Fourier Transform Infrared Spectra and Molecular Orientation of Black

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Fourier Transform Infrared Spectra and Molecular Orientation of Black Lipid Films in Air Interacting with Metal Ions T. Tano and J. Umemura* Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan Received February 12, 1997. In Final Form: July 8, 1997X Polarized Fourier transform infrared spectra of black lipid films in air withdrawn from aqueous dispersions of dimyristoylphosphatidylcholine (DMPC) have been recorded at various electrolyte concentrations. The spectra revealed that monovalent cations gave little effect, whereas polyvalent cations except Mg2+ gave remarkable effects on vibrational wavenumbers of both the acyl chain and phosphate ester group of DMPC in black lipid films. In the case of Mg2+, the effect was discerned only for the phosphate ester group. By use of thin film optics, molecular orientation of DMPC in the black lipid film in air was quantitatively evaluated as a function of electrolyte concentration. In the case of monovalent and Mg2+ cations, the orientation angle of the acyl chain axes from the surface normal was little affected by the electrolyte addition. On the other hand, the other polyvalent cations caused decrease of the average orientation angle from ca. 40° to 30°. The affinity of the cations for the DMPC lipid films in air are in the order of Zn2+ > La3+ ∼ Ca2+ ∼ Pb2+ > Mg2+ > Na+ ∼ Li+.

Introduction Just 15 years ago, formation of a black lipid film in air stabilized by double-chained phospholipids was reported for the first time.1,2 After that, the nature of the black lipid film composed of insoluble surface-active agents such as fatty acids,3 alkanols,4 lysophospholipids,5-9 and especially double-chained phospholipids10-17 has widely been studied. The reasons that black lipid films in air stabilized by phospholipids have been extensively studied are as follows. First, phospholipids are major and essential membrane lipids. Second, black lipid films in air possess physicochemical characteristics that are intrinsically similar to those of the biomembranes.18 Third, the black lipid films in air have the advantage of their well-defined geometry and relatively well-known structure.19 For example, the black lipid film in air is a good model to * Corresponding author: fax, 81-774-38-3076; e-mail, umemura@ scl.kyoto-u.ac.jp; URL, http://kaimen.kuicr.kyoto-u.ac.jp/JU/. X Abstract published in Advance ACS Abstracts, September 15, 1997. (1) Yamanaka, T.; Hayashi, M.; Matuura, R. J. Colloid Interface Sci. 1982, 88, 458. (2) Yamanaka, T. Maku (Membrane) 1982, 7, 359. (3) Lal, J.; di Meglio, J.-M. J. Colloid Interface Sci. 1994, 164, 506. (4) Exerowa, D.; Cohen, R.; Nikolova, A. Colloids Surf. 1987, 24, 43. (5) Cohen, R.; Exerowa, D.; Kolarov, T.; Yamanaka, T.; Muller, V. M. Colloids Surf. 1992, 65, 201. (6) Yamanaka, T.; Tano, T.; Tozaki, K.; Hayashi, H. Chem. Lett. 1994, 1143. (7) Yamanaka, T.; Tano, T.; Kamegaya, O.; Exerowa, D.; Cohen, R. D. Langmuir 1994, 10, 1871. (8) Cohen, R.; Exerowa, D. Colloids Surf. A 1994, 85, 271. (9) Cohen, R.; Exerowa, D.; Yamanaka, T. Langmuir 1996, 12, 5419. (10) Cohen, R.; Koynova, R.; Tenchov, B.; Exerova, D. Eur. Biophys. J. 1991, 20, 203. (11) Exerowa, D.; Nikolova, A. Langmuir 1992, 8, 3102. (12) Nikolova, A.; Exerowa, D.; Lalchev, Z.; Tsonev, L. Eur. Biophys. J. 1994, 23, 145. (13) Lalchev, Z. I.; Wilde, P. J.; Clark, D. C. J. Colloid Interface Sci. 1994, 167, 80. (14) Lalchev, Z. I.; Wilde, P. J.; Mackie, A. R.; Clark, D. C. J. Colloid Interface Sci. 1995, 174, 283. (15) Vassilieff, C. S.; Manev, E. D. Colloid Polym. Sci. 1995, 273, 512. (16) Nikolova, A.; Koynova, R.; Tenchov, B.; Exerowa, D. Chem. Phys. Lipids 1996, 83, 111. (17) Tano,T.; Umemura, J. Chem. Lett. 1996, 801. (18) Ivanov, I. B. Thin Liquid Film; Marcel Dekker: New York, 1988. (19) Clunie, J. S.; Goodman, J. F.; Ingram, B. T. In Surface and Colloid Science; Matijevic, E., Ed.; Wiley-Interscience: New York, 1971; Vol. 3, p 167.

S0743-7463(97)00150-9 CCC: $14.00

study the adhesion (aggregation), fusion, and lateral packing change along the surface. Thus studies of black lipid films in air composed of phospholipids attract the attention of many investigators mainly from the view point of the role in biomembrane function. Recently, Matsumura et al.20 have reported that flocculation phenomena of phosphatidylcholine liposomes caused by Ca2+ ions depend on the liposome diameter. This system is of great interest because of its relevance to biomembrane systems. The diameter dependence of flocculation means that the interaction of the phospholipid with cations strongly relates to the size and/or the curvature of liposomes. The black film in air should be considered as the special case of large liposomes whose curvature is equal to zero. As is well-known, in black lipid films, the polar groups of the stabilizing molecules are located toward the aqueous core.19 Such films also have the defined flat surface in marked contrast to liposomes in dispersions where the size of liposomes usually have a broad distribution.21,22 Therefore black lipid films in air give a good example for studying the interaction of the phospholipid with a third component like electrolyte in the aqueous core. For the proposal mentioned above, however, systems of Langmuir films,23-26 (20) Matsumura, H.; Watanabe, K.; Furusawa, K. Colloids Surf. A 1995, 98, 175. (21) Hauser, H. In Phospholipids Handbook; Cevc, G., Ed.; Macel Dekker: New York, 1993; p 603. (22) van Zanten, J. H. In Vesicles; Rosoff, M., Ed.; Marcel Dekker: New York, 1996; p 239. (23) Shah, D. O.; Schulman, J. K. J. Lipid Res. 1967, 8, 227. (24) Hauser, H.; Phillips, M. C.; Levine, B. A.; Williams, R. J. P. Eur. J. Biochem. 1975, 58, 133. (25) Ohshima, H.; Ohki, S. J. Colloid Interface Sci. 1985, 103, 85. (26) Hunt, R. D.; Mitchell, M. L.; Dluhy, R. A. J. Mol. Struct. 1989, 214, 93. (27) Marra, J.; Israelachvili, J. Biochemistry 1985, 24, 4608. (28) Chapman, D.; Peel, W. E.; Kingston, B.; Lilley, T. H. Biochim. Biophys. Acta 1977, 464, 260. (29) Akutsu, H.; Seelig, J. Biochemistry 1981, 20, 7366. (30) Chrzeszczyk, A.; Wishnia, A.; Springer, C. S., Jr. Biochim. Biophys. Acta 1981, 648, 28. (31) Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand. R. P. Biochemistry 1981, 20, 1771. (32) Chowdhry, B. Z.; Lipka, G.; Dalziel, A. W.; Sturtevant, J. M. Biophys. J. 1984, 45, 633. (33) Altenbach, C.; Seelig, J. Biochemistry 1984, 23, 3913. (34) Herbette, L.; Napolitano, C.; McDaniel, R. V. Biophys. J. 1984, 46, 677.

© 1997 American Chemical Society

Molecular Orientation of Black Lipid Films

supported bilayers (Langmuir-Blodgett films),27 and uniand multilamellar vesicles20,28-42 have commonly been employed. On the other hand, Fourier transform infrared (FT-IR) spectroscopy is a new and powerful tool to explore the nature of the black lipid film in air.17,43,44 Also, the application of FT-IR spectroscopy to the study of black films in air offers many advantages.45 First, lipid monolayers in the black lipid film are macroscopically very well oriented at the air-aqueous core interface. Therefore they present a suitable system for using polarized IR techniques to study the orientation of the transition moments of the IR-active groups of lipid molecules. Second, one can measure the same sample at different angles of beam incidence. Then the obtained accuracy of any orientational information is expected to be very high. However, only a limited number of studies have been applied to black films because of their instability.17,43,46-48 In a previous study,17 we devised a new temperature-controlled cell and established a method to obtain stable black lipid films in air. Using this strategy, we have succeeded in measuring a gel to liquid-crystalline phase transition temperature of black lipid films in air by FT-IR spectroscopy. Also, we have revealed that the phase transition temperature of the black lipid film fairly agrees with that of a large vesicle in aqueous dispersions.17 In the present study, FT-IR spectroscopy was applied to black lipid films in air not only to clarify the orientational change quantitatively but also to elucidate the interactions between cations and phospholipid bilayers. Despite the fact that NMR spectroscopy as well as FT-IR spectroscopy is a particularly promising method to study the interaction of cations with phospholipid dispersions, NMR techniques have suffered from the poor detection limits because of the minute amount of constituents in the black lipid film. Therefore, our study using FT-IR spectroscopy will provide valuable information on the cation binding to this system. Experimental Section Materials. Synthetic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (purity g99%) was purchased from Sigma Chemical Co., St. Louis, and used without further purification. Water was purified with a modified Mitamura Riken Model PLSDFR automatic lab still consisting of a reverse osmosis module, an ion-exchange column, and a double distiller. PbCl2 (g99.99%) and NaCl (g99.9%) were purchased from Aldrich Chemical Co., Inc., Milwaukee, and Nacalai Tesque, Inc., Kyoto, respectively. Other chlorides of La, Ca, Zn, Mg, and Li were of the super-pure grades (g99.9%) from Wako Pure Chemical Ind., Ltd., Osaka. Measurements. A vertical black film was prepared by withdrawing a rectangular platinum frame (33 mm width, 14 (35) Tatulian, S. A. Eur. J. Biochem. 1987, 170, 413. (36) Conti, J.; Halladay, H. N.; Peterheim, M. Biochim. Biophys. Acta 1987, 902, 53. (37) Haverstick, D. M.; Glaser, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4475. (38) Petersheim, M.; Halladay, H. N.; Blodnieks, J. Biophys. J. 1989, 56, 551. (39) Akutsu, H.; Nagamori, T. Biochemistry 1991, 30, 4510. (40) Sapia, P.; Sportelli, L. J. Phys. (Paris) 1994, 4, 1107. (41) Kwon, K. O.; Kim, M. J.; Abe, M.; Ishinomori, T.; Ogino, K. Langmuir 1994, 10, 1415. (42) Minami, H.; Inoue, T.; Shimozawa, R. Langmuir 1996, 12, 3574. (43) Umemura, J.; Matsumoto, M.; Kawai, T.; Takenaka, T. Can. J. Chem. 1985, 63, 1713. (44) Mendelsohn, R.; Davies, M. A. In Fourier Transform Infrared Spectroscopy in Colloid and Interface Science; Scheuing, D. R., Ed.; ACS Symposium Series No. 447; American Chemical Society: Washington, DC, 1991; p 24. (45) Lewis, R. N. A. H.; McElhaney, R. N. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Eds.; Wiley-Liss: New York, 1996; p 159. (46) Tian, Y. Langmuir 1992, 8, 1354. (47) Liang, Y.; Zhang, Z. J. Dispersion Sci. Technol. 1995, 16, 339. (48) Zhang, Z.; Liang, Y. J. Colloid Interface Sci. 1995, 169, 220.

Langmuir, Vol. 13, No. 21, 1997 5719 mm height, and 1 mm diameter) from an aqueous dispersions of DMPC containing various concentrations of electrolytes (0-1 M) in the cell. For the preparation of aqueous dispersions, 50 mg of DMPC either in 100 mL pure water or in each electrolyte solution was agitated by a Vortex mixer (∼3 min) at 40 °C, which is above the gel to liquid-crystalline phase transition temperature (Tc ) 24 °C49). After that, each solution was sonicated by a Branson Model SONIFIER 250 until the suspension became clear and bluish (3-10 min) at the same temperature.1 The platinum frame in the cell was designed to be rotary around its central axis, which is vertical to the surface of the solution so that the angle of incidence can be easily changed. The precision in the angles of incidence was within (0.5°. The platinum frame was attached to a device which could be moved vertically at a controllable speed. To ensure the supply of DMPC molecules from the bulk reservoir and to get the stable film, we obeyed a new protocol as given below. After the frame was withdrawn by 1-2 mm from the solution within 2 min, we waited for 15-20 min to obtain a black film, and again withdrew 1-2 mm from the solution within 2 min and so on. The film, being attached to the dispersed solution through the meniscus, was fragile. Therefore, we completely withdrew the frame out of the solution and the black lipid film thus obtained was subjected to FT-IR measurements. In order to investigate the reproducibility of the observed absorbances, FT-IR measurements were applied to four distinct solutions without electrolytes. The dispersion of the observed absorbances was less than (4% with respect to the average value. The sample cell used in this work was the same as that reported previously.17 The infrared beam normally passed through the lipid film and two CaF2 windows (17 mm diameter) mounted on the cell wall. In the case of normal incidence to the film surface, the spot of the infrared beam on the film was as small as about 9 mm in diameter. The cell consists of a double wall, except for the CaF2 windows, and thermostated water was circulated through it by a Neslab RTE-8 bath circulator to achieve the equilibrium of temperature and vapor pressure inside the cell. Temperature was monitored by a copper-constantan thermocouple inserted into the cell. The overall accuracy of temperature control and reading was within (0.1 °C. All the measurements were performed at 28 °C. Infrared spectra were recorded on a Nicolet 710 FT-IR spectrophotometer equipped with an MCT detector. A Hitachi wire grid polarizer was used for the polaization measurements. Typically 1000-3000 interferograms were collected with a resolution of 4 cm-1.

Results and Discussion Figure 1 shows the FT-IR spectra in the 3800-2800 cm-1 region of the lipid films withdrawn from the aqueous dispersions of DMPC containing various concentrations of CaCl2. In this frequency region, we can observe the antisymmetric and symmetric CH2 stretching bands (νa(CH2) and νs(CH2)) of DMPC at ca. 2920 and 2850 cm-1, respectively.43,45,50 The broad band at ca. 3400 cm-1 is ascribed to the OH stretching vibration of the core water.43,51 The features of the 3400 cm-1 band (peak wavenumber and contour) are very similar to those of bulk liquid water, which indicates that the core water of the black lipid film is in almost random orientation. Since the spectra have been recorded with a high signal-to-noise ratio, we can reliably read the wavenumber of a particular band and calculate the orientation of its transition moment based on the observed band intensity. In Figure 2, the activity of various electrolytes is plotted against the concentration of electrolyte. From now on, we use the activity of electrolyte instead of the concentration of electrolyte. The reasons are as follows. First, by the use of association constant, activity is connected to (49) Kodama, M.; Miyata, T.; Takaichi, Y. Biochim. Biophys. Acta 1993, 1169, 90. (50) Umemura, J.; Cameron, D. G.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 602, 32. (51) Grdadolnik, J.; Kidricˇ, J.; Hadzˇi, D. J. Mol. Struct. 1994, 322, 93.

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Tano and Umemura

Figure 1. Infrared spectra in the 3800-2800 cm-1 region of black lipid films in air withdrawn from aqueous dispersions of DMPC containing various concentrations of CaCl2.

Figure 3. Activity dependence of the νa(CH2) wavenumber of DMPC (a) and of the tilt angles (γ) of the acyl chain axes (b) in the black lipid film for various electrolytes: (9) LaCl3; (b) CaCl2; (2) ZnCl2; ([) PbCl2; (]) MgCl2; (O) NaCl; (4) LiCl. The lines are a guide to the reader’s eye.

Figure 2. (top) Activity against concentration of various electrolytes calculated from the approximate equations. (bottom) Enlargement of top with electrolyte concentration between 0.1 and 1 M.

the cation binding energy, which can determine the binding affinity to a bilayer surface.52,53 Second, we want to discuss the effect of various cations over the wide concentration range where the activity cannot be replaced by the bulk concentration. Activity of electrolytes was (52) Cevc, G.; Marsh, D. Phospholipid Bilayers; Wiley-Interscience: New York, 1987. (53) Cevc, G. Biochim. Biophys. Acta 1990, 1031-3, 311.

calculated by y2c2, 4y3c3, and 27y4c4, for 1:1, 2:1, and 3:1 types of electrolytes, respectively. Here c is the molar concentration and y is the corresponding activity coefficient of electrolytes. For the estimation of y covering the wide range of electrolyte concentration, we used the approximate equations which are appropriate for the concentrations below 1 M with less than 1% error (eqs 3, 6, 9, and 11 in ref 54). In Figure 3a, the wavenumber of νa(CH2) is plotted against the activity of electrolytes. At activities below ca. 10-6 (which corresponds to the concentration of ca. 0.1 M for CaCl2), the wavenumbers in the case of di- and trivalent cations remain constant at ca. 2921 cm-1. At activities above 10-6, the wavenumbers gradually decrease down to 2918 cm-1 for Ca2+ and 2917 cm-1 in the case of Zn2+. Such observed shift in wavenumber upon the increase of the electrolyte concentration not only indicates the conformational change of acyl chains of DMPC from the gauche to trans isomers50 but also illustrates the surfacecharge-induced phase transition that occurred in the black lipid film.55 For La3+ above 0.1 M (activity of 3.5 × 10-5), we could not conduct measurements because the lipid film showed behavior as a rigid film, indicating strong affinity of the cation to the lipid layers. On the other hand, no wavenumber change upon the increase of the electrolyte concentration was observed in the case of monovalent and Mg2+ cations. Figure 4 shows the FT-IR spectra in the polar head group region of lipid films which were withdrawn from aqueous dispersions of DMPC containing various concentrations of CaCl2. In this frequency region, we can observe the CdO stretching band (ν(CdO)), the CH2 scissoring band (δ(CH2)), and also the antisymmetric and (54) Pitzer, K. S.; Mayorga, G. J. Phys. Chem. 1973, 77, 2300. (55) Tian, Y. J. Phys. Chem. 1991, 95, 9985.

Molecular Orientation of Black Lipid Films

Langmuir, Vol. 13, No. 21, 1997 5721

Figure 6. Schematic representation of the uniaxial orientation of the acyl chain and of a polar head group of the DMPC molecule. Figure 4. Infrared spectra in the 1800-1000 cm-1 region of black lipid films in air withdrawn from aqueous dispersions of DMPC containing various concentrations of CaCl2.

aqueous core/DMPC/air in which only DMPC phases are anisotropic.43 By use of the thin layer optics developed by Hasegawa et al. for the uniaxially anisotropic polylayered system,59,60 the absorbances for the s-polarized light (As) and for the p-polarized light (Ap) can be calculated by the relation

A ) -log(T/T0)

(1)

where T and T0 are the transmittances of the DMPC layers with and without absorption, respectively. Here, the transmittances for the p- and s-polarized light are given by

Figure 5. Activity dependence of the νa(PO2-) wavenumber of DMPC in the black lipid film for various electrolytes: (9) LaCl3; (b) CaCl2; (2) ZnCl2; ([) PbCl2; (]) MgCl2; (O) NaCl; (4) LiCl. The lines are a guide to the reader’s eye.

symmetric PO2- stretching bands (νa(PO2-) and νs(PO2-)) at ca. 1735, 1468, 1228, and 1085 cm-1, respectively.38,45,50 In Figure 5, the wavenumber of νa(PO2-) is plotted against the activity of the electrolytes. In the presence of cations, νa(PO2-) of DMPC was shifted to higher frequency due to the counterion binding occurring at the polar head group region and/or to the rupture of hydrogen bonds between phosphate oxygen and water by the loss of bound water from the DMPC phosphate group as well.34,56-58 The frequency change in the polyvalent cations, especially in Zn2+, is larger than that for the monovalent ones. Calculation of Molecular Orientation. We assumed a uniaxial orientation of the transition moments of the acyl chains and the polar region of DMPC around the surface normal (z axis, Figure 6), because no dichroism was found in the polarized transmission spectra of the black lipid film. To quantitatively determine the orientation of the DMPC molecules in a lipid film, we consider a black lipid film as a five-phase plane-bounded system, air/DMPC/ (56) Kawai, T.; Umemura, J.; Takenaka, T. Colloid Polym. Sci. 1984, 262, 61. (57) Wong, P.; Mantsch, H. Chem. Phys. Lipids 1988, 46, 213. (58) Grdadolnik, J.; Kidricˇ, J.; Hadzˇi, D. Chem. Phys. Lipids 1991, 59, 57. (59) Hasegawa, T.; Takeda, S.; Kawaguchi, A.; Umemura, J. Langmuir 1995, 11, 1236. (60) Hansen, W. N. J. Opt. Soc. Am. 1968, 58, 380.

Tp )

Re(q5) 2q1 | |2 q1 (m11 + m12q5)q1 + (m21 + m22q5)

(2)

Ts )

Re(p5) 2p1 | |2 p1 (m11 + m12p5)p1 + (m21 + m22p5)

(3)

where

qj )

pj )

(nˆ je2 - n12 sin2 θ1)1/2

(4)

nˆ jo2/nˆ je2 (nˆ je2 - n12 sin2 θ1)1/2

(5)

nˆ je2/nˆ jo2

Here, θ1 and nˆ j are the angle of incidence and the refractive index of the jth layer, respectively. The subscripts e and o refer to extraordinary (out of plane) and ordinary (in plane), respectively. mvw is the vwth element of the following 2×2 matrices

with elements

Mp ) M2pM3pM4p

(6)

Ms ) M2sM3sM4s

(7)

[ [

i sin βj qj -iqj sin βj cos βj i - sin βj cos βj Mjs ) pj -ipj sin βj cos βj

Mjp )

Here

cos βj

-

] ]

(8)

(9)

5722 Langmuir, Vol. 13, No. 21, 1997

βj )

2 2 2 1/2 2π (nˆ je - n1 sin θ1) hj λ nˆ je2/nˆ jo2

Tano and Umemura

(10)

where λ is the wavelength of the incident light in vacuum and hj is the thickness of the jth layer. Moreover

n1 ) n5

(11)

nˆ 2e ) nˆ 4e ) n2e + ik2e

(12)

nˆ 2o ) nˆ 4o ) n2o + ik2o

(13)

nˆ 3 ) n3 + ik3

(14)

where k is the extinction coefficient. Because of the optical anisotropy of the uniaxial film, the extinction coefficient k can be represented by the following equations;59,61

3 sin2 φ k 2 bulk

(15)

k2e ) k2z ) k4z ) 3 kbulk cos2 φ

(16)

k2o ) k2y ) k4y )

where φ is the orientation angle of the transition moment from the surface normal and kbulk is the extinction coefficient of the isotropic bulk medium. Although the all-trans chain is depicted in Figure 6, the actual molecule may contain some gauche conformations. In such cases, the orientation angle concerning the chain is the average of all the methylene segments in a molecule. In other words, φ corresponds to an average angle φav which satisfies 〈tan2 φ〉 ) tan2 φav. By comparison of the theoretical dependence of A with the experimentally observed values Aobs, the most appropriate orientation angles (φ) were determined. The used optical constants are θ1 ) 0°, 30°, 45°, and 60°, n1 ) 1.0 (air), n2o ) 1.464 (DMPC), n2e ) 1.486 (DMPC),62 nˆ 3 ) 1.415 + 0.0163i (water at 2920 cm-1), nˆ 3 ) 1.398 + 0.00941i (water at 2850 cm-1), nˆ 3 ) 1.253 + 0.0417i (water at 1090 cm-1),63 and h2 ) h4 ) 16.0 Å (DMPC).64 Every film withdrawn from DMPC dispersions containing various concentrations of electrolytes has its own thickness (h3) of the aqueous core, estimated from infrared measurement.7,43 We calculated the mean value of 2.2 nm, which is close to the result obtained by supported DMPC bilayers deposited on mica surfaces, 2.4-2.5 nm.27 Generally, one can calculate molecular orientation angles using the results obtained only from either p- or s-polarized light measurements, if we know kbulk. In our case, however, the conformation of the acyl chain of DMPC molecule and the lateral surface density in the black lipid film are feasible to change due to the presence of cations (cf. Figure 3a), which affects kbulk of the νa(CH2) and νs(CH2) bands of DMPC. If we take the ratio Ap/As to obtain orientation angles, they are practically independent of kbulk values if kbulk is at least less than 0.3 and φ is greater than 60°. Therefore, first, we tried to evaluate φ values. Ap/As obtained from eq 1 for the νa(CH2) band of the black lipid film containing 0.87 M CaCl2 (activity of 0.29) is plotted in Figure 7 against the angle of incidence at various orientation angles (φa) as kbulk being constant at 0.24. This kbulk value is estimated from that of octadecanethiolate.65 The black circles represent the results obtained by the (61) Hasegawa, T.; Umemura, J.; Takenaka, T. J. Phys. Chem. 1993, 97, 9009. (62) den Engelsen, D.; de Koning, B. Photochem. Photobiol. 1975, 21, 77. (63) Downing, H. D.; Williams, D. J. Geophys. Res. 1975, 80, 1656. (64) Pearson, R. H.; Pascher, H. I.; Sundel, S. Nature 1981, 281, 499.

Figure 7. Calculated absorbance of p- (top) and s-polarization (bottom) for νa(CH2) of the black lipid film containing 0.87 M CaCl2 (activity of 0.29) against the angle of incidence at various orientation angles (φa) as kbulk being constant at 0.24. The black circles show the results obtained from measurements.

measurement. By comparison of the observed absorbance ratio with the theoretical one, the orientation angle can be estimated to be 70°. By using the obtained φa value, we could derive kbulk values as 0.26 directly from the observed absorbances (Ap or As). Note that if φ is less than 60°, we replot the curves in Figure 7 by using the obtained kbulk and re-evaluate φa. Several iterative steps can fix both kbulk and φ. In Figure 8, the theoretical absorbances of the (top) pand of the (bottom) s-polarized light obtained from eq 1 for the νa(CH2) band of the black lipid film containing 0.87 M CaCl2 are plotted against the angle of incidence at various orientation angles (φa) by using the obtained kbulk value, 0.26. The black circles represent the results obtained from the measurements. For the s-polarization, the theoretical absorbance increases with an increase of the angle of incidence regardless of the orientation angle. In the case of the p-polarization, on the other hand, the relationship between the absorbance and the angle of incidence is not simple. When the orientation angle is between 50° and 90°, curves cross at near θ1 ) 58° as indicated by eq A9 (see Appendix for details). The observed values fit well with the theoretical curve for 70° in either case of the p- or s-polarization. This fact also supports the appropriateness of the obtained kbulk value. In the same way, other φa values at various activities of CaCl2 were calculated and are shown in Figure 9. Similarly, we calculated the orientation angles φs of the transition moment of νs(CH2). From φs and φa, the tilt angle γ of the acyl chain axes from the surface normal could be calculated by using eq 17, since these three directions are mutually orthogonal (see Figure 6)

cos2 φa + cos2 φs + cos2 γ ) 1

(17)

Figure 3b shows the tilt angles (γ) of the acyl chain axes of DMPC in black lipid films plotted against the activity. When the film is in the liquid-crystalline state which (65) The extinction coefficients (kbulk) of the νa(CH2) bands of DMPC in the gel state could be determined by taking into consideration the number of methylene groups between DMPC and octadecanethiolate.59,66 By comparing the transmission intensities of the KBr pellet of DMPC at various temperatures with the transmission intensity in the gel state, we could obtained kbulk of the νa(CH2) bands of DMPC in the liquidcrystalline phase ranging from 0.20 to 0.25. (66) Popenoe, D. D.; Stole, S. M.; Porter, M. D. Appl. Spectrosc. 1992, 46, 79.

Molecular Orientation of Black Lipid Films

Langmuir, Vol. 13, No. 21, 1997 5723

Figure 10. Activity dependence of the orientation angle (φp) of the νs(PO2) transition moment of DMPC in black lipid film for various electrolytes: (9) LaCl3; (b) CaCl2; (2) ZnCl2; ([) PbCl2; (]) MgCl2; (O) NaCl; (4) LiCl. The lines are a guide to the reader’s eye.

Figure 8. Calculated values of Ap/As for νa(CH2) of the black lipid film containing 0.87 M CaCl2 against the angle of incidence at various orientation angles (φa) as kbulk being constant at 0.26. The black circles show the results from measurements.

Figure 9. Obtained extinction coefficients (kbulk) of the νa(CH2) bands of the black lipid film as a function of activity: (9) LaCl3; (b) CaCl2; (2) ZnCl2; ([) PbCl2; (]) MgCl2; (O) NaCl; (4) LiCl.

contains more gauche conformers, an averaged orientation of all axes of chain segments is obtained in the time scale of the spectroscopic experiment, as mentioned above. Therefore, it is better to discuss the orientational order parameter of the acyl chain f which is given by

1 f ) (3〈cos2 γ〉 - 1) 2

(18)

The averaged tilt angle obtained is about 40° from the surface normal at the small activity range, yielding an order parameter f ) 0.38. This value implies that the acyl chains of DMPC still maintain some degree of order of molecular orientation even in the liquid-crystalline state. As activity increases, the angle in the polyvalent cations, except Mg2+, decreases down to about 30°. This value is in good agreement with earlier investigations of the lamellar gel phase of fully hydrated DMPC dispersions.67,68 On the other hand, in the case of monovalent and Mg2+ cations, the angle remains almost unaltered against the activity.

The obtained extinction coefficients (kbulk) of the νa(CH2) bands of the black lipid film are also shown in Figure 9 as a function of activity. As seen from Figure 9, the obtained kbulk values range from 0.19 to 0.26. This range fully corresponds with the one (0.20-0.25) we estimated from another way,65 which indicates that our other method to obtain kbulk values is still valid. In the case of monovalent and Mg2+ cations, the observed values are almost independent of activity. This means that the electrolyte addition has little effect not only on the molecular conformation, indicated by the change of wavenumber in Figure 3a, but also on the surface density because the change in kbulk with electrolyte concentration is contributed by both changes. In the case of La3+, Pb2+, Ca2+, and Zn2+, on the other hand, the change in kbulk values with the increase of activity are explained by the changes in molecular conformation and surface density. Especially, at maximum difference in the case of Ca2+ and Zn2+, the obtained kbulk value is about 1.3 times larger than that shown by the one without electrolyte. This strongly indicates that while the average chain tilt angle changes from ca. 40° to 30°, the molecules on the surface are packed denser than that without electrolyte. The orientation angle (φp) of the transition moment of νs(PO2-) are also evaluated and plotted against the activity in Figure 10. The angle is about 50° from the surface normal without electrolytes. Despite dispersion of the data, the polyvalent cations apparently increase the orientation angle more than the monovalent cations do. The orientation angles approach almost 60° at high activity in the case of polyvalent cations. This orientation angle is consistent with that obtained for lamellar gel phase of fully hydrated DMPC dispersions.67,68 Such conformational changes of the lipid polar head groups have been reported by many workers cited in ref 57. On the other hand, in the case of monovalent cations, the orientation angles also increase with activity, but the change is relatively small. Summarizing the results mentioned above, we can say that the affinity of the cations for the DMPC lipid films in air are in the order of Zn2+ > (La3+) ∼ Ca2+ ∼ Pb2+ > Mg2+ > Na+ ∼ Li+. The position of La3+ in the order is meaningful only below 0.1 M, because we could not obtain the stable film above this concentration where the film became rigid, indicating the ion’s too strong affinity for the black lipid film.32 This may correspond to the (67) Ter-Minassian-Saraga, L.; Okamura, E.; Umemura, J.; Takenaka, T. Biochim. Biophys. Acta 1988, 946, 417. (68) Hu¨bner, W.; Mantsch, H. H. Biophys. J. 1991, 59, 1261.

5724 Langmuir, Vol. 13, No. 21, 1997

Tano and Umemura

Conclusion With our new method to make the stable film, polarized FT-IR spectra of black lipid films of DMPC in air with and without electrolyte were recorded at various electrolyte concentrations. By use of the formulas for the anisotropic polylayered films to the five-phase system (air/DMPC/ water layer/DMPC/air), the orientation angle of the acyl chain axis of DMPC was quantitatively estimated. In the case of polyvalent cations, the orientation angle from the surface normal decreased from ca. 40° to 30° with the electrolyte addition. On the other hand, the addition of mono- and Mg2+ cations caused almost no orientational change. The affinity of the cations for the DMPC lipid films in air are in the order of Zn2+ > La3+ ∼ Ca2+ ∼ Pb2+ > Mg2+ > Na+ ∼ Li+.

Figure 11. Schematic view of DMPC arrangements in black lipid films in the presence of electrolytes. For an explanation, see the text. Symbols for the molecules are not drawn to scale.

phenomenon that the single-shelled vesicles were apparently no longer stable and formed aggregates at high concentrations of La3+.69 This order almost follows the magnitudes of the association constants of metal ions with simple phosphates as already mentioned in the study of liposomes by Chapman.28 Many workers have mentioned that phospholipid affinity for cations seems to follow the sequence lanthanides > transition metals > alkali earth metals > alkaline metals.70 Taking into consideration the strong La3+-DMPC interaction above 0.1 M, our results follow this sequence. Concerning the relatively weak Mg2+-DMPC interaction in our case, we have to point out several reasons for that. First, we have performed measurements above the gel to liquid-crystalline phase transition temperature. According to Marra et al.,27 the cation binding constants to phospholipids are much lower in the fluid state than in the gel phase and Mg2+ binding to phospholipids is weaker than Ca2+ binding. Second, the results shown in Figures 5 and 10 suggest that Mg2+ does interact with the polar region of DMPC lipid films in air. However, Mg2+ has no influence on the acyl chain regions (Figure 3). These conclusions seem to be consistent with the observations that addition of Mg2+ to phosphatidylserine (PS) vesicles has no crystallization action of the acyl chains at above 20 °C.71 The explanation of the obtained results can be summarized as follows (Figure 11). In the case of monovalent cations (a), an increase in concentration of electrolyte shows almost no effect because of the lack of interaction with the phosphate region due to the strong hydration or lower charge density of these cations. On the other hand, in the polyvalent cations except for Mg2+ (b), the molecules in the film aggregate with the increase of electrolyte because of the decrease of the cross section of the polar region due to its conformational change caused by the presence of cation. In addition, the bridging effect of the cations can help the aggregation of the film. Thus the orientation of the lipid molecules and hence the structure of the film is changed. In the case of Mg2+, the interaction is intermediate between (a) and (b), only affecting the head group. (69) Westman, J.; Eriksson, L. E. G. Biochim. Biophys. Acta 1979, 557, 62. (70) Tatulian, S. A. In Phospholipids Handbook; Cevc, G., Ed.; Macel Dekker: New York, 1993; p 511. (71) Portis, A.; Newton, C.; Pangborn, W.; Papahadjopoulos, D. Biochemistry 1979, 18, 780.

Acknowledgment. We thank Professor Masaru Nakahara of our laboratory for his warm encouragement during this work. We are grateful to Professor Teruko Yamanaka of Chiba University for her helpful advice. We also acknowledge a reviewer for the suggestion to get kbulk values directly from the present data. Appendix The Fresnel coefficients of the interface between two uniaxial phases j and k, whose optical axes are perpendicular to the interface, for parallel (p) polarized radiation are given by59

rjk )

tjk )

ξj )

ξj/nˆ jo2 - ξk/nˆ ko2 ξj/nˆ jo2 + ξk/nˆ ko2 2ξj/nˆ jo2 ξj/nˆ jo2 + ξk/nˆ ko2

nˆ jo 2 (nˆ - n12 sin2 θ1)1/2 nˆ je je

(A1)

(A2)

(A3)

where parameters are the same as mentioned earlier. Another parameter, βj, is also defined as βj ) 2πnˆ jhjξj/λ, where λ is the vacuum wavelength and hj the thickness of the jth layer. Now, we can express the transmission coefficient for a five-layer system72

t5 ) t12t23t34t45 exp[i(β2 + β3 + β4)]/D

(A4)

D ) 1 + r12r23 exp(2iβ2) + r23r34 exp(2iβ3) + r34r45 exp(2iβ4) + r12r34 exp[2i(β2 + β3)] + r23r45 exp[2i(β3 + β4)] + r12r23r34r45 exp[2i(β2 + β4)] + r12r45 exp[2i(β2 + β3 + β4)] The transmittance for parallel polarization of the fivephase system (phase 1 is isotropic) is

T)

µ5Re(ξ5/nˆ 5o2) µ1 cos(θ1/n1)

|t5|2

(A5)

We assume that the phases 1 and 5 and phases 2 and 4, respectively, are the same (symmetrical five-phase system like black lipid films), phases 1 and 5 are nonabsorbing, absorption of phase 3 is small (k3 , 1), and only phases 2 and 4 are uniaxial phases. In addition, we make an assumption that thicknesses of phases 2-4 are (72) Fujiyama, T.; Herrin, J.; Crawford, B. L., Jr. Appl. Spectrosc. 1970, 24, 9.

Molecular Orientation of Black Lipid Films

Langmuir, Vol. 13, No. 21, 1997 5725

much less than the wavelength of the incident radiation on the surface. If the exact equation for transmission, A5, in a symmetrical five-phase system is expanded to first order in terms of the thicknesses of the phases 2 and 3, we obtain

T)1-

[

4πn12n3k3h3 cos2 θ1 (ξ1/n12)λ

[

n14

8πn12h2 n2ok2o cos2 θ1 (ξ1/n12)λ 2

2

4π n1 h2h3 (ξ12/n14)λ

[

(n12 - n32) 2

]

(

(n2e2 + k2e2)2 2

2

cos θ1

] )

[

[

(ξ12/n14)λ2

n2ok2o cos2 θ1

n32

[

cos2 θ1 n14

(n2e2 + k2e2)2

+

]

][

n34

-

n34

n14

(ξ1/n12)λ

]

]

n2ek2e sin2 θ1 +

n14

-

(n2e2 + k2e2)2

(A7)

]

[

cos2 φ

×

(ξ12/n14)λ2 ×

{

2n2z sin2 θ1

(n2z2 + k2z2)

2

]

]}

n2x cos2 θ1

{

n14

)

2 n2x cos2 θ1 12π n1 h2kbulk 1 + ln 10 λ ξ1/n12 n14

]

n2ek2e sin2 θ1 -

][

2 n2x cos2 θ1 12π n1 h2kbulk 1 + AT ) ln 10 λ ξ1/n12 n14

16π2n14n3k3h2h3

sin2 θ1 n2ok2o cos2 θ1 -

]

sin2 θ1

n2ek2e sin2 θ1 +

n14

n14

n34

When eqs 15 and16 are substituted in A8, we obtain

×

(n2o2 - k2o2) cos2 θ1 (n2e2 - k2e2) sin2 θ1 1 n12 n14 (n2e2 + k2e2)2 32π2n14n3k3h2h3 cos2 θ1

[

n14

8πn12h2 n2ok2o cos2 θ1

[

+

sin θ1 -

n12

(ξ1/n12)λ

sin2 θ1 +

2 2 2 1 8πn1 h2 n2xk2x cos θ1 n2zk2z sin θ1 AT ) + ln 10 (ξ /n 2)λ n14 (n2z2 + k2z2)2 1 1 (A8)

+

n2ek2e sin2 θ1 +

n14

n34

[

4πn12n3k3h3 cos2 θ1

By converting eq A7 to absorbance, we get eq A8

sin2 θ1 +

T)1-

(n2e2 + k2e2)2 (A6)

In the black lipid films in air, the length of DMPC (phases 2) and water core thickness (phases 3) are a few nanometers. Therefore we can ignore the terms including h2h3 in (A6) and have

[

cos2 φ

2n2z sin2 θ1

(n2z2 + 9kbulk2 cos4 φ)2

]}

n2x cos2 θ1 n14

(A9)

From eq A9, one can easily understand the reason why curves cross around the angle of incidence, θ1 ) 58°. Around such an angle, the factors in brackets of eq A9 become almost zero, if φ is between 50° and 90°. Therefore, the curves cross at this angle of incidence. Registry No. Supplied by Authors: DMPC, 13699-48-4.

LA970150W