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Surface Topography of Acetylcholinesterase in Langmuir and Langmuir-Blodgett Films Leila Dziri,† Salah Boussaad, Shaopeng Wang, and Roger M. Leblanc* Department of Chemistry, UniVersity of Miami, Cox Science Building, 1301 Memorial DriVe, P.O. Box 249118, Miami, Florida 33124-0431 ReceiVed: April 7, 1997; In Final Form: June 7, 1997X
The surface topography of the enzyme acetylcholinesterase was studied at the air/aqueous and the air/solid interfaces using the Brewster angle and the atomic force microscopies, respectively. Surface potentials of the enzyme monolayer have been measured in conjunction with the surface pressure. The surface potential and the surface dipole moment data show that the orientation of the molecular dipoles occurs before the orientation of the hydrophobic groups of the acetylcholinesterase monolayer. The variations of the surface potential observed at large molecular area suggest the presence of domains in the film. The Brewster angle images confirm the formation of domains at the air/aqueous interface. The size of these domains increases with decreasing the molecular area. Furthermore, the Brewster angle microscopy allowed us to detect a reversible formation of the domains upon the compression and the decompression of the monolayer. On the other hand, the atomic force microscope images of the Langmuir-Blodgett films show that the enzyme molecules are more close-packed at a surface pressure of 25 mN/m than at 20 mN/m. Size measurements of the enzyme particles indicate that acetylcholinesterase has an ellipsoidal shape and that the tetramer form of this enzyme is the most abundant.
Introduction Acetylcholinesterase (AChE) belongs to the class of R/β proteins, consisting of a large central mixed β-sheet surrounded by 15 R-helices,1 and exists in multiple conformations.2-4 The forms of AChE are asymmetric and globular, and they are distinguished on the basis of their quaternary structure and solubility characteristics.3,4 The asymmetric form, or collagen-tailed form, is characterized by the presence of a collagen-like tail and aggregates at low ionic strength in the presence of a polyanionic compound (e.g., glycosaminoglycan).5 They may contain one, two, or three catalytic subunit tetramers while the globular forms may be identified as monomer G1, dimer G2, or tetramer G4.6 The globular forms are characterized by the absence of a collagenlike tail. They can be divided into two different groups: amphiphilic and nonamphiphilic on the basis of their capacity to associate with micelles of nondenaturing detergents.7,8 There are three types of AChE amphiphilic forms: type I amphiphilic dimers which are glycophosphatidylinositol (GPI)anchored dimers and type II amphiphilic forms which may be distinguished from the type I forms by their insensitivity to GPIspecific phospholipases (PI-PLC) and by the fact that they do not aggregate in the absence of detergent. The third type of these forms is the hydrophobic-tailed tetramer which is a heterooligomeric form and is anchored in plasma membranes by a hydrophobic domain. The globular AChE, whether hydrophobic or nonhydrophobic, are known to be the most abundant and widely distributed forms of the enzyme. Hydrophobic AChE was found abundant in the electric organ of Torpedo. It has been shown that about 70% of electric AChE are a globular form.6 We have showed in our earlier work9 that AChE forms a highly stable monolayer when it is spread on the surface of an aqueous subphase containing an electrolyte. This great stability was attributed to its polar and nonpolar nature and also to its * To whom correspondence should be addressed. † E-mail address:
[email protected]. X Abstract published in AdVance ACS Abstracts, July 15, 1997.
S1089-5647(97)01191-7 CCC: $14.00
high molecular weight. Thus, the AChE monolayer (weight 255 000) was compressed to relatively high surface pressures (up to 40 mN/m) without any loss of the enzyme into the subphase.9 This is in agreement with the previous studies10-12 which reported that, effectively, molecular size is an important parameter to study the desorption of macromolecules when they are compressed at the air/aqueous interface. In fact, it was demonstrated that rates of desorption decreased markedly with increasing molecular weight at a given surface pressure; i.e., desorption for insulin (weight 6000) was measured at low surface pressure (15 mN/m), whereas for catalase (weight 230 000) the desorption was measured at 42 mN/m.11,12 Few questions arise now. Is the adsorbed enzyme at the air/ aqueous interface homogeneous or heterogeneous? Does the adsorbed enzyme exist in one or multiple states at the interface? Is there any formation of congregated groups or domains, or do we observe a uniform monolayer formed gradually during the compression without forming any domains at the interface? Several works had reported that adsorbed proteins may exist in more than one state. There are several mechanisms that could lead to these multiple states of adsorbed proteins at an interface, including the nonuniform distribution of amino acids residues on the exterior of the protein molecule or the existence of domains at the exterior of the protein enriched in amino acid residues of a particular type (e.g., negatively or positively charged). The purpose of the present paper is, first, to measure simultaneously the surface potential and the surface pressure of the enzyme monolayer at the air/aqueous interface and second to study the surface topography of the enzyme using two microscopic techniques: Brewster angle microscopy (BAM) at the air/aqueous interface and atomic force microscopy (AFM) of a Langmuir-Blodgett film of the enzyme. It is most likely that, at the air/aqueous interface, AChE monolayer forms domains during the compression. The domains start growing at higher surface pressures. Furthermore, the monolayer, transferred onto a graphite substrate, shows also © 1997 American Chemical Society
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domains formation at the same surface pressures. The AFM images show that the enzyme is a tetramer having a spherical form. Material and Methods Acetylcholinesterase (EC 3.1.1.7: V-S from electric eel) was purchased from Sigma Chemical Co. (St. Louis, MO). The enzyme was used as purchased without further purification. Fresh AChE solutions were prepared at the day of experiments at a concentration of 1 mg/mL; the buffer solution was 0.1 M KH2PO4 and 0.1 M NaOH (pH 6.5). The solid enzyme was weighed using a vacuum electrobalance system (ATI-Cahn C-2000, ATI Mattson, Unican, & Cahn, Madison, WI). Surface potential and surface pressure measurements were carried out simultaneously using a subphase solution containing 10 mM KCl as an electrolyte, 0.1 M KH2PO4, and 0.1 M NaOH as buffer (pH 6.5). The water was purified by a Modulab 2020 water purification system (Continental Water Systems Corp., San Antonio, TX); it has a specific resistivity of 18 MΩ cm and a surface tension of 72.6 mN/m at 20 ( 1 °C. The injected volume of the enzyme solution was 150 µL for the surface potential, the surface pressure, and the Langmuir-Blodgett films preparation and 80 µL for the Brewster angle experiments. The compression rate was set up at 5 Å2 molecule-1 min-1. All the experiments were conducted in a clean room class 1000 where the temperature (20 ( 1 °C) and the humidity (50 ( 1%) are controlled. Different Langmuir troughs have been used in this work depending on the type of experiments performed. The Langmuir trough used for the surface pressure and the surface potential measurements is a homemade trough. Two symmetrically movable barriers controlled by the computer are used to regulate the surface area. The dimensions of this trough are 0.6 × 12 × 100 cm3. The surface pressure was measured by the Wilhelmy method. The surface potential was measured using the ionizing electrode method as described previously.13 A reference platinum electrode was immersed in the reference trough compartment and an americium electrode (241Am) was placed about 1-2 mm above the water subphase. The ionization was concentrated in the air gap between the subphase and the electrode surface in order to provide conduction between the platinum and the americium electrodes. The measurements of the surface potential in conjunction with the surface pressure were reproduced using different solutions of AChE. The isotherm curves shown in the result section represent an average of five measurements. For the Brewster angle measurements, a Nippon trough, equipped with a moving wall system (NL-LB140S-MWC, Nippon Laser & Electronics Lab., Nagoya, Japan), was used, to which is connected a Brewster angle microscope (BAM) (EMM633S, Nippon Laser & Electronics Lab., Nagoya, Japan), a helium-neon laser (wavelength 632.8 nm and power 10 mW), and a CCD camera. The images from the CCD were captured and digitized into a computer using a digital video capture mode (Snappy video Snapshot, Rancho Cordova, CA) for further analysis or printout. The Nippon trough has dimensions of 0.8 × 5 × 44 cm3. The surface morphology of the enzyme monolayer was visualized and recorded during the compression and the decompression using the BAM. Then, the images were analyzed at two surface pressures (0 and 20 mN/m) using the digital video capture mode mentioned above. The threedimensional (3D) representations of the BAM images were also investigated using a software NIH 1.62 image that allowed us to reduce noise using a low-pass filter. These 3D images were considered as an evidence of the presence of different brightness that could be observed on the surface. Thus, the dark areas of
Figure 1. Surface pressure (π)-molecular area (A) and surface potential (∆V)-A isotherms of AChE monolayer at the air/aqueous interface.
Figure 2. Surface potential-surface density isotherm of AChE monolayer.
the image will be translated by low picks whereas the bright area will be represented by high picks in the 3D figures. The BAM images were reproduced using three different samples of the same solution. For the Langmuir-Blodgett (L-B) film experiments, a homemade double trough was used, and it has dimension of 0.6 × 15.3 × 54.7 cm3. The deposition of one monolayer of the enzyme on graphite substrate, HOPG (highly oriented pyrolytic graphite), was made at two different surface pressures (20 and 25 mN/m) and at a dipping speed of 2 mm min-1. The AFM images were recorded 2 h later after making the deposition of the monolayer. A Nanoscope II microscope (Digital Instrument Inc., Santa Barbara, CA) was used to image the L-B AChE monolayer which was scanned in air and in constant force mode with microfabricated Si3N4 cantilevers (spring constant 0.09 N/m). The probing force was between 1 and 5 nN. The results obtained with the AFM were reproduced with three different samples prepared using the same experimental conditions. Results and Discussion Surface Pressure and Surface Potential. The surface pressure-area (π-A) isotherm of AChE is shown in Figure 1. An apparent limiting molecular area of 1.1 × 104 Å2 and a collapse surface pressure of 35 mN/m are observed. These results agree with our previous work.9 The surface potential isotherm measured simultaneously with the π-A isotherm of the AChE monolayer is presented also in Figure 1. As the monolayer is compressed and the area decreases, the surface potential increases to reach a maximum. Three different phases can be distinguished on the surface potential isotherm. During
Surface Topography of Acetylcholinesterase
Figure 3. Surface dipole moment-molecular area curve of AChE monolayer.
J. Phys. Chem. B, Vol. 101, No. 34, 1997 6743 the compression and at large area, the surface potential measured is nil. Then, at a molecular area of about 3.1 × 104 Å2, a rapid increase up to 75 mV is observed. As the monolayer is compressed, the surface potential increases gradually from 70 to 130 mV (molecular area of 1.75 × 104 Å2) where a change of the slope is observed. This change of slope is due to a sudden reorientation of the polar dipoles in the monolayer. Further compression shows that the surface potential reaches a maximum at 230 mV. At a surface potential of 140 mV, the surface pressure starts increasing. The surface potential measured reaches its maximum during the liquid phase of the monolayer and remains constant until the monolayer is compressed to its collapse surface pressure (up to 35 mN/m) where the enzyme molecules are closely packed and organized. The stability of the surface potential at this level indicates the presence of a uniform monolayer. However, upon further compression, the
Figure 4. Brewster angle images and their three-dimensional (3D) representations recorded during the compression of the AChE monolayer: (a) image recorded at 0 mN/m; (b) 3D picture of image (a); (c) image recorded at 20 mN/m; (d) 3D picture of image (c).
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Figure 5. Brewster angle images and their 3D representations recorded during the decompression of the AChE monolayer. (a) and (b) images recorded at minimum trough area; (c) and (d) 3D representations of (a) and (b), respectively; (e) image recorded at maximum trough area, and (f) 3D picture of image (e).
collapse of the monolayer and the possibility of formation of agglomerates at the interface causes a destabilization of the surface potential, leading to its decrease during the collapse. A plot of the surface potential of AChE as a function of the
surface density (Figure 2) shows that, at large molecular area (nil surface pressure), the surface potential depends on the density of molecules at the interface. At surface densities between 0.3 × 1012 and 1 × 1012, the increase of the surface
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Figure 6. AFM images of AChE LB film deposited at a surface pressure of 20 mN/m onto HOPG. The L-B films were scanned in air and the force was set up to 1-5 nN. The images size and scan rate are respectively (a) 12 × 12 µm, 5.8 Hz; (b) 6 × 6 µm, 3.5 Hz; (c) 3 × 3 µm, 3.5 Hz; and (d) 6 × 6 µm, 9 Hz.
potential with the number of molecules present at the interface is almost linear. In this region of the curve, the surface potential measurements are dependent on the number of the enzyme molecules spread on the aqueous surface. However, at a surface density of 1.2 × 1012, which corresponds to a molecular area of 8330 Å2 and a surface pressure of 14 mN/m, the surface potential reaches a plateau and remains constant upon further compression. The surface potential is no more dependent on the molecular density at the air/aqueous interface. The effective dipole moment expressed in mD (µ⊥) perpendicular to the surface has been calculated using the Helmholtz equation14
µ⊥ ) A∆V/12π where A is the area per molecule expressed in Å2, ∆V the surface potential given in mV, and µ⊥ the surface dipole moment expressed in mD. Figure 3 shows the surface dipole moment as a function of the molecular area. The dipole moment µ⊥ varies under compression of the monolayer. Thus, as it can be seen in this figure, it increases gradually as the area decreases,
and at molecular area between 1.1 × 104 and 1.3 × 104 Å2, it passes through a maximum (+60 D). Upon further compression, the effective dipole moment decreases to +20 D at 3500 Å2. The positive values of the surface dipole moment indicate that the positive ends of the dipoles were being oriented upward. The surface potential corresponds to measurements of the orientation of dipoles at the interface. It is more likely that, during the compression, all the molecular dipoles of the enzyme were being oriented more vertically; then, the side chains of the molecules start organizing themselves on the surface by orienting their polar side chains to the aqueous subphase and the nonpolar groups directed into the air to form a closely packed and stable monolayer. Furthermore, the observed trend of the surface dipole moment with the molecular area indicates that the changes in the tilt angle (the angle with respect to the vertical) and the hydrophobic part of the monolayer do not occur simultaneously. The fluctuations of the surface potential observed at large area can be explained by the presence of dispersed domains at the interface. After further compression, the domains become larger, leading, therefore, to formation of a closely packed and
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Figure 7. AFM images of AChE LB film deposited at a surface pressure of 25 mN/m onto HOPG. The L-B films were scanned in air and the force was set up to 1-5 nN. The images size and the scan rate are respectively (a) 6 × 6 µm, 4 Hz; (b) 3 × 3 µm, 4 Hz; (c) 1.2 × 1.2 µm, 4 Hz; and (d) 600 × 600 nm, 9 Hz. The arrows show the ellipsoidal shape of the enzyme.
condensed AChE monolayer. Consequently, the surface potential reaches a plateau, indicating that the film is becoming more uniform at low molecular area. Brewster Angle Microscopy. The images recorded using the BAM during the compression of the enzyme are shown in Figures 4 and 5. The images were analyzed at two different surface pressures (0 and 20 mN/m) and during the decompression of the monolayer. In all images, we note areas with different brightness in the images due to the different molecular density which correlates with the thickness of the monolayer at the interface. During the compression of the monolayer (from a molecular area of 1.5 × 104 to 1 × 104 Å2) and at nil surface pressure (Figure 4a), we observe different domains with different sizes dispersed at random on the aqueous surface. Thus, while compressing the enzyme monolayer at large molecular area, the spread molecules start regrouping and form small domains, but dark areas are still present. The 3D representation (Figure 4b) of the image in Figure 4a confirms the presence of the different brightness observed. The highest picks correspond to the presence of the enzyme clusters on the surface while the lower part in the 3D Figure results from the presence of the subphase
only or a low surface density. These BAM data agree with the surface potential results in the fact that at large molecular area, i.e., nil surface pressure, there is formation of islands on the aqueous subphase leading to the variations observed on the surface potential isotherm. The image shown in Figure 4a was taken at nil surface pressure and at a molecular area around 1.3 × 104 Å2, which corresponds to a surface potential value of 185 mV (see Figure 1). The number of molecules present on the surface at this area was calculated and found to be similar to the number present when the surface potential is reaching its maximum. This may confirm the fact that the AChE molecule dipoles are all oriented on the surface, and the monolayer starts to form before detection of a surface pressure. Upon further compression, the domains start growing and the patches become larger. At a surface pressure of 20 mN/m (molecular area 0.7 × 104 Å2), the growth of solid domains is observed (Figure 4c). The dark areas disappeared, and large domains covered almost all the surface. Therefore, a surface pressure of 20 mN/m, which corresponds to the plateau value of the surface potential (Figure 1), can be considered as the end point of the liquid expanded phase and the beginning of
Surface Topography of Acetylcholinesterase formation of a more condensed enzyme monolayer. As it can be seen in Figure 4d, the 3D representation of the image in Figure 4c confirms the fact that the domains are growing as the monolayer is compressed to low molecular area; the layer becomes thicker on the surface, and the dark areas disappeared. Thus, at 20 mN/m, a large part of the aqueous surface is covered by the enzyme particles, and the polar groups are all oriented at the air/aqueous interface. BAM images have also been recorded during the decompression of the monolayer. As it is shown in Figure 5a,c, we observe that the formed domains of larger size start to break down on the surface upon decompression of the monolayer. The large domains formed at high surface pressure (20 mN/m) dissociate into different clusters and spread on the surface when the AChE monolayer was decompressed to nil surface pressure and maximum trough area (Figure 5e). Figure 5b,d,f shows, in 3-dimensionnal representation, the process of separation of the domains during the decompression. At maximum trough area, the domains disaggregated completely and form very small patches which are dispersed on the surface. These data indicate that the BAM allows us to detect a reversible formation of domains upon compression and decompression of the enzyme monolayer at the air/aqueous interface. Atomic Force Microscopy. The AChE L-B films examined with the AFM in this study were deposited at two different surface pressures, i.e., 20 and 25 mN/m. The surface topography of the L-B films deposited at 20 mN/m is shown in Figure 6. The AFM images (a) 12 × 12 µm, (b) 6 × 6 µm, and (c) 3 × 3 µm show a punched L-B films. The gaps formed in these L-B films are wide, and their shapes tend to resemble the ellipse. The gap dimensions with respect to the ellipse axis are 610-2345 × 305-1804 nm, and the distance between two gap boundaries varies between 302 and 1053 nm. The monolayer thickness was measured using the height difference between the top of the L-B monolayer and the substrate surface which is accessible through the gap area. To our surprise, we found that the monolayer thickness changes with the scan size of the AFM images. The thickness of the AChE monolayer estimated from image (a) ranges between 3.0 and 4.0 nm, whereas the one estimated from images (b) and (c) is much greater, 6.0 and 7.0-8.0 nm, respectively. This increase of the L-B film thickness is probably caused by the lateral movement of the AFM tip. We believe that it is the S raster scanning of the tip that causes the agglomeration of the L-B film, resulting in the increase of the thickness. Therefore, the shape of the gaps and the large distribution of the distance between two gaps can be considered as a consequence of the L-B film elasticity. The merging of two gaps observed at the bottom and top of the AFM image (d) (6 × 6 µm) can also be considered as evidence of the L-B film elasticity. These AFM results (Figure 6) are in agreement with the BAM images recorded at 20 mN/m (Figure 4c). Thus, at this surface pressure (molecular area 0.5 × 104 Å2), there is formation of large domains of the enzyme monolayer on the surface. In Figure 7, the AFM images present the surface topography of AChE Langmuir-Blodgett films deposited at a surface pressure of 25 mN/m. In images (a) (6 × 6 µm), (b) (3 × 3 µm), and (c) (1.2 × 1.2 µm), the organization of AChE particles is not as well good as one expects in an L-B film. However, their distribution in the film is uniform. Also seen in these images are the graphite steps which have ∼4.0 nm height. We should mention that this type of structure has been observed recently in the case of the protein cytochrome f L-B films deposited at 15 mN/m.15 Size measurement carried out in Figure 7c indicates that
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Figure 8. AFM images of AChE LB films deposited at a surface pressure of 25 mN/m onto HOPG. The L-B films were scanned in air and the force was set up to 1-5 nN. The images size and the scan rate are respectively (a) 6 × 6 µm, 3.5 Hz and (b) 3 × 3 µm, 3.5 Hz.
AChE particles can be grouped in three different categories. The larger and medium sized particles are shaped like an ellipsoid, and their dimensions with respect to the ellipsoid axis are 68-84 × 32-44 × 7-8 and 36-48 × 21-26 × 2-3 nm, respectively. The small particles of AChE are the most abundant population of the L-B film, and their average size is 25 × 18 × 1.6 nm. Compared to the former categories, the shape of the AChE particles in the last category is also ellipsoidal. The above values represent the apparent dimensions of the particles and tip broadening must be considered in order to estimate their dimensions. In this study, we have used standard AFM tips with radius of 20-40 nm, and using the following dependence
d′ ) x2rd where d′ is the apparent dimension of the particle, d is the dimension of the particle, and r is the tip radius, the dimensions of the larger and medium sized particles would be 8 × 15 × 7
6748 J. Phys. Chem. B, Vol. 101, No. 34, 1997 and 5 × 9 × 3 nm. On the other hand, the dimensions of the small AChE particles are 5 × 7 × 2 nm. Even AChE monolayer transferred at high surface pressure (25 mN/m), we observe surface defects (dark areas) still present in the L-B film (see Figure 7d, 600 × 600 nm). Moreover, only few L-B films that were prepared at 25 mN/m are punched. In Figure 8, the AFM images (a) (6 × 6 µm) and (b) (3 × 3 µm) show a sparse distribution of small gaps in the L-B film. In this case, the shapes of the gaps resemble a circle, and their average diameter ranges between 72 and 214 nm. Also, the average thickness of the monolayer obtained using the method mentioned above is ∼2.4 nm. This value is within the range of the thickness estimated from the AFM image represented in Figure 6a. The ellipsoidal shape of AChE particles observed with the AFM is in agreement with the three-dimensional structure determined by X-ray analysis.16 As we already mentioned, globular AChE molecules, in solution, might exist in the form of monomer, dimer, or tetramer.8 Considering this observation, the sizes of the small particles observed by the AFM are within the range of the dimensions estimated by X-ray data for an AChE monomer (175 000 Å3).16 On the other hand, the dimensions obtained with the AFM (720 000 Å3) for the large and medium size of AChE particles are within the range of the estimated dimension of an AChE tetramer (700 000 Å3), using the monomer dimensions obtained by the X-ray analysis.16 Therefore, we consider that the most abundant population of the L-B film is the globular AChE monomer. Moreover, the larger and medium sized particle of AChE represent the tetramer form of this enzyme. Conclusion The surface potential and surface dipole moment show that a reorientation of the polar dipoles occur before any orientation of the hydrophobic groups on the subphase is observed. Furthermore, the surface potential data indicate that there is formation of domains at the air/aqueous interface. Microscopic surface topography of acetylcholinesterase has been also investigated using the Brewster angle and the atomic force
Dziri et al. microscopies. The BAM measurements enable us, by visualizing the surface of the monolayer, to conclude that there is formation of domains while the monolayer is compressed. The size of these domains increases with decreasing the molecular area. Furthermore, the domains formed are shown to be reversible which is expressed by the fact that a decompression of the monolayer shows a disaggregation of the large domains observed during the compression. At the end of the decompression, we observe small clusters. Using AFM, we were able to show that the shape of the enzyme is globular. Also, the size of the particles has been measured and found to be comparable to a monomer and tetramer forms of AChE. Acknowledgment. This work was supported by the Department of the Army under Contract 36479-CH. References and Notes (1) Skladal, P.; Pavlik, M.; Fiala, M. Anal. Lett. 1994, 27, 29-40. (2) Stoytcheva, M. Anal. Lett. 1994, 27, 3065-3080. (3) Stoytcheva, M. Electroanalysis 1995, 7, 660-662. (4) Mionetto, N.; Marty, J. L.; Karube, I. Biosens. Bioelectron. 1994, 9, 463-470. (5) Stein, K.; Schwedt, G. Anal. Chim. Acta 1993, 272, 73-81. (6) Wollenberger, U.; Setz, K.; Scheller, F. W.; Loffler, U.; Gopel, W.; Gruss, R. Sens. Actuators, B 1991, 4, 257-260. (7) Wang, J.; Lin, Y.; Eremenko, A. V.; Kurochkin, I. N.; Mineyeva, M. Anal. Chem. 1993, 65, 513-516. (8) Silman, I.; Futerman, A. H. Eur. J. Biochem. 1987, 170, 11-22. (9) Dziri, L.; Puppala, K.; Leblanc, R. M. J. Colloid Interface Sci., in press. (10) MacRitchie, F. J. Colloid Interface Sci. 1985, 105, 119-123. (11) Chudinova, G. K.; Pokrovskaya, O. N.; Savitskii, A. P. Russ. Chem. Bull. 1995, 44, 1958-1962. (12) Maksymiw, R.; Walter, N. J. Colloid Interface Sci. 1991, 147, 6777. (13) Lamarche F.; Max, J. J.; Leblanc, R. M. Surface Characterization of Biomaterials; Ratner, B. D., Ed.; Elsevier Science Publishers B. V.: Amsterdam, 1988; pp 117-133. (14) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interface; Interscience Publishers: New York, 1966; p 75. (15) Tazi, A.; Boussaad, S.; Leblanc, R. M. Langmuir, submitted for publication. (16) Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 253, 872-879.
