815–825 mD, which is in according with values obtained by independent measurements. 4.1.2. Methanol By the analysis of the measurement of the Maxwell diplacement current in relation with area per molecule, we calculated the dependence of the dipole moment projection on the area per molecule of the DPPC monolayer on the subphase methanol–water [19]. At DPPC monolayer on the surface of subphase methanol–water no significant changes of the dipole moment projection of the DPPC molecule appear when compared to the dipole moment projection of the DPPC molecule on water, even though the effect of subphase methanol–water is obvious. We can observe the time shift of the area per DPPC molecule to higher values. We assume that DPPC molecules move away from each other leading to incorporation (penetration) of methanol molecules into the air–liquid interface [16,31–34]. During incorporation (penetration) into DPPC molecules layer, no significant influence on to DPPC is observed, as we do not observe any changes of the dipole moment projection. Incorporation (penetration) of the methanol molecules into the layer is manifested by the shift of p–A isotherm to higher area values. From the shape of p–A isotherm we can assume a smooth transition from liquid expanded to liquid condensed phase [20]. DPPC monolayer on subphase methanol–water has no distinct electrical properties in comparison to the DPPC monolayer on water. We suppose that incorporation (penetration) of methanol molecules into DPPC layer causes no changes in electrical properties of this layer when compared to electrical properties of DPPC on water [35]. Consequently, we assume no electrical interactions between ions of the polar head and methane ions. Due to moving molecules away from each other, increased membrane permeability can occur [36,37]. 4.1.3. Ethanol Measurement of the dipole moment projections of the DPPC monolayer on the surface of subphase ethanol–water in relation
to area per molecule show two order transitions. As we observe no time shift of the dipole moment projection, we assume that the distance between molecules does not increase. Unlike DPPC monolayer on the subphase methanol–water, adsorption of ethanol to this layer might occur. Yamamoto et al. [38] investigated interaction with DPPC and dihexadecyl phosphate with ethanol. They found different behaviour of ethanol–water solution depending on its concentration. Based on their results, it can be concluded that at low concentration of ethanol, hydrates adsorb on the monolayer–water interface and saturate on the interface. The increase of ethanol concentration causes multilayer formation of hydrates and/or penetration of hydrates into the monolayer core. From NMR results Barry and Gawrisch [6] showed ethanol binding in the lipid–water interface. The interaction of ethanol in the lipid–water interface changed order parameter. The amount of cholesterol influenced the phase transition to the liquid-ordered phase and ethanol binding decreased with increasing amounts of cholesterol. Moreover, measurements show the time shift of the dipole moment projection of the DPPC molecule to lower values. Values of the dipole moment projection reach negative values. Major part of the dipole moment of DPPC molecule consists of the phosphatocholin (PC) group in the polar head. Interaction of DPPC–ethanol influenced bonds in this group. Due to the adsorption of ethane molecules to monolayer consisting of DPPC molecules, reversion of the dipole moment projection of the DPPC molecule is observed. We suppose that either change of bond orientation or disruption of a bond occured [39] resulting in significant change of electric properties of PC group. The change of electric charge on the membrane surface can appears which will influence the diffusion of ions. The change of electric charge on the membrane surface (in natural state the membrane surface is weakly negatively charged due to the PC group) will change the ion diffusion through the membrane (reduction of the negative charge might occur resulting in easier transition of negative ions). Measurements of MDC in dependence of area per molecule on the subphase ethanol–water suggest, that no time-dependent
M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431
significant changes occur as it was in case of DPPC monolayer on the subphase methanol–water. We suppose that electric properties of this monolayer have not changed significantly. 4.2. Membrane rigidity From graph representing the relation between elasticity modulus and area per molecule of DPPC monolayer on the surface of both subphases (ethanol–water and methanol–water) we found that elasticity modulus of the monolayer decreases. Based on our results, we assume a gradual ‘‘solidification’’ of the membrane and loss of its elasticity [40,41]. Increase of rigidity is proportional to alcohol length, what is in agreement with other studies [16]. Inequality of influence of alcohol molecules on phospholipid monolayer is caused by the different alcohol chain length. Bending rigidity depends on chain length and adsorption rate in complex form. The measurements of the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols revealed that the order in decreasing mechanical properties was butanol > propropanol > ethanol > methanol [42]. Goldstein [43] desribed that animals after chronical treatment of ethanol showed stiffer membranes. This stiffer effect of ethanol may be reduced by cholesterol or saturated fatty acids. Goldstein and Chin [44] examined the influence of ethanol on a cell membrane. The mice were treated with ethanol for 8 days. It was found that ethanol disorders mouse cell membranes, making the lipid matrix more fluid. The consequent disruption of the function of integral membrane proteins may be the cause of ethanol’s central actions. The tolerance to the disordering effect of ethanol was accompanied by an increased proportion of cholesterol in the membranes. 5. Conclusion Behaviour of DPPC molecules on two different subphases is substantialy different. In DPPC monolayer on the subphase of methanol–water, a gradual absorption (incorporation, penetration) of methanol molecules into the layer can appear leading to ‘‘dilution’’ of the layer and thus to the change of monolayer permeability. In DPPC monolayer on the subphase of ethanol– water adsorption of ethanol molecules to the layer can be observed leading to the change of electric properties of the layer surface. Consequently, the membrane permeability might change. At both subphases (ethanol–water and methanol–water) the elasticity modulus of the monolayer decreases leading to the loss of membrane elasticity.
References [1] [2] [3] [4] [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] [30] [31] [32] [33] [34] [35]
[36] [37] [38] [39] [40]
Acknowledgement This work was supported by grant of Science and Technology Assistance Agency nos. APVT-20-003104 and APVT-51-013904.
2431
[41] [42] [43] [44]
G. Mandel, W. Clark, J. Immunol. 120 (1978) 1637. E.S. Rowe, T.A. Cutrera, Biochemistry 29 (1990) 10398. P. Nambi, E.S. Rowe, T.J. McIntosh, Biochemistry 27 (1988) 9175. C.L. Kowalczyk, J.J. Stachecki, J.F. Schultz, R.E. Leach, D.R. Armant, Alcohol Clin. Exp. Res. 20 (1996) 566. B.T. Hutchinson, P.A. Hyslop, C.E. Kuhn, R.D. Sauerheber, Biochem. Pharmacol. 34 (1985) 1079. J.A. Barry, K. Gawrisch, Biochemistry 34 (1995) 8852. H. Komatsu, E.S. Rowe, Biochemistry 30 (1991) 2463. J. Mou, J. Yang, C. Huang, Z. Shao, Biochemistry 33 (1994) 9981. J. Zhang, H. Cao, B. Jing, S.L. Regen, J. Am. Chem. Soc. 128 (2006) 265. J.A. Barry, K. Gawrisch, Biochemistry 33 (1994) 8082. L.L. Hote, K. Gawrisch, Biochemistry 36 (1997) 4669. S.E. Feller, C.A. Brown, D.T. Nizza, K. Gawrisch, Biophys. J. 82 (2002) 1396. W.G. Wood, F. Schroeder, Life Sci. 43 (1988) 467. P. Celec, P. Jani, L. Smrekova, A. Mrlian, M. Kudela, J. Hodosy, P. Boor, V. Kristova, J. Jakubovsky, D. Jezova, L. Halcak, P. Bozek, J. Slamova, O. Ulicna, D. Hojsik, I. Jurkovicova, Life Sci. 74 (2003) 419. D.B. Goldstein, Ann. Rev. Pharmacol. Toxicol. 24 (1984) 43. L. Foret, A. Wu¨rger, Phys. Rev. Lett. 86 (2001) 5930. V. Luzzati, L. Mateu, G. Marquez, M. Borgo, J. Mol. Biol. 286 (1999) 1389. G.Y. Sun, A.Y. Sun, Alcohol Clin. Exp. Res. 9 (1985) 164. M. Weis, M. Kopani, P. Michalka, Cs. Biro, P. Celec, L. Danisovic, J. Jakubovsky, J. Biochem. Biophys. Methods 65 (2005) 81. V.M. Kaganer, H. Mo¨hwald, P. Dutta, Rev. Modern Phys. 71 (1999) 779. M. Iwamoto, Y. Majima, Thin Solid Films 178 (1989) 67. M. Iwamoto, Y. Majima, J. Chem. Phys. 94 (1991) 5135. D. Barancˇok, J. Cira´k, P. Tomcˇ´ık, J. Vajda, Phys. Stat. Sol. 169 (1998) 267. A.V. Zakharov, M. Iwamoto, Phys. Rev. E66 (2002) 0616051. K. Sulaiman, W.H.A. Majid, M.R. Muhamad, Appl. Surf. Sci. 252 (2006) 2875. J. Cira´k, D. Barancˇok, P. Tomcˇ´ık, J. Vajda, Mater. Sci. Eng. C 8–9 (1999) 13. R. Lipowsky, Nature 349 (1991) 475. F.C. Goodrich, in: J.H. Schulman (Ed.), Proceedings of the Second International Congress of Surface Activity, vol. 15, Butterworth, London, 1957, p. 85. J. Vajda, M. Weis, D. Barancˇok, J. Cira´k, P. Tomcˇ´ık, Appl. Surf. Sci. 229 (2004) 183. V. Vogel, D. Mo¨bius, J. Colloid Interface Sci. 126 (1988) 408. H. Nakahara, S. Nakamura, T. Hiranita, H. Kawasaki, S. Lee, G. Sugihara, O. Shibata, Langmuir 22 (2006) 1182. A. Takahara, S. Ge, K. Kojio, T. Kajiyama, J. Biomater. Sci. Polym. Ed. 11 (2000) 111. I. Ohtsuka, S. Yokoyama, Chem. Pharm. Bull. (Tokyo) 53 (2005) 42. W. Caetano, M. Ferreira, O.N. Oliveira Jr., R. Itri, Colloids Surf. B Biointerfaces 38 (2004) 21. R.O. McCracken, A.W. Carr, W.H. Stillwell, K.B. Lipkowitz, R. Boisvenue, G.O. O’Doherty, D.I. Wickiser, Biochem. Pharmacol. 45 (1993) 1873. T. Katsu, Biochem. Int. 23 (1991) 413. B.A. Prior, J. Fennema, O. Pate, Appl. Microbiol. 29 (1975) 685. Y. Yamamoto, K. Taga, T. Yoshida, H. Kamaya, I. Ueda, J. Colloid Interface Sci. 298 (2006) 529. J.G. Petrov, E.E. Polymeropoulos, H. Mo¨hwald, J. Phys. Chem. 100 (1996) 9860. L.M. Chi, W.G. Wu, K.L. Sung, S. Chien, Biochim. Biophys. Acta 1027 (1990) 163. B.G. Vertessy, T.L. Steck, Biophys. J. 55 (1989) 255. H.V. Ly, M.L. Longo, Biophys J. 87 (2004) 1013. D.B. Goldstein, Ann. Emerg. Med. 15 (1986) 1013. D.B. Goldstein, J.H. Chin, Fed. Proc. 40 (1981) 2073.