6540
Langmuir 1997, 13, 6540-6546
Choline Oxidase Associated with Behenic Acid LB Films. Reorganization of Enzyme-Lipid Association under the Conditions of Activity Detection A. P. Girard-Egrot, R. M. More´lis, and P. R. Coulet* Laboratoire de Ge´ nie Enzymatique-UPRESA CNRS 5013-UCBL, 43, Bd du 11 novembre 1918, 69622 Villeurbanne Cedex, France Received February 4, 1997. In Final Form: July 29, 1997X This study deals with the structural modifications of enzyme-lipid Langmuir-Blodgett films under conditions required for enzyme activity detection. As a model, choline oxidase was associated with behenic acid LB films either through a direct adsorption onto the head groups of the lipidic LB films or through an adsorption followed by the transfer of a behenic acid layer onto the protein molecules, which sandwiched the enzyme between the polar groups of two lipidic layers. The structure, homogeneity, and stability of the protein-lipid LB stacking has been studied using FTIR spectroscopy and Nomarski differential interference contrast microscopy before and after immersion of the proteo-lipidic multilayers in the buffer used for the enzymatic activity detection. In the absence of enzyme, a pH variation induces a structural reorganization of the lipid stacking with formation of lipidic vesicles, most probably in reversed phase. The association of choline oxidase with the fatty acid LB films acts as an accelerating factor of this lipidic reorganization. These results can be explained by the effects of both hydration of the multilayers and the establishment of repulsive interactions either between protein and lipid molecules or between lipid molecules themselves.
Introduction The Langmuir-Blodgett technique is an attractive approach for obtaining artificial biomimetic models with a well-characterized molecular organization. Insertion of enzyme molecules in LB films has been reported1 and several bioactive protein-lipid LB films have been studied with regard to their potential applications in biosensing devices.2-11 Structural informations on the enzyme-LB films association are available from studies of the structure of protein-incorporating LB films through IR-ATR spectroscopy12,13 or AFM.14,15 However, these studies are usually performed immediately after enzyme association, and to our knowledge, no structural details are available on the conditions of detection of enzyme activity, i.e., after immersion of enzyme-containing LB films in the adequate reaction medium. Recently, it has been shown in our * To whom correspondence should be addressed. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, November 1, 1997. (1) Zhu, D. G.; Petty, M. C.; Ancelin, H.; Yarwood J. Thin Solid Films 1989, 176, 151. (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. (3) Moriizumi, T. Thin Solid Films 1988, 160, 413. (4) Li, J. R.; Cai, M.; Chen, T. F.; Jiang, L. Thin Solid Films 1989, 180, 205. (5) Okahata, Y.; Tsuruta, T.; Ijiro, K.; Ariga, K. Langmuir 1988, 4, 1373; Thin Solid Films 1989, 180, 65. (6) Sriyudthsak, M.; Yamagishi, H.; Moriizumi, T. Thin Solid Films 1988, 160, 463. (7) Anzaı¨, J.-I.; Lee, S.; Osa, T. Makromol. Chem., Rapid Commun. 1989, 10, 167. (8) Zaitsev, S. Yu.; Kalabina, N. A.; Zubov, V. P. J. Anal. Chem. USSR 1991, 45, 1054. (9) Arisawa, S.; Arise, T.; Yamamoto, R.Thin Solid Films 1992, 209, 259; Arisawa, S.; Yamamoto, R.Thin Solid Films 1992, 210/211, 443. (10) Fiol, C.; Valleton, J. M.; Delpire, N.; Barbey, G.; Barraud, A.; Ruaudel-Teixier, A. Thin Solid Films 1992, 210/211, 489. (11) Pal, P.; Nandi, D.; Misra, T. N. Thin Solid Films 1994, 239, 138. (12) Ancelin, H.; Zhu, D. G.; Petty, M. C.; Yarwood, J. Langmuir 1990, 6, 1068. (13) Subirade, M.; Salesse, C.; Marion, D.; Pe´zolet, M. Biophys. J. 1995, 69, 974. (14) Fujiwara, I.; Ohnishi, M.; Seto, J. Langmuir 1992, 8, 2219. (15) Fiol, C.; Alexandre, S.; Delpire, N.; Valleton, J. M.; Paris, E. Thin Solid Films 1992, 215, 88.
S0743-7463(97)00115-7 CCC: $14.00
group16 that the enzyme glutamate dehydrogenase adsorbed on behenic acid LB films plays a protecting role on the organized lipidic multilayer structure, preserving it from a disorganizing effect by the alkaline buffer used for the enzyme activity detection. In the present work, investigating the effects of proteinlipid interactions, our goal is to determine through FTIR spectroscopy and Nomarski microscopy the structural evolution of the behenic acid multilayers incorporating choline oxidase when they are immersed in the alkaline buffer required for the enzymatic activity detection. Experimental Section Reagent and Substrate Preparation. Behenic acid (docosanoic acid) and choline oxidase (EC 1.1.3.17) from Alcaligenes species were both obtained from Sigma (St Quentin Fallavier, France) and were used without further purification. Chloroform (analytical-reagent grade, purity ) 99%) was purchased from Chimie plus (France), potassium dihydrogen phosphate (for analysis) from Merck (Germany) and analytical-grade potassium chloride from Prolabo (France). Ultrapure water (resistivity g 18.2 MΩ.cm) was obtained with a Milli-Q four cartridge purification system (Millipore, France). The calcium fluoride (CaF2) substrates (35 mm × 9.5 mm × 2 mm) were purchased from Sorem (France), they were cleaned with TFD4 detergent (Franklab, France) as described elsewhere.17 Langmuir-Blodgett (LB) Film Deposition. A LangmuirBlodgett trough, model LB-105 from ATEMETA, Paris [licence CEA, Patent 83 19770 (12-09-83)] with a Wilhelmy balance was used. It was filled with 7 liters of Milli-Q water in equilibrium with atmospheric CO2. The temperature was kept at 21 ( 1 °C. The monolayer was obtained as previously described.17 Briefly, the water surface was cleaned by suction, and a known volume of 10-3 M behenic acid solution was spread. The monolayer was compressed by applying a discontinuous compression by steps of 2 mN‚m-1 and by controlling the parameters of the feedback servoloop of the surface pressure. While the surface pressure was maintained at 32 ( 0.3 mN‚m-1, the compressed monolayer was transferred onto the hydrophilic CaF2 substrate with the vertical dipping technique at a rate of 2 cm‚min.-1. The (16) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Thin Solid Films, in press. (17) More´lis, R. M.; Girard-Egrot, A. P.; Coulet, P. R. Langmuir 1993, 9, 3101.
© 1997 American Chemical Society
Behenic Acid LB Films monolayer deposition rate precisely defined elsewhere18,19 was poised at a low value. Then 12 layers in the Y-type form were deposited with a transfer ratio close to 1. In order to obtain the hydrophilic head groups at the surface of the multilayers, the monolayer was removed after the transfer of the last layer and the substrate was rapidly withdrawn. Enzyme Association. Before enzyme association, the behenic acid multilayers were immersed for one night in 0.1 M phosphate buffer/0.1 M KCl pH 8.0 and then dried under a purified air-dried flow. Choline oxidase adsorption was performed by immersion of the coated substrate in a 2 mg‚mL-1 choline oxidase solution in 0.1 M phosphate buffer for 2 h at 20 °C either at pH 8.0 when the enzyme was only adsorbed or at pH 6.0 when the enzyme was sandwiched between two lipidic layers. Each face of the coated substrate was further rinsed with 10 mL of phosphate buffer and thoroughly dried at room temperature under a purified air-dried flow. For sandwiching the enzyme between the heads of the lipidic layers (referred to as inclusion by sandwiching), the substrate with the adsorbed enzyme was rapidly immersed at the rate of 10 cm‚min.-1 (to avoid any transfer of molecules) through the behenic acid monolayer compressed at 2 mN‚m-1. The monolayer compression was next pursued as described above. When the surface pressure reached 32 mN‚m-1, one layer was transferred at the upstroke onto the substrate with a transfer ratio equal to 1.16. In order to study the enzyme retention in the conditions used for the enzymatic activity detection, the coated substrates were immersed in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0, for different times and were dried under a purified air-dried flow for FTIR studies. Structure and Homogeneity of Protein-Lipid LB Films. The structure and homogeneity of the proteo-lipidic LB films were controlled at each step of their preparation. The homogeneity of the LB films was checked through a Nomarski differential interference contrast microscope (Carl Zeiss, Jena, Germany). FTIR spectroscopy was used to detect the presence of the protein and to assess both the chemical composition and the structure of the protein-lipid LB films. The infrared absorption spectra were recorded using a 510 M FTIR interferometer (Nicolet instruments, Trappes, France). The transmission spectra were obtained with a resolution of 4 cm-1. The thickness of the multilayers was controlled after different times of immersion in the buffer through the integration value (through Nicolet’s software) of the intensities of the CH stretching vibrations between 2803 and 2984 cm-1. Three zones of the immersed area (top, middle, bottom) were defined on the substrate in order to check the topographic homogeneity of the multilayer structure.
Results and Discussion 1. Effects of Phosphate Buffer at pH 8.0 on Behenic Acid Multilayer Structure. To be in accordance with the experimental conditions required for the detection of the choline oxidase activity retained in the LB films,20 the enzyme association was performed after a one-night immersion of the multilayers in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0. Table 1 presents the band assignments from 12 behenic acid layers deposited on each face of a calcium fluoride substrate, before and after the one-night immersion. The FTIR spectrum collected before immersion was characteristic of the crystallized form of behenic acid multilayers. They are constituted by carboxylic dimers of behenic acid (ν CdO at 1700 cm-1) in predominant trans configuration (ν(CO)/δ(OH) at 1300 cm-1) with a all-trans conformation of hydrocarbon chains (νa(CH2) at 2917 cm-1; νs(CH2) at 2850 cm-1; presence of the band progression arising from the CH2 wagging and the CH2 twisting vibrations).21 The (18) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Langmuir 1993, 9, 3107. (19) Girard-Egrot, A. P.; More´lis, R. M.; Coulet, P. R. Langmuir 1996, 12, 778. (20) Girard-Egrot, A. P., Ph. D. Thesis, University of Lyon, Lyon, France, 1995. (21) Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986, 2, 96.
Langmuir, Vol. 13, No. 24, 1997 6541 Table 1. Band (cm-1) and Band Assignments from 2 × 12 Behenic Acid Multilayers before and after One-Night Immersion in 0.1 M Phosphate Buffer/0.1 M KCl, pH 8.0a band nature
before
νa(CH3) νa(CH2) νs(CH2) ν(CdO)
2956 2917 2849 1701
νa(COO-) δ(OH‚‚‚O)/νa(COO-) δ′′(CH2) δ′(CH2) δ(CH2)R ν(C-O)/δ(OH) δω(CH2), δγ(CH2)
N. P. N. P. 1472 1464 1410 1304 1350-1100
after 1 night 2954 2915 2849 attenuated, centered at 1717-1701 centered at 1648 traces (1576-1559) 1472 sh. split at 1418 and 1406 N.P. attenuated
a The underlined values indicate a shift in the wavenumber; N.P., non present; sh., shoulder (ν, stretching; νa, antisymmetric stretching, νs, symmetric stretching; δ, bending; δω, wagging; δγ, twisting; δ′′ CH2 and δ′ CH2, the two components of the CH2 scissoring vibration in the crystallized state of the multilayers; δ (CH2)R, CH2 scissoring vibration of the group adjacent to the carboxylic or the carboxylate group; ν (C-O)/δ(OH), coupled mode of the C-O stretching and the OH in-plane bending vibrations of the trans configuration of the carboxylic dimers).
Figure 1. 1850-1200 cm-1 region of FTIR transmission spectra of 2 × 12 behenic acid multilayers transferred onto a CaF2 substrate before (a) and after (b) a one-night immersion in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0.
splitting of the CH2 scissoring vibration in two components δ′(CH2) and δ′′(CH2) indicates that the multilayers crystallize with two dimers per unit,22 and the behenic acid LB films are in a monoclinic crystal form.23 As shown in Figure 1, after one night in the alkaline phosphate buffer, both the composition and the structure of the multilayers are modified. The intensity of the ν(CdO) band is reduced; two bands assignable to the vibrations of the carboxylate groups appear: νa(COO-) near 1648 cm-1 and the coupled mode of δ(OH‚‚‚O)/νa(COO-) centered at 1570 cm-1 owing to hydrogen bonds with the COO- groups;24 this partial transformation of the carboxylic groups comes with the disappearance of the ν(C-O)/δ(OH) band. The modifications of the head groups have an influence on the order of the hydrocarbon chains and their vibrations are also modified: after immersion, the δ(CH2) band becomes a singlet (with a shoulder at 1464 cm-1), the δ(CH2)R band is split and the intensity of the CH2 wagging and CH2 twisting band progression is attenuated; the bands due to the νa(CH3) and νa(CH2) vibrations are shifted downwards at 2954 and 2915 cm-1 respectively (Table 1). Figure 2 shows the microscopic details of the structure of the multilayers (22) Chollet, P.-A.; Messier, J. Thin Solid Films 1983, 99, 197. (23) Bonnerot, A.; Chollet, P.-A.; Frisby, H.; Hoclet, M. Chem. Phys. 1985, 97, 365. (24) Vogel, C.; Corset, J.; Dupeyrat, M. J. Chim. Phys. 1979, 76, 909.
6542 Langmuir, Vol. 13, No. 24, 1997
Figure 2. Photographs of behenic acid multilayers before (a) and after (b) a one-night immersion in 0.1 M phosphate buffer/ 0.1 M KCl, pH 8.0. (The stripes are the consequence of the ultrasonic cleanup treatment of the substrate.)
observed through a Nomarski differential interference contrast microscope before (a) and after immersion (b). This technique is convenient to observe the stress relief pattern and the scattering defect centers.17,25 Before immersion, the behenic acid multilayers are homogeneous; after immersion, they appear granulous. The structural modifications have been attributed to an hydration of the polar groups leading to a modification of the ionic state of the LB films which induces a minor rearrangement of the molecules. According to Chollet,26 the splitting of the scissoring vibration of the methylene groups (δ(CH2)) depends on the crystallized state of the multilayers, and a possible transition from the perfectly crystallized (splitting) to the amorphous state (no splitting) can be induced by an inclusion of water molecules. In our case, with behenic acid multilayers, the modification of the splitting of the CH2 scissoring band is an indication of the hydration of the polar groups by the alkaline phosphate buffer, leading to a partial transformation of the carboxylic groups into carboxylate forms. After one night of immersion, the multilayers are composed of a mixing of behenic acid and behenate salt in an amorphous state; they are characterized by the presence of the vibrations due to the carboxylic and the carboxylate groups, and by the appearance of the δ(OH‚‚‚O)-νa(COO-) coupled mode characteristic of mixed multilayers constituted of acid and salt forms.24 In addition, we noticed the disappearance of the ν(C-O)/δ(OH) band indicating the loss of the trans configuration of the dimeric acids in these multilayers. To explain all the modifications, we propose a peculiar structure for the polar groups of the mixed multilayers, named behenate-acid multilayers. In this structure, one carboxylic acid interacts with one carboxylate salt. Such an interaction explains in particular, both the disappearance of the ν(CsO)/δ(OH) band and the shifting to 1717 cm-1 of the ν(CdO) vibration since this latter position is characteristic of the CdO stretching vibration in interaction with salt molecules.24 In the mixed layers, the arrangement of hydrocarbon chains is modified. First, the appearance of the salt form is responsible for the attenuated intensities of the band progression due to the δω(CH2) and δγ(CH2) modes, as previously reported by other authors for the multilayers of calcium stearate or sodium palmitate.21 Second, the simultaneous presence of the carboxylic and carboxylate groups and their interaction may explain the splitting of the scissoring (25) Tippmann-Krayer, P.; Meisel, W.; Mo¨hwald, H. Adv. Mater. 1990, 2, 589. (26) Chollet, P.-A. Thin Solid Films 1978, 52, 343.
Girard-Egrot et al.
Figure 3. FTIR spectra (1800-1200 cm-1 region) of (a) behenate-acid multilayers (2 × 12 behenic acid multilayers after one night in 0.1 M phosphate/0.1 M KCl, pH 8.0), (b) behenate-acid multilayers after 2 h in 0.1 M phosphate buffer pH 6.0 (control spectrum), and (c) behenate-acid multilayers after choline oxidase inclusion by sandwiching (inclusion spectrum).
vibration of the CH2 group adjacent to the carbonyl group (δ(CH2)R band). Finally, the hydration of the polar groups and the appearance of the salt form may explain the shift of the CH stretching vibration by a spacing of the polar groups leading to a rearrangement of the CH dipole of the methylene groups along the hydrocarbon chain. It is noteworthy that a longer immersion in alkaline phosphate buffer does not induce additional modifications. The mixed multilayers are stable. Moreover, the integration value of the CH stretching vibration intensities representative of the multilayer thickness was the same before and after a one-night immersion of the multilayers, the transformation of the behenic acid into behenateacid does not provoke a desorption of the lipidic molecules (data not shown). Thus, the enzyme adsorption occurred with behenate acid multilayers when the association is performed under the conditions of the enzyme activity detection. 2. Choline Oxidase Inclusion by Sandwiching between the Head Groups of Two Lipidic Layers. The inclusion by sandwiching of choline oxidase molecules was performed by immersion of the behenate-acid multilayers in the enzyme solution (adsorption step) followed by the transfer of one behenic acid monolayer onto the adsorbed protein molecules (covering step). In order to avoid a pH variation, these steps were performed in phosphate buffer at pH 6.0, close to that of ultrapure water of the subphase. 2.1 FTIR Characterization of the Protein-Lipid Organization Immediately after Enzyme Association. Figure 3 shows the IR spectra collected after a onenight immersion in alkaline buffer (behenate-acid multilayers, spectrum a), after a 2-h immersion in 0.1 M phosphate buffer pH 6.0 containing (spectrum c) or not containing (spectrum b) the choline oxidase molecules. The immersion of the behenate-acid multilayers in phosphate buffer at pH 6.0 reverses the salt formation (spectrum b). The νa(COO-) vibration disappears, the intensity of the δ(OH‚‚‚O)-νa(COO-) band is largely attenuated, and both the ν(CdO) and ν(CsO)/δ(OH) modes are detected in the form of a doublet located at 1713 and 1694 cm-1 and at 1304 and 1290 cm-1, respectively. The vibrations of the hydrocarbon chains are the same as for the behenic acid multilayers (Figure 1a). (The δ(CH2) band is split, the δ(CH2)R band is a singlet, the band progression of the δω(CH2) and δγ(CH2) vibrations is markedly more intense, and the νa(CH2) vibration is
Behenic Acid LB Films
Langmuir, Vol. 13, No. 24, 1997 6543
located at 2917 cm-1). According to Higashi et al.,27 the splitting of the ν(CdO) band at 1710 and 1680 cm-1 reflects that the cis configuration of the acid dimers is of major importance in the structure, whereas the positions of δ(CH2)R and ν(C-O)/δ(OH) bands at 1410 and 1300 cm-1, respectively, indicate the trans configuration is of major importance. So, we may conclude that the multilayers which get back their acidic form may be constituted of dimeric acids in both cis and trans configurations. Moreover, it cannot be excluded that other associations different from the dimeric acids could be formed, explaining the splitting of the ν(C-O)/δ(OH) band. The slight splitting of the δ(CH2) band and the presence of the weak δ(OH‚‚‚O)-νa(COO-) vibration are attributable to the traces of buffer retained in the multilayer structure. Thus, the structure of the reversed behenic acid multilayers seems different from that of the behenic acid multilayers just after transfer. After sandwiching of choline oxidase molecules, the bands characteristic of the peptidic bond, the amide I (ν(CdO)), amide II (δ(NH)) and amide III (ν(CN)) bands are present (spectrum c), and the ν(NH) vibration is observed at 3286 cm-1 (data not shown). The ν(CdO) band of the carboxylic groups reappears with the same intensity as for the control layers (spectrum b); however, this band is not split but is shifted to 1686 cm-1 with a shoulder at 1717 cm-1; the ν(C-O)/δ(OH) vibration characteristic of the trans dimeric acid configuration is not detected, the δ(CH2) band stays in a singlet line, and the intensities of the progression bands and the position of the CH stretching vibrations are the same as for the behenate-acid multilayers before protein association (spectrum a). It can be noticed that the enzyme inclusion does not imply covalent bonds with the polar groups since they are reversed in their acid form, and the shift of the CO stretching vibration of the carboxylic groups toward lower wavenumbers suggests the occurrence of hydrogen-bonded interactions between the protein molecules and the polar groups (in addition to the electrostatic forces involved in the protein adsorption process).1,12 Moreover, the presence of the protein prevents the trans dimeric acid formation of the carboxylic groups of the underlaying layers and allows the hydrocarbon chains to keep the characteristics of behenate-acid structure with the antisymmetric CH stretching vibration located at its lowest wavenumber. Therefore, the presence of the enzyme maintains the spacing of the hydrocarbon chains induced by the hydration of the polar group in the behenate-acid multilayers. 2.2 FTIR Characterization of the Protein-Lipid LB Films after Immersion in the Phosphate Buffer at pH 8.0. To study the enzyme retention and the stability of protein-lipid organization in the conditions of the enzyme activity detection, the coated substrates were immersed in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0, during different times. The spectra collected on the control layers (Figure 4A) show that a short immersion in the alkaline buffer (1 h 45 min) induces important modifications in the lipidic structure (spectrum b). Mainly, a very broad band centered at 3300 cm-1 arises from the OH-bonded stretching vibration which can be assigned to the OH stretching mode of the carboxylic groups (involved in hydrogenbonded intermolecular interactions). It must be stressed that a longer immersion yields only an intensification of these modifications (spectrum c). In the presence of the enzyme (Figure 4B), the same modifications appear and they are amplified (spectrum b), no intensification of the
phenomenon occurs during a longer immersion time (spectrum c); the νa(CH2) stretching vibration is shifted to 2913 cm-1 (this shift never occurred in the control layers). Figure 5A shows the 1800-1100 cm-1 region of the control spectra. It clearly appears that after reimmersion in the alkaline buffer (spectra b and c), both νa(COO-) and ν(CdO) bands of the behenate-acid multilayers merged into the same broadened and intensified region; in fact, there is an additional absorption at 1670 cm-1 which may be attributed to the OH bending mode of water vibration.28 The δ(OH...O)/νa(COO-) band is more intense and appears with two predominent locations at 1576 and 1547 cm-1. The band progression of the δω(CH2) and δγ(CH2) vibrations are attenuated and the δ(CH2)R band is not split; moreover, the band at 1259 cm-1 assigned to the phosphate stretching vibration (ν(PdO)), suggests that buffer molecules are retained in the layers. So, the reimmersion in the alkaline buffer leads to a further hydration of the lipidic structure. It is likely that the OH stretching vibration of the bound water molecules normally located at 3300 cm-1 28 was superimposed to that of the carboxylic groups, and the establishment of new hydrogen-bonded interactions between water and carboxylic groups could explain the strong intensity of the OH stretching band. The appearance of the hydrated carboxylate groups also explained the intensification of the coupled vibration (δ(OH‚‚‚O)/νa(COO-)). Figure 5B shows modifications found in the 1800-1100 cm-1 region when the protein is included in the LB films. The ν(CdO), amide I, and νa(COO-) bands are also merged into a broad and a very intensified peak between 1760 and 1590 cm-1 (spectrum b). As the protein does not covalently interacts with the carboxylic groups, immersion in the alkaline buffer leads to the formation of carboxylate and the νa(COO-) band intensifies in this region. As for
(27) Higashi, A.; Czarnecki, M. A.; Ozaki, Y. Thin Solid Films 1993, 230, 203.
(28) Okamura, E.; Umemura, J.; Takenaka, T. Vib. Spectrosc. 1991, 2, 95.
Figure 4. FTIR spectra of control layers without enzyme (A) or of LB films with choline oxidase associated by inclusion (B) before and after different immersion times in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0: (a) before; (b) after 1 h 45 min immersion; (c) after a one-night immersion.
6544 Langmuir, Vol. 13, No. 24, 1997
Girard-Egrot et al. Table 2. Quantitative Effects of Choline Oxidase Association on Structural Modification at 1650 cm-1 (Given in Absorbance Unit × 10-3)
substrate control inclusion
Figure 5. 1800-1200 cm-1 region of FTIR spectra collected on the control (A) and on the choline oxidase inclusion substrate (B) after different times of immersion in 0.1 M phosphate buffer/ KCl 0.1 M, pH 8.0: (A/a) behenate-acid multilayers; (b) after 1 h 45 min immersion; (c) after a one-night immersion. (B/a) after inclusion by sandwiching of the protein; (b) after 1 h 45 min immersion; (c) control layers at the same immersion time; (d) behenic acid multilayers (as reference for the location of the original bands).
the control layers, the OH bending vibration of water molecules at 1670 cm-1 can be suspected to expand this region. The amide II band (sharp peak immediately after protein inclusionsspectrum a), is modified between 1580 and 1540 cm-1 after immersion (spectrum b). Compared with the control spectrum (spectrum c), this region corresponds to the δ(OH...O)/νa(COO-) band. The band localized at 1246 cm-1, assigned to the PdO stretching vibration, proves the presence of phosphate buffer and the further hydration of the layers containing the protein. The fact that the integration of the CH stretching vibrations intensities gives the same values after the two immersion times indicates that no lipid molecule desorption occurred during the structural modification with or without enzyme. It clearly appears from this study that successive changes of the buffer pH have a drastic effect on the structural lipidic organization. As expected for an immersion in phosphate buffer at pH 8.0, the carboxylate groups and all the bands characteristic of the behenateacid mixed organization are present. However, additional modifications, such as the appearance of the OH-bonded stretching vibration, suggest that the successive pH variations induce a structural reorganization where the lipidic molecules are packed in a much hydrated structure different from the orderly behenic acid multilayers. In the presence of the protein, the same modifications are induced, but the structural reorganization is higher and a lengthy immersion does not amplify the phenomenon; on the contrary, after a one-night immersion, the intensity of the ν(OH) band was slightly decreased and the intensity of the broad band (between 1760 and 1590 cm-1) was slightly increased (spectrum 4B-c). Consequently, the presence of the protein seems to accelerate the structural
substrate zones
after 1h45
after 1 night
difference between 1 h 45 and 1 night
top middle bottom top middle bottom
5.65 7.47 7.98 10.65 15.28 21.64
8.12 11.46 12.39 16.10 20.99 28.88
2.47 3.99 4.41 5.45 5.71 7.24
reorganization which ends in the first hour of the immersion. To check this hypothesis, we have quantified the structural reorganization of the lipidic layers in the presence or absence of the enzyme, by measuring the absorption intensity at 1650 cm-1 which corresponds to the νa(COO-) band (superimposed on the amide I band when the protein is present). This location was chosen because the νa(COO-) vibration appeared only after immersion in the alkaline buffer, and it was particularly intensified during the structural reorganization even in the presence of the protein. To check the homogeneity of the structural reorganization, we measured, at the bottom, at the middle, and at the top of the substrate, the intensities of the νa(COO-) peaks after 1 h 45 min and one-night immersions. The results are presented in Table 2. The values obtained for the control layers after a onenight immersion are approximately of the same order of magnitude as those obtained for the incorporated-protein layers after a 1 h 45 min immersion. The difference between 1 h 45 min and 1 night corresponds to one-third of the total modifications for the control substrate whereas it corresponds to one-fourth for the inclusion one. Then, at 1 h 45 min, two-thirds of the structural modifications are achieved in the control layers whereas, with the protein, three-fourths of the modifications are already made. So, these measurements demonstrate that the kinetic of the structural reorganization induced by the reimmersion in the alkaline buffer is quantitavitely accelerated by the presence of the protein. It must be underlined that for the same immersion time, the reorganizations are nonhomogeneous all over the substrate: it decreases from the bottom to the top particularly for the inclusion one. For the control layers, it could be possible that the buffer diffuses by capillarity between the layers, thus explaining the slowest kinetic of the modifications. For the inclusion substrate, the difference in the amplitude of the modifications could be explained by the nonhomogeneous distribution of the protein on the substrate. Indeed, immediately after enzyme association, the amide I band is more intense at the bottom than at the top of the substrate (data not shown). This nonhomogeneous distribution of the protein molecules perfectly fits with the amplitude of the structural reorganization induced by the buffer. So, we concluded that the presence of the protein has two effects on the structural lipidic reorganization; first, the reorganization is more intense and appears to depend on the amount of enzyme retained, and second, the reorganization occurs more rapidly. This confirms the hypothesis of the accelerating role of the protein molecules on the structural lipidic reorganization induced by the variation of the buffer pH. 3. Adsorption and Retention of Choline Oxidase onto the Behenate-Acid Multilayers in Alkaline Phosphate Buffer (pH 8.0). In this study, we investigated what is happening when both the adsorption and retention of the enzyme are performed at the same alkaline
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Langmuir, Vol. 13, No. 24, 1997 6545
Figure 6. FTIR spectra collected on behenate-acid multilayers before (a) and after enzyme adsorption (b and c, respectively, collected at the top and the bottom of the substrate) and (d) after a one-night immersion in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0.
pH. Choline oxidase was adsorbed onto the behenateacid multilayers at pH 8.0. The stability of the proteinlipid LB films was then studied at the same pH. Figure 6 shows the spectra collected on the behenate-acid multilayers (spectrum a) and after enzyme association either at the top (spectrum b) or at the bottom of the substrate (spectrum c). At the top of the substrate (spectrum b), the ν NH stretching vibration of the peptide bond is visible, the νa(COO-), amide I and amide II bands appear to be well separated. At the bottom of the substrate (spectrum c), the broad band of the OH-bonded stretching vibration coming with the intensification of the bands located between 1760 and 1590 cm-1 appears as early as the enzyme adsorption step; the νa(CH2) stretching vibration is already shifted at 2913 cm-1. So, we concluded that at the bottom of the substrate, choline oxidase is adsorbed in large quantities, which initiates the reorganization as early as the adsorption step. After a one-night immersion in the alkaline buffer (spectrum d), the structural modifications become detectable whatever the adsorbed amount of enzyme. However, the kinetic of the modifications depends on this amount. For the largest quantity (at the bottom of the substrate), the modifications are only amplified with time whereas for the smallest quantity (at the top of the substrate), the modifications appear after a one-night immersion (the spectrum collected after an immersion of 1 h 30 min stayed identical to that collected immediately after adsorption). It must be stressed that the constant integration of intensities of the CH stretching vibrations at the different immersion times indicated that no desorption of the lipidic molecules occurred during the structural reorganization. This study shows that the structural reorganization occurs systematically when choline oxidase is present in (or on) the behenate-acid multilayers at pH 8.0. The amplitude and the kinetic of the rearrangement are directly related to the amount of choline oxidase retained in the LB films. It is noteworthy that, in the reorganized structures with the protein, the hydrocarbon chains are in a more condensed state, as revealed by the specific shift of the CH2 antisymmetric stretching vibrations. 4. Nomarski Microscopic Observations. The homogeneity of the protein-lipid LB films was investigated through Nomarski microscopic observations. Figures 7 and 8 refer, respectively, to the observations performed during the studies of the inclusion or the adsorption of choline oxidase. After immersion of the behenate-acid multilayers in the phosphate buffer at pH 6.0 (Figure 7a), the reversed behenic acid multilayers kept the granulous aspect of the behenate-acid multilayers (Figure 2b). After
Figure 7. Nomarski microscopic observations during the study of the inclusion/retention of choline oxidase in the behenateacid multilayers: (a, b) surface relief of the control layers after 2 h in 0.1 M phosphate buffer pH 6.0 (a) and after one night in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0 (b); (c, d) surface relief of the protein-lipid LB films immediately after sandwiching (c) and after one night in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0 (d).
Figure 8. Nomarski microscopic observations during the study of the adsorption/retention of choline oxidase on the behenateacid multilayers at pH 8.0. The photograph was taken after a one-night immersion in 0.1 M phosphate buffer/0.1 M KCl, pH 8.0.
a one-night immersion in the alkaline buffer, several droplets appeared in the structure of the layers (Figure 7b). These droplets were compared to lipidic globules. After choline oxidase inclusion (Figure 7c), the presence of the protein could be checked through the presence of fiberlike structures because these structures were never observed when the enzyme was not present. After a onenight immersion of the protein-lipid LB films (Figure 7d), the filaments were always present and a lot of lipidic globules appeared; obviously the number of the lipidic globules was higher when the choline oxidase molecules were sandwiched in the lipidic multilayers (comparing Figure 7b with Figure 7d). Moreover, the observations
6546 Langmuir, Vol. 13, No. 24, 1997
performed at the same times suggest that these lipidic globules did not appear with the same kinetics in the presence or absence of the enzyme, which corroborates the previous conclusion about the accelerating role of the choline oxidase molecules on the structural lipidic reorganization. When the enzyme was adsorbed at pH 8.0, the protein filaments and the lipidic globules are also present after a one-night immersion (Figure 8). From these microscopic observations, we can postulate that the structural reorganization of the lipidic layers corresponds to the formation of the lipidic vesicles. 5. General Discussion. This study shows that successive pH variations weaken the well-ordered and homogeneous structure of LB films. The change from alkaline to an acidic buffer induces the reverse formation of behenic acid multilayers where the dimeric acid associations (other than the main trans configuration) take place in granulous structures including some aqueous phase. So, immersion of these multilayers in the alkaline buffer induces a complete intermolecular reorganization owing to a further hydration which leads to both the appearance of a large quantity of carboxylate groups and to the formation of lipidic vesicles. The structural reorganization may be the outcome of the strong repulsive interactions between the negative charges of carboxylate groups which appeared in a weakened structure. It is conceivable that the appearance of negative charges in a structure where the polar group association is different from the well ordered dimeric acid of the behenic acid LB films leads to interactions less favorable than in the behenate-acid structure; indeed, in this structure, the repulsive interactions can be stabilized by the interaction of one carboxylate with one carboxylic group. Thus, the reorganization allows one to minimize the unfavorable interactions, and in order to stabilize the charges, the vesicles were more probably constituted of a mixing of acid and salt molecules. As the FTIR and Nomarski microscopy studies were performed on a dried substrate, it can be noticed that the lipidic vesicles formed were likely in the reverse phase, i.e., in the form of reversed micelles with the polar groups inside the structure trapping the aqueous phase. Therefore, the successive pH variations lead progressively to the transformation of the original perfectly crystallized behenic acid LB films into a much more hydrated structure in the form of a reverse phase. When choline oxidase molecules were associated with the behenate-acid LB films, two phenomena occurred according to the pH of the enzyme association: when the
Girard-Egrot et al.
association is performed at pH 6.0, the protein intensifies and accelerates the structural reorganization initiated by the pH variation; when the association is perfomed at pH 8.0, the protein directly provokes the reorganization. Taking into account the value of the isoelectric point of the choline oxidase (pHi ) 4.529 ), the reorganization may be explained by the repulsive effect of the interactions between the negative charges of the lipid (carboxylate groups) and of the protein (negatively charged too). Similar to the lipidic layers, the structural modifications allowed one to minimize these unfavorable interactions. When the reorganization was initiated by the pH variation, the repulsive interactions between the lipid and the protein accelerated and intensified the structural lipidic reorganization because these interactions could be more unfavorable than the repulsions existing between the carboxylate groups themselves. It must be stressed that the effect of the protein on the structure of the LB films depends on the proper nature of the protein itself. As shown previously,16 the adsorption of glutamate dehydrogenase on behenic acid multilayers plays a protecting role on the cristallized and homogeneous LB structure against the rearrangement induced by the alkaline buffers. Obviously, the protecting or the disorganizing role of the proteinic molecules in the LB films immersed in buffer seems directly dependent on the ionization state of the protein at a fixed pH. Conclusion What clearly appears from this study is that the conditions of the enzyme activity detection can be responsible for a drastic modification of the proteo-lipid LB film structure. Indeed, this work first reports that a pH variation can induce a tridimensional restructuring of the LB films and that the associated enzyme protein can enhance this destructuring of the organized multilayer stacking. The structural reorganization is due to both the polar group hydration and the unfavorable repulsive interactions between proteinic and lipidic molecules or between lipidic molecules themselves, which directly depend on the ionization state of the molecules and consequently on the buffer pH. Acknowledgment. The research was partly supported by CNRS-ULTIMATECH and Re´gion Rhoˆne-Alpes. LA970115P (29) Toyo Jozo Enzymes; Toyo Jozo, Co., Ltd.: Tokyo, Japan, 1982.
