J . Phys. Chem. 1990, 94, 5073-5078
spectrometer and the standard pulse sequence “Kinet” for acquisition of kinetic data. Rate constants were calculated by integration of the signals and exponential fitting with the software of the GE-500 instrument. 3sCI line width and cmc measurements were performed as described.
5073
Acknowledgment. Support by the National Science Foundation (Organic Chemical Dynamics Program) and the Aluminum Company of America is gratefully acknowledged. We are grateful to Dr.Ata Shirazi for his help with the NMR measurements. Registry No. Methyl naphthalene-2-sulfonate,5 138-53-4.
Lipid and Lipid-Protein Monolayers Spread from a Vesicle Suspension: A Microfluorescence Film Balance Study S.-P. Heyn, M. Egger, and H. E. Gaub* Technische Vniversitat Miinchen, Physikdep E22, 8046 Garching, FRG (Received: November 6. 1989)
The process of the formation of a monolayer from a vesicle suspension for the two lecithins DMPC and DPPC, for the phosphatidic acid DMPA, and for a synthetic proteolipid was investigated. Exchange kinetics between vesicles and a monolayer as well as equilibrium spreading pressure of the above substances were determined for different temperatures. The resulting monolayers were separated from the vesicle phase and characterized by film balance microfluorescence techniques. Pressure-area diagrams were taken. Simultaneously the lateral distribution of a fluorescent probe that was a minor constituent of the monolayer was recorded. The data of the pure lipid films were compared with those obtained by the equivalent monolayers spread from an organic solution. The vesicle-spread monolayers were transferred onto solid supports, and the teKture and the height distribution of the monolayers were determined. I n the case of the lipid-protein monolayer a fluorescence binding assay was carried out after the spreading and transfer procedure. It is shown (1) that the vesicle-spread monolayers are separable from the vesicles, (2) that within the framework of our study their properties do not differ from the equivalent monolayers that were spread from an organic solution, (3) that their composition is the same as that of the vesicles, and ( 4 ) that the technique employed here enables one to transfer certain membrane-bound proteins from the vesicle phase via the air-water interface onto solid supports retaining their functionality. Furthermore, a model for the lipid exchange between the vesicles and the monolayer is suggested.
Introduction Monomolecular lipid films at the air-water interface have been studied extensively in recent years. The interest in these films has on the one hand been stimulated by the relatively easy access to the physical and chemical processes in a twc+dimensional system that they offer.Is2 On the other hand, such monolayers are relevant model systems of biological membranes. The possibility of a monolayer transfer onto a solid support makes them of interest from the point of view of novel material^.^ These systems have also been demonstrated to be useful in cell surface modeling especially in studies of molecular recognition at cell ~urfaces.~For certain types of problems the use of an artificial membrane system on a solid support offers significant advantages compared to suspended bilayers. Surface sensitive measuring techniques may then be employed. These techniques enable one to distinguish extremely sensitively between events close to the surface and those occurring in the bulk phase.s For example, if one of the membranes in a cell-cell interaction can be mimicked by a supported membrane, these techniques offer insight into just the region where the interaction takes place. By means of this the signal to noise ratio of the measurement might be drastically increased.6 Monolayers of pure or mixed lipids are spread in general from an organic solution allowing the solvent to evaporate at the airwater interface. This spreading procedure is, however, the main obstacle in the application of the film balance technique to biological systems. Most molecules with biological functions like proteins denature under those conditions. In order to incorporate proteins into such a monomolecular film, other ways of spreading have to be employed. Verger and Pattus’ formed monolayers by adding dropwise a vesicle suspension onto a vertical glass rod that had its lower end in contact with an aqueous phase. Heck1 et al.33 dropped small amounts of protein in a detergent solution onto a lipid monolayer. Schindler and co-workers investigated the *To whom correspondence should be addressed.
0022-3654/90/2094-5073$02.50/0
self-assembly of a lipid monolayer at the air-water interface of a vesicle suspension.* This latter spontaneous monolayer formation as well as the glass rod method has been used to reconstitute functional channel proteins in black lipid membranes (BLM).9Jo Since most of the studied molecules were transmembrane proteins, it remained unclear if indeed only a monomolecular film was formed or if a densely packed bilayer structure close to the surface would facilitate the environment for the protein to stay intact. In a more recent work of Kolomytkinll it has been shown by measuring current-voltage characteristics that the ion channel amphotericin B can be reconstituted in a black membrane. The author furthermore showed indirectly that this BLM was greatly reduced of attached or partly fused liposomes if the two monolayers obtained according to Schindler’s method had to pass a wet bridge of etched glass before the bilayer was formed. With the motivation to create supported planar lipid bilayers containing intact proteins, we have investigated the monolayer formation from an aqueous suspension of pure lipid vesicles and such vesicles containing the Fab’ fragment of an antibody cova( I ) McConnell, H. M.; Keller, D.; Gaub, H. E. J . Phys. Chem. 1986, 90, 1.71 7-1. 721. -.
(2) Gaub, H. E.; Moy, V. T.; McConnell, H. M. J . Phys. Chem. 1986,90, 1721-1725. (3) Barlow, W. A., Ed. Langmuir-Blodgett Films; Elsevier: New York, 1980. (4) Watts, T. H.; Gaub, H. E.: McConnell, H. M. Nature 1986, 320, 179-1 8 I . (5) Schmidt. C. F.; Zimmermann, R.; Gaub, H. E. Biophys. J . , in press. (6) Rothenhausler, B.; Knoll, W. Nature 1988, 332, 615-617. (7) Verger. R.; Pattus, F. Chem. Phys. Lipids 1976, 16, 285-291. ( 8 ) Schindler, H.Biochim. Biophys. Acta 1979, 555, 3 16-336. (9) Schindler, H.; Quasf,U. Proc. Natl. Acad. Sci. U.S.A. 1980, 77 ( 5 ) , 3052-3056 .
(IO) Schindler, H.; Rosenbusch, J. P. Proc. Natl. Arad. Sci. U.S.A.1978, 75, (8), 3751-3755. ( 1 1 ) Kolomytkin, 0. V . Biochim. Biophys. Acta 1987, 900, 145-156.
0 1990 American Chemical Society
Heyn et al.
5074 The Journal of Physical Chemistry, Vol. 94, No. 12, I990 lently bound to a lipid anchor. For the reconstitution of a bilayer onto a solid support by means of lipid-protein monolayers at the air-water interface, it is essential to ensure that no vesicles or lipid structures are attached. This is also a strict requirement if one wishes to control the thermodynamic state of the monolayer before the transfer onto the solid support is carried out. Therefore, film balance and microfluorescence techniques were used to characterize the vesicle-spread monolayers, After the transfer onto solid supports, the texture and height distribution of the monolayers were determined by ellipsometry and surface plasmon microscopy (SPM). As an example of the potential application of this combination of techniques, we have reconstituted an asymmetric supported bilayer containing oriented Fab-lipid and used it as an immunoassay
,-recorder
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photomultiplier
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Materials and Methods
Materials. Dipalmitoylphosphatidylcholine(L-a-DPPC, 99+%; D-a-DPPC, 99+%), dimyristoylphosphatidylcholine(L-CX-DMPC 99+%), and dimyristoylphosphatidic acid, (L-CX-DMPA, 98%) were bought from Sigma and used without further purification. The fluorescent probe I-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecyl]phosphatidylcholine (NBD-PC) was purchased from Avanti-Polar-Lipids. N-(Texas Red sulfonyl)dipalmitoyl-L-a-phosphatidylethanolamine (TxRed-PE) was bought from Molecular Probes and used as an alternative fluorescence probe. The lipids were either dissolved in pure chloroform (DMPC, DPPC) or in a mixture chloroform/methanol (3:l) (DMPA). Monolayers were spread from organic solutions of I mg of lipid/mL containing either 2 mol 5'% of NBD-lipid or 0.1% of TxRed-lipid. Cholesterol was purchased from Sigma and used without purification as a chloroformic solution. For vesicle preparation and as a monolayer subphase for pure lipids, double-distilled water of Millipore quality (Milli-Q-System, Millipore Corp., El Paso, TX) of pH 5.5 was used, and in the case of proteolipids 0.01 M 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid buffered salt solution (1 50 mM NaCI) of pH 7.4 was used. Experiments with DMPA/DPPC mixtures were performed in 0.1 M glycine buffer, I 0-5 M ethylenediaminetetraacetic acid, pH 11.4. Monoclonal antibodies of subtype IgGl derived from the cell line AN02.I2 Fluorescence-labeled F(ab'), fragments were prepared according to ref 13 and 14. The conjugation between antibody fragment and the lipid moiety, dipalmitoylphosphatidylethanolamine (L-a-DPPE, Sigma), was done with the heterobifunctional cross-linker N-succinimidyl 3-(2-pyridyldithi0)propionate (SPDP, Pharmacia/LKB). Vesicle Preparation. To prepare unilamellar vesicles, the organic lipid solutions containing fluorescent lipid and cholesterol were either used directly or were mixed in order to obtain the desired compositions. Then in a test tube the organic solvent was evaporated with dry N2. Residual traces of organic solvent were then evaporated in a vacuum chamber. After that Millipore water was added resulting in a suspension of a concentration of typically 1.5 mg of lipid/mL. Swelling of the liposomes was allowed by keeping the suspension for at least 1 h at a temperature higher than the phase transition temperature of any of the single lipid components. To obtain unilamellar vesicles, the suspension was finally sonicated for 15 min with a Microtip Sonifier (Branson). The preparation of the proteolipid vesicles was described previ0us1y.~~Briefly, the reactive spacer lipid, DPPE-DTP, which carries a (pyridy1dithio)propionate moiety at the head group. was synthesized, and vesicles of it were reacted with freshly reduced monovalent Fab' fragments. As the spacer lipid does not form stable vesicles, it is mixed with DMPC in a molar ratio of 1 :20. After covalent linkage the resulting Fab' vesicles were purified from unreacted products by size exclusion chromatography and (12) Balakrishnan, K.;Hsu, F. J.; Hafeman, D. G.; McConnell, H. M. Biochim. Biophys. Acla 1982, 721, 30-38. (13) Titus, J. A,; Haughland, R.; Sharrow, S. 0.;Segal, D. M. J. Immunol. Methods 1982, SO, 193-204. (14) Parham, P. J. Immunol. 1983, 131, 2895-2902. (15) Egger, M.:Heyn, S. P.; Gaub, H. E. Submitted for publication in Biochim. Biophys. Acta.
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density gradient centrifugation. Self Assembly of Monolayer and Wet-Bridge Transfer. The procedure of spreading a monolayer was carried out as follows: A monolayer was allowed to form by spontaneous self-assembly at the air-water interface of a vesicle suspension in a spreading well. To separate the monolayers from attached vesicles or other lipid structures, the spreading well communicated with a Langmuir-Blodgett (LB) trough through a thin Millipore water or buffer film, supported by a clean, wet strip of filter paper. Due to the gradient in lateral pressure the monolayer expanded over the wet bridge and was thus transferred onto the subphase of the adjacent trough. After the removal of the wet bridge the thermodynamic state of the monolayer could be controlled, while observation was possible by a fluorescence microscope. Finally, the monolayer could be transferred onto a solid support. Experimental Setup. The experimental setup is shown in Figure 1. Lipid and proteolipid monolayers were spread in the described way. The spreading well was made from Plexiglas that was siliconized and had the dimensions 5 mm X I O mm X 5 mm. Its temperature could be controlled by electrical heating up to 50 OC with an accuracy of 0.3 'C. The wet-bridge paper strip had a width of 10 mm and a length of 20 mm and was suspended by a PTFE-coated silver wire. Ashless filter paper (Schleicher & Schiill, FRG) turned out to be most suitable. A new CHC13washed paper strip was used for each spreading. To prevent drying out, it was kept in a chamber of humid atmosphere. The miniature LB trough consisted of a PTFE frame mounted on a copper plate that was covered with 0.15-mm-thick PTFE foil. At full expansion the surface dimension of the trough was 20 mm X 160 mm. This maximum area dimension resulted in comparatively small amounts of vesicle solution needed to spread a monolayer. Convection was minimized by a trough depth of only 2.5 mm. During spreading the PTFE barrier was kept at a fixed position and the surface pressure was measured by a Wilhelmy system integrated into the barrier. Filter paper of width 8 mm was used as a Wilhelmy plate attached to a displacement transducer (Trans-Tek Inc., USA). In a first class of experiments the surface of the LB trough was withdrawn by suction two or three times prior to removing the wet bridge. Then the spreading pressure was allowed to rise again until the desired surface pressure was reached. This procedure proved to remove remaining surface-active impurities from the LB trough. In a second class of experiments with Fab-lipid vesicles in a pure DMPC monofilm was spread beforehand from an organic solution to a lateral pressure of between 5 and 20 m N / m . At the same time a vesicle solution in the spreading well was allowed to communicate with the buffer solution in a waste well. After 20 min the wet bridge was removed from the waste
Lipid and Lipid-Protein Monolayer Spread well and positioned into the LB trough. This procedure ensured ( I ) that any traces of surface-active impurities from the filter paper were removed and (2) that the protein parts of the Fab-lipid molecules transferred into the LB trough were kept in the aqueous subphase by lateral packing in the monolayer. To drive the PTFE barrier, a linear actuator (Isert, FRG) was used that could be positioned to an accuracy of 5 Km and was controlled by a computer program. The program allowed compression and expansion of the monolayer with a variable barrier speed. The program also recorded pressurearea diagrams as well as the time course of the lateral pressure. For pressurearea diagrams and the slow compressions the barrier speed was chosen in the range of 50-100 pm/s. Heating and cooling of the trough was performed by four Peltier elements (Peltron, FRG). The temperature was measured by a PtlOO thermoresistor and could be varied in the range of 5-40 OC to an accuracy of 0.3 OC. In order to observe microfluorescence at the air-water interface of the LB trough, an epifluorescence microscope was mounted on a motorized x-y stage. To scan the monolayer, the stage position was controlled via a track ball (Teldix, FRG). The microscope consisted of an Olympus 40X long working distance objective which was attached to an epifluorescence condensor (Zeiss, IV FI). The condensor was equipped with two sets of optical filters allowing observation of fluorescence after excitation at either 490 or 580 nm. A 50-W mercury lamp was used as the excitation light source (HBO 50, Osram, FRG). Focusing of the microscope was achieved roughly by regulating the water surface level. The fine adjustment was performed by a Piezo actuator (Owis, P675 15, FRG) which could move the microscope by a maximum length of 150 pm in the (vertical) z direction by applying a voltage of maximal 1 kV. The microscope was interfaced to a SIT camera (Hamamatsu, C2400). Details of the setup are given elsewhere.I6 Supported Bilayer. As supports, cleaned quartz slides were alkylated by dipping them into a solution 0.1% solution of octadecyltrichlorasilane (Petrach Systems, Inc., USA) for about l min. As solvent, a 8: 12:80 mixture of CHCI3/CCl4/hexadecane was used. The slides were rinsed extensively with CHC13 immediately after removal from the silanization bath and then baked for 1 h at 200 OC. The forward contact angle toward water was measured to be between 1 loo and 115'. The monolayer was transferred onto the support by the so-called horizontal approach" resulting in a reconstituted membrane with one leaflet being chemically linked to the support. The lateral mobility of the upper layer is known to be drastically reduced in these structures,'* resulting in a conservation of phase pattern which has previously been formed at the air-water interface. Ellipsometry and Surface Plasmon Microscopy. The height of the pure lipid films was measured by null ellip~ometry.'~The films were transferred from the LB trough onto Si wafers with thermally grown oxide (1 50 nm) using the LB technique. The texture and height distribution of the Fab-lipid films was determined with a SPM.6 Here the films were transferred onto one side of a quartz glass prism which was coated with a 50-nm gold layer. The prism was horizontally dipped through the air-water interface. When kept submerged, the previously induced phase pattern proved to be stable on the gold layer for more than 24 h.
Results and Discussion Lipid Monolayer Formation from the Vesicle Suspension. In Figure 2 the time course of the lateral surface pressure during monolayer formation by self-assembly at the air-water interface is shown for DMPC at different temperatures. For this purpose a spreading well of dimensions 3 cm X 1 cm X 1 cm was used. The lateral pressure was measured directly in the well. For (16) Heyn, S.-P.;Tillmann, R. W.; Gaub, H. E. Submitted for publication in Sci. Instrum. (17) Tscharner, V.V.; McConnell, H. M. Biophys. J . 1981, 36, 421-427. (18) Merkel, R.; Sackmann, E.; Evans, E. J . Phys. Fr. 1989, 50, 1535-1555. (19) Azzam, R. M. A.; Bashra, N. M . Ellipsometry and Polarized Light; North-Holland, Amsterdam, 1977.
The Journal of Physical Chemistry, Vol. 94, No. 12, 1990 5075
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Figure 2. Time course of the lateral pressure rise at the air interface of a vesicle suspension. The lateral pressure rise during the self-assembly of a monolayer on a DMPC vesicle suspension (1.5 mg/mL) was measured for different temperatures. The equilibrium pressure of ?re = 44 m N / m was reached within the time scale of I h only for temperatures above u, = 23 O C .
temperatures above the main phase transition temperature u, = 23 OC, the equilibrium surface pressure of A, = 44 mN/m ( f l mN/m) is reached within about 1 h or less and no significant change is observed for different temperatures. For temperatures smaller than u, the equilibrium pressure is not reached within the same time scale. At vesicle concentrations as high as 1 mg/mL this process does not significantly depend on the concentration. For DPPC similar time-surface-pressure curves were obtained and the lateral equilibrium pressure was the same as above when the temperature was higher than u, = 43 "C. In both cases vesicle suspensions which were not strictly kept above u, after their swelling and subsequent sonification did not show a reproducible rise of the surface pressure. In the case where a lipid monolayer was allowed to form at the air-interface of a vesicle solution in the spreading well which communicated with the LB-trough subphase via the wet bridge, the results were the same when the surface pressure was measured in the LB trough. Furthermore, the pressure-time course did not depend on the LB-trough temperature in the range between 10 and 25 "C. Monolayer Characteristics. Once a measurable lateral pressure had built up in the LB trough, the bridge to the spreading well was removed and the film was slowly compressed. Figure 3 shows typical epifluorescence micrographs that were taken during the compression of a DPPC-film spread from a vesicle solution. The micrographs clearly characterize the gas/fluid coexistence region (Figure 3a), the fluid region phase (Figure 3b), and the fluid/solid coexistence region (Figure 3c) of a DPPC monolayer and show no difference with the ones of an equivalent monolayer spread from an organic solution (see refs 2, 20, and 21). It is also important to note that the fluorescence intensities measured from both a vesicle-spread and an organic-solution-spread film are under equivalent conditions the same within the experimental error of
