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New Functional Proteo-glycolipidic Molecular Assembly for Biocatalysis Analysis of an Immobilized Enzyme in a Biomimetic Nanostructure Ste´phanie Godoy,† Jean-Paul Chauvet,‡,| Paul Boullanger,§ Loı¨c J. Blum,† and Agne`s P. Girard-Egrot*,† Laboratoire de Ge´ nie Enzymatique et Biomole´ culaire, UMR 5013-CNRS/UCBL, Universite´ Claude Bernard Lyon 1, 43 Bvd du 11 novembre 1918, F-69622 Villeurbanne Cedex, France, Inge´ nierie et Fonctionnalisation des Surfaces, UMR 5621-CNRS/ECL, Ecole Centrale de Lyon, 36 Avenue Guy de Collongue, F-69134 Ecully Cedex, France, and Laboratoire de Chimie Organique 2, UMR 5622-CNRS/ UCBL, Universite´ Claude Bernard Lyon 1, Ecole Supe´ rieure de Chimie Physique Electronique de Lyon, 43 Bvd du 11 novembre 1918, F-69622 Villeurbanne Cedex, France Received March 25, 2003 A new organized biomimetic nanostructure embedding a monoclonal antibody in a lipidic matrix has been designed to sequester a hydrophilic enzyme in an oriented position and allowed to preserve the enzyme activity over a few months. The nanostructure was constituted of a glycolipid and a noninhibitory monoclonal IgG directed against the soluble form of acetylcholinesterase (AChE). A mixed monolayer (IgG-glycolipid) was obtained by spreading mixed IgG-glycolipid vesicles at the air/buffer interface. Several measurements (π-A isotherms, surface potential measurements, and compression-decompression cycles) allowed us to demonstrate the presence of IgG in the monolayer, as well as a reorientation of IgG molecules during the compression. After transfer on solid supports by the Langmuir-Blodgett technique, the presence of IgG in the mixed monolayer was characterized by ATR FTIR spectroscopy. Linking of the AChE on the IgG-glycolipid matrix was realized by immunoaffinity, and the enzyme was shown to retain its activity. The opportunity to detect a strong enzymatic activity, even after transfer at high surface pressures, suggested a preferential orientation of the antibody, favorable to retain the enzyme active at the surface of the nanostructure. The homogeneity of the transferred monolayer before and after immunoassociation, observed by Nomarski microscopy, did not display any structural modification. The enzyme kinetics was typical of the biocatalytic behavior of an immobilized enzyme, with a decrease of reaction rates due to the lower accessibility to the substrate at higher enzyme content. With the advantages of stability and favorable orientation of IgG, this new active matrix induces, in turn, a favorable orientation of the enzyme bound by immunoaffinity. The typical enzymatic behavior of the ternary nanostructure (glycolipid-IgG-AChE) demonstrates the usefulness of such a functional molecular assembly for biocatalysis study in a biomimetic situation.
Introduction The orientated insertion of proteins in lipid layers is of outstanding interest for fundamental investigations of lipid/protein interactions as well as for applications in analytical biosensors. Therefore, the building of organized proteolipidic nanostructures possessing properly orientated recognition sites could constitute a promising biomimetic model. Associated with a sensitive transducer, it could allow investigating biocatalytic behavior in a biomimetic environment. The Langmuir-Blodgett (LB) technique is one of the most powerful methods to prepare multilayered films, and many papers report on association of proteins to lipidic LB films.1-14 However, such associations most often lack defined orientation of the protein, which results in a decrease of activity. Furthermore, the absence of strong * Corresponding author. E-mail:
[email protected]. † Universite ´ Claude Bernard Lyon 1. ‡ Ecole Centrale de Lyon. § Ecole Supe ´ rieure de Chimie Physique Electronique de Lyon. | Deceased (January 12, 2003). (1) Moriizumi, T. Thin Solid Films 1988, 160, 413. (2) Anzaı¨, J.-I.; Furuya, K.; Chen, C.-W.; Osa, T.; Matsuo, T. Anal. Sci. 1987, 3, 271. Anzaı¨, J.-I.; Hashimoto, J.-Y.; Osa, T.; Matsuo, T. Anal. Sci. 1988, 4, 247. Anzaı¨, J.-I.; Lee, S.; Osa, T. Makromol. Chem., Rapid Commun. 1989, 10, 167. Anzaı¨, J.-I.; Osa, T. Sel. Electrode Rev. 1990, 12, 3.
interactions between the lipid matrix and the protein results in a rapid desorption of the latter. Several approaches have been recently proposed to overcome these problems, such as the immobilization of proteins onto lipid monolayers able to chelate metallic ions15 or the covalent coupling of antibody fragments to a linker lipid embedded in phospholipid monolayers.16-18 In this paper, we propose another alternative: the insertion in LB films of a monoclonal antibody specific to the enzyme and able to bind the latter at the surface of the lipid matrix, in an orientated position. (3) Tsuzuki, H.; Watanabe, T.; Okawa, Y.; Yoshida, S.; Yano, S.; Koumoto, K.; Komiyama, M.; Nihei, Y. Chem. Lett. 1988, 1265. Tatsuma, T.; Tsuzuki, H.; Okawa, Y.; Yoshida, S.; Watanabe, T. Thin Solid Films 1991, 202, 145. (4) Okahata, Y.; Tsuruta, T.; Ijiro, K.; Ariga, K. Langmuir 1988, 4, 1373-1375; Thin Solid Films 1989, 180, 65. (5) Li, J. R.; Cai, M.; Chen, T. F.; Jiang, L. Thin Solid Films 1989, 180, 205. (6) Miyauchi, S.; Arisawa, S.; Arise, T.; Yamamoto, R. Thin Solid Films 1989, 180, 293. Arisawa, S.; Arise, T.; Yamamoto, R. Thin Solid Films 1992, 209, 259. Arisawa, S.; Yamamoto, R. Thin Solid Films 1992, 210/211, 443. (7) Zaitsev, S. Yu.; Kalabina, N. A.; Zubov, V. P. J. Anal. Chem. USSR 1991, 45, 1054. Zaitsev, S. Yu.; Hanke, Th.; Wollenberger, U.; Ebert, E.; Kalabina, N. A.; Zubov, V. P.; Scheller, F. Bioorg. Khim. 1991, 17, 767. Hanke, Th.; Wollenberger, U.; Ebert, B.; Scheller, F.; Zaitsev, S. Yu. In Biosensors, Fundamentals, Technologies and Applications; Scheler, F., Schmid, R. D., Eds.; GBF monographs; VCH Publishers: New York, 1992; Vol. 17, p 43.
10.1021/la034517a CCC: $25.00 © 2003 American Chemical Society Published on Web 05/10/2003
Proteo-glycolipidic Molecular Assembly
Spreading vesicles at an air/water interface has been proposed as an alternative way to form interfacial films.19-23 In our group, we have recently used such a procedure to obtain mixed enzyme-lipid interfacial films.24,25 With the immunological proteins of an ascitic fluid, the method appeared very promising to bring and to retain soluble antibodies at the air/buffer interface.26 This paper is devoted to the preparation of a new proteolipidic assembly based on vesicle spreading and the LB technique. The protein is a noninhibitory monoclonal immunoglobulin, directed against the nonamphiphilic monomer of acetylcholinesterase (AChE, EC 3.1.1.7) extracted from Bungarus fasciatus venom, and the lipid is a neutral synthetic glycolipid able to form vesicles at room temperature.27 In previous studies, we have shown that these vesicles were able to embed a purified immunoglobulin (IgG) and that they could disrupt at an air/ buffer interface to form a true mixed IgG-glycolipid interfacial film.28 In this paper, we report on (i) the stability of the proteolipidic interactions in the interfacial film through compression-expansion monolayer cycles, (ii) the immunoassociation of AChE onto mixed IgG-glycolipid layers after transfer onto solid support, and (iii) the kinetic behavior of the immobilized enzyme in a biomimetic environment (AChE-IgG-glycolipid) close to the structural lipid membrane. Materials and Methods Materials. The glycolipid (10-undecyloxymethyl-3,6,9,12tetraoxatricosyl 2-acetamido-2-deoxy-β-D-glucopyranoside) was synthesized as previously reported.29
Phosphate buffer subphases and other buffer solutions were prepared with ultrapure water (resistivity ) 18.2 MΩ‚cm) (8) Fiol, C.; Valleton, J.-M.; Delpire, N.; Barbey, G.; Barraud, A.; Ruaudel-Texier, A. Thin Solid Films 1992, 210/211, 489. Fiol, C.; Alexandre, N.; Delpire, N.; Valleton, J. M. Thin Solid Films 1992, 215, 88. (9) Pal, P.; Nandi, D.; Misra, T. N. Thin Solid Films 1994, 239, 138. (10) Wan, K.; Chovelon, J. M.; Jaffrezic-Renault, N. Talanta 2000, 52, 663. Chovelon, J. M.; Wan, K.; Jaffrezic-Renault, N. Langmuir 2000, 16, 6223. Chovelon, J. M.; Gaillard, F.; Wan, K.; Jaffrezic-Renault, N. Langmuir 2000, 16, 6228. (11) Yasuzawa, M.; Hashimoto, M.; Fujii, S.; Kunugi, A.; Nakaya, T. Sens. Actuators, B 2000, 65, 241. (12) Ramsden, J. J.; Bachmanova, G. I.; Archakov, A. I. Biosens. Bioelectron. 1996, 5, 523. (13) Ancelin, H.; Zhu, D. G.; Petty, M. C.; Yarwood, J. Langmuir 1990, 6, 1068. Zhu, D. G.; Petty, M. C. Thin Solid Films 1989, 176, 151. (14) Fujiwara, I.; Ohnishi, M.; Seto, J. Langmuir 1992, 8, 2219. (15) Kent, M. S.; Yim, H.; Sasaki, D. Y. Langmuir 2002, 18, 3754. (16) Ihalainen, P.; Peltonen, J. Langmuir 2002, 18, 4953. (17) Vikholm, I.; Viitala, T.; Albers, W. M.; Peltonen, J. Biochim. Biophys. Acta 1999, 1421, 39. (18) Vikholm, I.; Albers, W. M. Langmuir 1998, 14, 3865. (19) Verger, R.; Pattus, F. Chem. Phys. Lipids 1976, 16, 285-291. Pattus, F.; Desnuelle, P.; Verger, R. Biochim. Biophys. Acta 1978, 507, 62. (20) Obladen, M.; Popp, D.; Scho¨ll, C.; Schwarz, H.; Ja¨hnig, F. Biochim. Biophys. Acta 1983, 735, 215. (21) Ivanova, Tz.; Georgiev, G.; Panaı¨otov, I.; Ivanova, M.; LaunoisSurpas, M. A.; Proust, J. E.; Puisieux, F. Prog. Colloid Polym. Sci. 1989, 79, 24. (22) Launois-Surpas, M. A.; Ivanova, Tz.; Panaı¨otov, I.; Proust, J. E.; Puisieux, F.; Georgiev, G. Colloid Polym. Sci. 1992, 270, 901. (23) Vassilieff, C. S.; Panaı¨otov, I.; Manev, E. D.; Proust, J. E.; Ivanova, Tz. Biophys. Chem. 1996, 58, 97. (24) Marron-Brignone, L.; More´lis, R. M.; Chauvet, J.-P.; Coulet, P. R. Langmuir 2000, 16, 498. (25) Pizzolato, F.; More´lis, R. M.; Coulet, P. R. Quı´m. Anal. 2000, 19, 32.
Langmuir, Vol. 19, No. 13, 2003 5449 obtained from a milli-Q four-cartridge purification system (Millipore, France). Germanium internal reflection parallelogram plates (25 × 10 × 3 mm3) with a 45° face angle purchased from Thermo Optek (France) or rectangular calcium fluoride plates (35 × 9.5 × 2 mm3) purchased from Sorem (France) were used as transfer substrates after a cleaning procedure already described.26 The immunoglobulin (monoclonal antibody directed against AChE of B. fasciatus venom), generously supplied by Dr. Grassi (SPI, CEA Saclay, Gif-sur-Yvette, France), was purified before use by Protein A HyperD F chromatography (BioSepra, France), as reported elsewhere.28 The nonamphiphilic watersoluble monomer of B. fasciatus’s AChE was a generous gift of Dr. Bon (Institut Pasteur de Paris, Unite´s des venins, France). S-Acetylthiocholine iodide (ATChI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent), and IgG free bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chimie (St Quentin Fallavier, France). Glycolipid and Immunoglobulin-Glycolipid Vesicle Formation. Mechanical dispersion was used to prepare glycolipid and proteoglycolipidic vesicles at room temperature according to the procedure previously described.28 Thus, glycolipid vesicles and IgG-glycolipid vesicles were respectively prepared by dispersion of a dry glycolipid film (2.5 mg) either in 250 µL of 10 mM phosphate buffer at pH 7.4 or in 250 µL of the antibody solution (3.5-4 mg/mL). The final glycolipid concentration and protein-lipid molar ratio were 10 mg/mL and 1/550, respectively. The proteoglycolipidic vesicles displayed the same multilamellar structure as that previously reported.28 The vesicle suspensions were used directly for the spreading procedure and could be stored at 4 °C over a 1 week period, without apparent modification of their spreading kinetics. Surface Pressure Measurements. The interfacial films were prepared with a computerized KSV 3000 Langmuir-Blodgett trough (KSV Instrument Ltd., Finland) working in a symmetrical compression mode. The surface pressure (π) was measured with a platinum Wilhelmy plate with an accuracy of (0.05 mN/m. The interfacial films were formed onto a 5-fold concentrated 10 mM phosphate buffered saline (PBS) solution, pH 7.4 (used as the subphase), and thermostated at 20 °C ( 0.5 °C. A 5 or 10 µL sample of vesicle suspension was spread over the subphase. Zero on the time scale corresponded to the beginning of the spreading procedure. The surface pressure variation consecutive to the vesicle spreading was recorded for 35 min. Then, the interfacial film was continuously compressed at a rate of 15 cm2/min and the surface pressure (π)-area (A) isotherm was recorded. The partial irreversible diffusion of the spread vesicles into the subphase did not allow the knowledge of the surface concentration (Γ); consequently, the abscissa in the π-A diagram was taken as the total surface film area (A, in cm2). The two-dimensional compressibility of the monolayer (Cs) was directly calculated from the slope of the π-A isotherm by KSV software, according to the following equation:30
Cs ) -1/A(∂A/∂π)T where A corresponds to the surface film area and π to the surface pressure. It is expressed in m/mN and can be related to the compressional elasticity (reciprocal of compressibility) of the interfacial film.30 To record π-A hysteresis diagrams, the interfacial monolayer was successively compressed and expanded three times at a rate of 15 cm2/min. The cycling time was 3740 s. A 1 min waiting period was imposed at the end of the compression step just before the following expansion. A 1 h lag time was respected between each compression-decompression cycle. Surface Potential Measurements. Surface potential measurements were performed with a Teflon (PTFE) homemade (26) Girard-Egrot, A. P.; More´lis, R. M.; Boullanger, P.; Coulet, P. R. Colloids Surf., B: Biointerfaces 2000, 18, 125. (27) Sancho, M.-R. Ph.D. Thesis, University of Lyon, Lyon, France, 1994. (28) Girard-Egrot, A. P.; Chauvet, J.-C.; Boullanger, P.; Coulet, P. R. Langmuir 2001, 17, 1200. (29) Boullanger, P.; Sancho-Camborieux, M. R.; Bouchu, M. N.; Marron-Brignone, L.; More´lis, R. M.; Coulet, P. R. Chem. Phys. Lipids 1997, 90, 63. (30) Davies, J. T.; Rideal, E. K. Interfacial phenomena, 2nd ed.; Academic Press: New York and London, 1963; Chapter 5.
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Figure 1. Schematic representation of the functional proteo-glycolipidic molecular assembly. Langmuir trough equipped with a temperature regulation. The asymmetric compression of the monolayer was achieved through a single PTFE mobile barrier. The surface pressure (π) was measured by the Wilhelmy plate balance method, using a Whatman paper (nο. 3). Surface potential (∆V) was recorded with an electrode (241Am-210Po) placed 5 mm above the liquid surface and connected to a voltmeter (STI 200, ScienTech International, Trois Rivie`res, Que´bec), against a platinum reference electrode dipped into the subphase, behind the compression barrier. The interfacial films were formed onto a 10 mM PBS subphase, pH 7.4, in the same conditions as described above. After a 35 min lag time, the monolayer was continuously compressed at a rate of 8.4 cm2/min. The surface potential diagram was then recorded as a function of the reciprocal total area (1/A in cm-2), as a reflection of the surface concentration Γ. Langmuir-Blodgett Film Deposition. The transfer of the interfacial film was performed using a vertical LangmuirBlodgett film deposition procedure, either on germanium or calcium fluoride. For ATR-FTIR measurements, one monolayer was deposited on a germanium plate. To avoid any protein adsorption on the latter,31 it was rapidly immersed into the aqueous subphase after the 35 min lag time following the spreading procedure. The monolayer was then compressed at a rate of 15 cm2/min, until reaching the transfer surface pressure. Then, one layer was deposited at the upstroke with a rate of 5 mm/min. The transfer ratio was calculated from the film surface removed. For the detection of antibody immunoaffinity and for Nomarski microscopy, two monolayers were transferred onto calcium fluoride substrates precoated with four behenic acid layers. This precoating, realized from a monolayer of behenic acid spread onto a 10-2 M NaCl, 10-4 M MnCl2 subphase,32 was shown to be essential to the transfer of a glycolipid bilayer with the hydrophilic headgroups pointing to the surface.33 The precoated plate was handled as previously, and two layers were deposited, one at the upstroke and the second one at the downstroke with a rate of 5 mm/min. To avoid respreading of the second transferred layer during the interfacial crossing, the plate had to be withdrawn from the aqueous subphase through the compressed monolayer, as recommended elsewhere.34 Under these conditions, no additional transfer was observed. Nomarski Microscopy. The homogeneity of LB films transferred on the CaF2 substrates was checked by Nomarski differential interference contrast microscopy35 with a lateral resolution of 1 µm and a thickness difference of ∼ (0.3 nm at a magnification of 500.36 Infrared Spectroscopy. Infrared spectra were recorded with a Fourier transform infrared (FTIR) spectrometer (Model 510M, Nicolet instruments, France) equipped with a DTGS room temperature detector. The instrument was continuously purged with dry air from a Balston air purifier. Germanium ATR plates, yielding eight internal reflections, were used as internal reflection elements. The germanium ATR plates, cleaned as described
above, were considered to be convenient for IR spectroscopic analysis if the ν(CH2) bands at ∼2920 and ∼2850 cm-1 completely disappeared in the ATR-FTIR spectrum (single beam mode). The crystal on which the carefully dried layers were transferred was placed in a variable angle vertical ATR accessory (Model 300, Spectra-Tech Inc., France). Each spectrum of 150 scans was collected in the single beam mode with a resolution of 4 cm-1. After cleaning of the germanium plate, backgrounds were recorded in the same way. All spectra reported here result from the subtraction of the background data from the experimental data measured on the same germanium plate. The integration of peak areas proposed by the Nicolet’s software allowed a quantitative comparison between the spectra. Immobilization of AChE onto the Transferred Monolayer. To avoid any back-transfer of the IgG-glycolipid film at crossing a liquid interface, an adapted procedure was developed to handle the plate in the horizontal position. The plate was laid on a Teflon ring positioned at the bottom of a 10 mL glass cell. The cell was then carefully filled with AChE solution (0.1 M phosphate buffer, pH 7.4, 0.15 M NaCl, 1 mg/mL BSA) until the plate was completely immersed and the immunoassociation was left completing for 18 h, at 4 °C, under gentle magnetic stirring. The plates were then washed twice, for 30 min, with the same buffer. Biocatalytic Behavior of the Immobilized Enzyme. The activity of immobilized AChE was detected by the colorimetric Ellman’s method37 with 0.75 mM ATChI as the enzyme substrate, in the presence of 0.25 mM DTNB in a 0.01 M phosphate buffer, pH 7.4. The cell containing the plate previously coated with AChE was carefully filled with a defined volume of the enzyme substrate mixture until complete immersion of the plate. The hydrolysis of the enzyme substrate was monitored at 25 °C during 2 min, by the production of a yellow compound (5-thio-2-nitrobenzoate) detected at 412 nm (Figure 1). The spontaneous hydrolysis of the substrate was also determined in the absence of the enzyme and was routinely subtracted. The AChE activity retained on the solid plate was expressed in Ellman units/cm2. The nonspecific adsorption of AChE onto glycolipid monolayers was also determined in the same way from layers of glycolipids transferred without antibody. (31) Subirade, M.; Salesse, C.; Marion, D.; Pe´zolet, M. Biophys. J. 1995, 69, 974. (32) Le´onard, M.; More´lis, R. M.; Coulet, P. R. Thin Solid Films 1995, 260, 227. (33) Marron-Brignone, L.; More´lis, R. M.; Coulet, P. R. J. Colloid Interface Sci. 1997, 191, 349. (34) Marron-Brignone, L. Ph.D. Thesis, University of Lyon, Lyon, France, 1997. (35) Morelis, R. M.; Girard-Egrot, A. P.; Coulet, P. R. Langmuir 1993, 9, 3101. (36) Vandevyver, M.; Barraud, A. J. Mol. Electron. 1988, 4, 207. (37) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88.
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Figure 3. π-A isotherms of the interfacial film formed 35 min after spreading 5 µL of glycolipid vesicles (dashed line, a) or IgG-glycolipid liposomes (full line, b). Inset: Two-dimensional Cs against surface pressure of the corresponding isotherms (a and b). Dotted lines correspond to the surface pressures used for the monolayer transfers.
Figure 2. Time-dependent variations of the surface potential and surface pressure after spreading 5µL (A) or 10 µL (B) of glycolipid (a) or IgG-glycolipid (b) vesicles (10 mg/mL) onto phosphate saline buffer, pH 7.4. Zero on the time scale corresponds to the beginning of the spreading procedure. Assays have been performed for several acetylthiocholine concentrations for both the soluble and the immobilized forms of the enzyme. For the kinetic studies on the soluble form of AChE, the reaction was directly realized in a thermostated cell by injection of 5.33 ng of enzyme in the substrate reaction mixture.
Results and Discussion IgG-Glycolipid Interfacial Film Formation. The formation of surface films from vesicle spreading at the air/water interface was followed by time-dependent surface pressure (∆π) and surface potential (∆V) variations (Figure 2). For a 5 µL spread volume (Figure 2A), increases in both surface potential and surface pressure were simultaneously observed just after spreading, as well for glycolipid (curves a) as for IgG-glycolipid (curves b) vesicles. After the first variation, the surface pressure rapidly decreases (arrow) to reach an equilibrium value while the surface potential stabilizes for a few minutes before reaching a second equilibrium plateau, after a substantial jump. For 10 µL spread volume (Figure 2B), surface and potential variations were very similar, except for IgG-glycolipid vesicles, reaching the surface potential equilibrium plateau immediately after spreading. The time-dependent surface pressure variations were previously demonstrated to be the result of an opening process of vesicules20-22,38,39 and formation of a true monolayer at the interface,41 as well for glycolipid as for IgG-glycolipid vesicles.24,26,28 New information is displayed by the surface potential measurements. The first ∆V increase could be attributed to the interfacial film formation, as already reported for
other spread liposomes.21,22,40 The second surface potential change, occurring a few minutes after spreading, without any surface pressure modification, could be attributed to a reorientation of the molecular dipoles in the glycolipidic interfacial film. This phenomenon, never reported to our knowledge, appeared to be a peculiarity of these spread glycolipid vesicles. In the presence of immunoglobulin, a similar behavior was observed for a 5 µL spread volume, but the opening process was both accelerated and emphasized (Figure 2A). For a 10 µL spread volume, a onestep opening process, without further reorientation, was observed (Figure 2B). This observation could be due to a destructuration of the vesicle membrane by the immunoglobulin, as already observed in TEM.26,28 Under such destabilizing conditions, the proteoglycolipid vesicles are less stable and, consequently, more efficiently opened at the air/liquid interface, mostly at higher surface coverage. After a 35 min lag time, the monolayers were symmetrically compressed. The resulting isotherms (Figure 3) display a shift toward larger areas for the IgG-glycolipid monolayer, thus confirming the presence of the immunoglobulin in the glycolipid film. This expanding effect was markedly evidenced by the increase (25-40 m/mN) of the two-dimensional monolayer compressibility (inset Figure 3), indicating an increase of the monolayer fluidity. An experimental evidence of molecular reorientation was brought by the surface potential measurements recorded during the interfacial film compression of the mixed and the pure glycolipid monolayers. For molecules endowed with an intrinsic dipole moment, the surface potential increases linearly during the compression when the dipoles keep their own orientations. Actually, the surface potential values are directly proportional to the perpendicular component (µ⊥) of the interfacial molecule dipole moment.42 The polar moment (µ) of the headgroup of the glycolipid has been calculated theoretically; it is (38) Panaı¨otov, I.; Ivanova, Tz.; Balashev, K.; Proust, J. Colloid Surf., A: Physicochem. Eng. Aspects 1995, 102, 159. (39) Vikholm, I.; Peltonen, J.; Teleman, O. Biochim. Biophys. Acta 1995, 1233, 111. (40) Ivanova, Tz.; Raneva, V.; Panaı¨otov, I.; Verger, R. Colloid Polym. Sci. 1993, 271, 290. (41) Salesse, C.; Ducharme, D.; Leblanc, R. M. Biophys. J. 1987, 52, 351. (42) Gaines, G. L., Jr. In Insoluble Monolayers at Liquid-Gas Interfaces; Interscience publishers; John Wiley & Sons: New York, 1966; Chapter 4.
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Figure 4. Mean orientation of the intrinsic dipole moment of the glycolipid molecule. (Calculus performed by PC Spartan Pro (AM1 Semiempirical calculation) version 1.0.3 (Wave function Inc.).)
Figure 5. ∆V-(1/A) isotherm diagrams of pure glycolipid (a) or mixed IgG-glycolipid (b) monolayers formed by vesicle spreading. (Compression performed 35 min after the spreading procedure.) The values indicated in brackets correspond to the surface pressure at which the slope rupture occurred.
orientated almost perpendicular to the triethylene glycol segment (Figure 4). Figure 5 shows the surface potential measurements performed during the compression of the glycolipid interfacial film, in the presence or in the absence of IgG, as a function of the surface film area reciprocal (1/A). The surface potential does not increase linearly with 1/A, which implies a reorientation of the dipole moment during the compression. For the pure glycolipid monolayer (Figure 5, curve a), a high value of the slope is observed at low surface pressure due to a high contribution of µ to the µ⊥ component, indicating that the polar headgroup remains almost parallel to the interface. For further compression, a decrease in slope is indicative of a reorientation of the polar headgroup to a more perpendicular position to the interface. For the mixed monolayer (Figure 5, curve b), the same behavior may lead to the same conclusions. Since IgG molecules present a significant contribution to the surface potential (potential values for the mixed monolayer are higher than those recorded for a pure glycolipid film), it can be postulated that the reorientation of the polar group of the glycolipid induces a reorientation of the IgG molecules in a more vertical position at the interface. Stability of the IgG/Glycolipid Interactions in the Mixed Interfacial Film. The stability of the IgG/lipid interactions in the interfacial film has been investigated
Figure 6. Successive dynamic compression-expansion isotherms of glycolipid (A) and IgG-glycolipid (B) monolayers formed by vesicle spreading. Inset: Two-dimensional Cs against surface pressure of the corresponding isotherms. Full line: Compression sweep. Dashed line: Expansion sweep. (Spread volume ) 5 µL.) Additional cycles (6 runs) perfectly overlapped the third curve. For clarity, only the first, second, and third isotherms were presented.
through dynamic cyclic π-A isotherms. Three compression-expansion cycles were recorded for glycolipid (A) and IgG-glycolipid (B) interfacial films (Figure 6). The upper value of the surface pressure (40 mN/m) was maintained lower than that of collapse (πc ) 45-46 mN/ m) but high enough to overlap the full range of compressibilities. The hysteresis loops observed in π-A diagrams of interfacial lipidic monolayers in compression-expansion
Proteo-glycolipidic Molecular Assembly
cycles have been previously ascribed to several independent events:43,44 (i) frictional drag between the monolayer and the liquid subphase (Marangoni’s effect),45 (ii) exchanges of molecules between the surface and the bulk phase, (iii) relaxation processes of the molecules in the monolayer, and (iv) monolayer collapse and respreading of collapsed phases. All these processes lead to hysteresis if their lifetimes are in the time scale of the cycling period. For the pure glycolipid, the cyclic π-A diagrams exhibited a small hysteresis (Figure 6A). The second isotherm was slightly shifted to higher areas, whereas the third one perfectly overlapped it. The shift between the first and the second cycle could be ascribed to vesicle transformation at the interface during the 1 h period between two runs, but no further transformation occurred in the third cycle. Taking into account the large cycling time (3740 s) and the maximal surface pressure (π < πc), the hysteresis phenomenon could be attributed neither to Marangoni effect (lifetime of 1-3 s44) nor to a collapsed phase respreading. Furthermore, the Cs curves (inset Figure 6A) perfectly overlapped during compression and expansion of the interfacial film. So, the small hysteresis observed could be mostly attributed to a reversible process, which might correspond to the monolayer relaxation. In the presence of the immunoglobulin, a significant hysteresis was observed on the π-A isotherm (Figure 6B). While the three expansion curves perfectly superimposed, the second compression isotherm was shifted toward smaller areas than the first one; the third one almost overlapped the second one. Contrariwise to what happened with the pure glycolipid monolayer, no interfacial vesicle transformation occurred during the 1 h period left between the first two cycles. This observation is in good agreement with the accelerated opening process of the IgG-glycolipid vesicles compared with the glycolipid ones. The shift observed in the second compression isotherm, without displacement of the expansion one, could indicate that some IgG molecules were expelled from the interface during the first compression step only. The overlap of the following dynamic compression-expansion curves (six further compression-expansion cycles did not display any other change) suggests that the protein ejection during the first compression was an irreversible phenomenon. In our opinion, these results clearly demonstrate that the IgG/lipid interactions are sufficiently stable to keep the IgG inserted in the interfacial film after several compression-expansion cycles. The analysis of the Cs modifications during the monolayer compression (inset Figure 6B) confirms this hypothesis. A reproducible behavior was observed from the second cycle, expressing a permanent effect of the presence of the IgG on the viscoelastic properties of the mixed monolayer. For the mixed IgGglycolipid film, the Cs curves showed a reversible compressibility modification with a maximum value around 18 mN/m and a constant magnitude for all the consecutive expansion steps (inset Figure 6B). In this case, the large hysteresis could be attributed to reversible relaxation processes of the mixed IgG-glycolipid monolayer. It is noteworthy that the experimental cycling time was sufficiently large to be compatible with the kinetics of such processes.43 Two parameters could be responsible for the strong IgG/ lipid interactions in the monolayer: (i) a favorable carbohydrate/carbohydrate interaction between the glycolipid (43) Taneva, S. G.; Keough, K. M. W. Biochemistry 1994, 33, 14660. (44) Ivanova, Tz.; Panaı¨otov, I.; Georgiev, G.; Launois-Surpas, M. A.; Proust, J. E.; Puisieux, F. Colloid Surf. 1991, 60, 263. (45) Dimitrov, D. S.; Panaı¨otov, I.; Richmond, P.; Ter-MinassianSaraga, L. J. Colloid Interface Sci. 1978, 65, 483.
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Figure 7. ATR-FTIR spectra of the glycolipid (dashed line) or the mixed IgG-glycolipid (full line) LB film monolayer transferred onto a germanium plate at 30 mN/m: A, 1800-1400 cm-1 region; B, 3000-2700 cm-1 region.
headgroup and the glycans of the IgG molecule, and/or (ii) hydrophobic interactions between the glycolipid and the hydrophobic Fc fragment of the immunoglobulin. In this hypothesis, the irreversible ejection of part of the immunoglobulin in the first compression step could be related to IgG molecules not properly orientated or embedded in the glycolipid monolayer. Presence of the Immunoglobulin and Immunoaffinity Associated with the Transferred Monolayer. After transfer on solid support at different surface pressures (dotted lines, Figure 3), the IgG-glycolipid organization in the monolayers was further investigated by ATR-FTIR spectroscopy and immunoaffinity studies. Two spectral regions have been more precisely analyzed in ATR-FTIR spectroscopy: (i) 3000-2800 cm-1 (stretching vibrations of the hydrocarbon chains) and (ii) 17501450 cm-1 (amide I and amide II stretching frequencies). The presence of amide I and amide II bands in the glycolipid film spectra (Figure 7A, dashed line) can be ascribed to the N-acetylated glucosamine headgroup, whereas their increase in the mixed IgG-glycolipid monolayer spectra (Figure 7A, full line) expresses the
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Table 1. Infrared Integration Values of the Amide I and ν(CH2)/ν(CH3) Bands after Transfer onto a Germanium Plate integration value (au) amide I
ν(CH2)/ν(CH3)
transfer pressure (mN/m)
with IgG (a)
without IgG (b)
ratio (RAI) (a/b)
relative evolution (REE)a
with IgG (a)
without IgG (b)
ratio (a/b)
18.5 25 30 36
0.158 0.311 0.288 0.047
0.043 0.197 0.137 0.037
3.7 1.6 2.1 1.3
+1.31 0 +0.31 -0.18
0.087 0.1 0.1 0.2
0.081 0.1 0.1 0.2
1.07 1 1 1
a The relative evolution of embedment (R EE ) (RAI1 - RAI0)/RAI0) is calculated using RAI0 ) 1.6 as the reference value, obtained at 25 mN/m (minimal condensed state favorable for a good transfer).
Table 2. Enzyme Activity Detected after Immunoassociation of AChE onto a Mixed IgG-Glycolipid Monolayer, Transferred at Several Surface Pressures enzyme activity (EU/cm2) transfer pressure (mN/m)
without IgG
with IgG (a)
relative evolution (REA)a
18.5 25 30 36
0 0 0 0
0.606 0.572 0.822 0.584
+0.06 0 +0.44 +0.02
a The relative evolution of activity (R EA ) (a1 - a0)/a0) is calculated using the enzyme activity value of 0.572 (a0) as a reference (value obtained at 25 mN/m).
presence of the protein in the transferred monolayer. The location of the stretching vibrations of the hydrocarbon chains in the glycolipid monolayer as well as in the IgGglycolipid one (Figure 7B) displays a gauche conformation for the glycolipid hydrocarbon chains,46 reflecting the fluidity of the glycolipid matrix at the experimental temperature. Similar results have been obtained at the other surface pressures investigated (data not shown). The integration of the peak areas allowed a quantitative analysis of the composition of the transferred monolayers. The results obtained at four different transfer pressures are given in Table 1. The integrated areas of the CH stretching vibrations are proportional to the number of deposited layers and constitute a reliable control of the transferred film thickness,47,48 a value of 0.1-0.2 au reflecting the thickness of one transferred monolayer.49 As can be seen from the results of Table 1, it can be considered that monolayers (with or without IgG) were properly transferred at surface pressures higher than 18.5 mN/m (columns 5 and 6) and that the same amount of surface film has been transferred in both cases (column 7). Therefore, the increase of amide I integration values, between the IgG-glycolipid and the glycolipid monolayers (RAI, column 3), can be unambiguously interpreted as the presence of immunoglobulin in the monolayers, even at high surface pressures. The immunoaffinity of IgG embedded in the transferred films was characterized at the four transfer surface pressures. After enzyme immunoassociation, the AChE activity associated with the layer was quantified by the colorimetric Ellman’s method. As can be seen in Table 2, no enzyme activity was detected with the pure glycolipid films (column 1), contrariwise with the results for mixed IgG-glycolipid films (columns 2 and 3). (46) Mantch, H. H.; MacElhaney, R. N. Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy. Chem. Phys. Lipids 1991, 57, 213. (47) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Langmuir 1996, 12, 778. (48) Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986, 2, 96. (49) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Thin Solid Films 1997, 292, 282.
The enzyme activity can be related to the amount of transferred IgG, at the different transfer pressures, by comparison of the results of Table 1 (columns 3 and 4) and Table 2 (columns 2 and 3). When the results at 25 mN/m are taken as references, it can be seen that a decrease of transfer pressure to 18.5 mN/m results in an increase of transferred IgG (REE ) +1.31), without increasing the enzyme activity (REA ) +0.06). This could be interpreted as an unfavorable orientation of IgG for immunoaffinity, in lowly condensed films. Contrariwise, an increase of the transfer pressure to 30 mN/m results in an increase of transferred IgG (REE ) +0.31) concomitant with an increase of enzymatic activity (REA ) +0.44), which could indicate a favorable orientation of the IgG for immunoaffinity, in highly condensed films. A further increase of the transfer pressure to 36 mN/m results in a small decrease of IgG embedment (REE ) -0.18, due to ejection occurring during the first compression) with conservation of the enzymatic activity (REA ) +0.02), showing that a higher condensed state probably supports a very favorable orientation of embedded IgG for immunoaffinity. These results are in good agreement with the hysteresis analysis and strongly suggest a reorientation of the immunoglobulin during the compression of IgG-glycolipid films. A surface pressure of 30mN/m was demonstrated to ensure an optimal IgG embedment in the glycolipid matrix, a good transfer of the IgG-glycolipid film, and a favorable antibody orientation in the transferred film for immunoaffinity. Functional Stability of the Molecular Assembly. To explore the functional stabilities of the AChE-IgGglycolipid molecular assemblies transferred at different surface pressures (18.5, 25, 30, and 36 mN/m), the enzyme activity has been regularly measured after storage in a buffer solution (0.1 M phosphate buffer pH 7.4, 0.15 M NaCl, 1 mg/mL BSA) at 4 °C, over a period of 82 days. As shown in Figure 8, the activity of the immobilized enzyme moderately decreases over this period (35% for layers transferred at 30mN/m, and 45% for the other transfer pressures). To our knowledge, such a high stability was never reported for immobilized enzyme onto Langmuir-Blodgett films. By comparison, Puu and Ohlsson’s group50,51 reported a half-life of 28 days only, for the activity of AChE immobilized by fusion of proteo-phospholipidic vesicles onto solid substrates. The micro-homogeneity of the AChE-IgG-glycolipid stacking has been checked through Nomarski microscopy, and no structural modification has been observed either after enzyme activity measurement or after successive immersions in the buffer (data not shown). When the immunoglobulin is properly orientated in the transferred glycolipid matrix, the AChE-IgG (50) Ohlsson, P.-A.; Tjarnhage, T.; Herbai, E.; Lofas, S.; Puu, G. Bioelectrochem. Bioenerg. 1995, 38, 137. (51) Puu, G.; Gustafson, I.; Artursson, E.; Ohlsson, P.-A. Biosens. Bioelectron. 1995, 10, 463.
Proteo-glycolipidic Molecular Assembly
Figure 8. Time stability of immobilized enzyme activity of an AChE-IgG-glycolipid molecular assembly obtained after AChE immunoassociation onto mixed IgG-glycolipid bilayers transferred at 30 mN/m (s), 36 mN/m (4), 25 mN/m (2), and 18.5 mN/m (0).
immune complex remains embedded into the film, thus allowing the nanostructure to retain AChE activity for several weeks. No enzyme desorption was observed when the plate was removed from the enzyme substrate medium. This strong retention can be attributed to the specificity of IgG-AChE recognition, to the strong proteolipidic interactions between the IgG and the glycolipid, and to the absence of charge in the glycolipid film, thus avoiding reorganization52,53 or denaturation54 due to charge repulsions, as previously reported. Immobilized Enzyme Biocatalysis. The biocatalytic behavior of AChE-IgG-glycolipid molecular assemblies was investigated for films transferred at 30 mN/m and for two different immobilized enzyme amounts. For the saturation of all the available antibody recognition sites in the nanostructure, an IgG-glycolipid coated support has been immersed in a 72 EU/mL AChE solution, thus ensuring an enzyme activity of 0.58 EU/cm2. Taking into account the surface occupied by the AChE-IgG complex (=60 nm2)55 in the orientated position and the turnover number of the enzyme, it could be estimated that 2% of the total monolayer surface is occupied by the complex. A support of lower enzyme activity has also been prepared by immersion in a 0.2 EU/mL AChE solution, thus ensuring an activity of 0.06 EU/cm2 (0.23% of monolayer area occupied by active IgG-AChE complexes). The normalized rates of reaction (V/Vm), catalyzed by the immobilized AChE, toward acetylthiocholine have been compared with those catalyzed by the soluble enzyme (Figure 9). It can be mentioned that AChE was inhibited at high substrate concentrations in the soluble as well as in the immobilized forms. The soluble AChE displayed kinetics (curve a) and a KM value (0.04 mM) in agreement with the results reported in the literature.56,57 The immobilized enzyme exhibiting the lower activity behaved similarly as the soluble form (curve b). On the other hand, curve c, corresponding to the kinetics of the immobilized enzyme (52) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Langmuir 1997, 13, 6540. (53) Tang, J.; Jiang, J.; Song, Y.; Peng, Z.; Wu, Z.; Dong, S.; Wang, E. Chem. Phys. Lipids 2002, 120, 119. (54) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Langmuir 1998, 14, 476. (55) Tronin, A.; Dubrovsky, T.; Nicolini, C. Langmuir 1995, 11, 385. (56) Frobert, Y.; Cre´minon, C.; Cousin, X.; Re´my, M.-H.; Chatel, J.M.; Bon, S.; Bon, C.; Grassi, J. Biochim. Biophys. Acta 1997, 1339, 253. (57) Cousin, X.; Cre´minon, C.; Grassi, J.; Me´flah, K.; Cornu, G.; Saliou, B.; Bon, S.; Massoulie´, J.; Bon, C. FEBS Lett. 1996, 387, 196.
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Figure 9. Activity of AChE versus acetylthiocholine concentration: (a) soluble AChE at 3.33 EU/mL; (b) immobilized AChE (nonsaturating enzyme) at 0.061 EU/cm2; (c) immobilized AChE (saturating enzyme) at 0.581 EU/cm2. Vm corresponds to the maximum enzymatic reaction rate for soluble as well as for immobilized enzyme.
exhibiting the higher activity, was shifted toward higher acetylthiocholine concentrations. This modification of the kinetics, typical of immobilized enzymes,58 has been previously analyzed as the result of retained enzyme activity and substrate diffusion from the bulk to the enzyme.59,60 For the immobilized AChE exhibiting the lower activity, the substrate diffusion was not a limiting parameter and the kinetics were comparable with those of the soluble enzyme. For the immobilized AChE exhibiting the higher activity, external diffusional resistances of substrates affected the kinetics of the reaction and lowered its apparent rate. Conclusion In a continuation of our preceding efforts devoted to the formation of mixed IgG-glycolipid assemblies, this work displayed several new features. (i) When mixed IgGglycolipid vesicles were spread at an air/buffer interface, a monolayer was formed which retained the antibody. (ii) During the compression of this monolayer, a very small amount of only the immunoglobulin was released to the bulk. The compression was accompanied by a reorientation of the strongly embedded IgG molecule. (iii) After LB deposition on solid support at 30 mN/m, a stable, homogeneous, and properly orientated matrix was obtained. (iv) The latter was able to bind the enzyme AChE by immunoaffinity, and the immobilized nanostructure (glycolipid-IgG-AChE) was able to retain enzymatic activity over 2 months. The main accomplishment of this work was the demonstration of the ability to sequester a hydrophilic enzyme onto a lipid layer environment while maintaining the enzymatic activity, which is typical of many signal transduction events. To our knowledge, only a few biocatalytic studies have been published to date, using LangmuirBlodgett films,61 and the results reported in this paper are really promising to perform biocatalysis studies in a biomimetic environment. (58) Engasser, J.-M.; Horvath, Cs. In Applied Biochemistry and Bioengineering; Wingard, L. B., Jr., Katchalski-Katzir, E., Goldstein, L., Eds.; Academic Press: New York, 1976; Vol. 1, p 127. (59) Engasser, J.-M.; Coulet, P. R. Biochim. Biophys. Acta 1977, 485, 29. (60) Engasser, J.-M. J. Biol. Chem. 1977, 252, 7919. (61) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Bioelectrochem. Bioenerg. 1998, 46, 39.
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Abbreviations A ) total film area AChE ) acetylcholinesterase ATChI ) S-acetylthiocholine iodide au ) arbitrary unit Cs ) compressibility DTNB ) 5,5′-dithiobis(2-nitrobenzoic acid) EU ) Ellman’s unit IgG ) immunoglobulin G LB ) Langmuir-Blodgett π ) surface pressure πc ) collapse pressure RAI ) amide I ratio REA ) relative evolution of activity REE ) relative evolution of embedment
Godoy et al. TEM ) transmission electronic microscopy TNB ) 5-thio-2-nitrobenzoate V ) enzymatic activity velocity Vm ) maximal enzymatic activity velocity
Acknowledgment. Thanks are due to Dr. J. Grassi (CEA, Saclay, France) for his generous gift of the ascitic fluid and to Dr. C. Bon (Institut Pasteur, Paris, France) for his generous gift of the acetylcholinesterase of Bungarus fasciatus venom, together with their interest in this work. We also thank Prof. P. Goekjian for molecular calculations. This work is dedicated to the memory of Pr. Jean-Paul Chauvet. LA034517A
