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Langmuir 2001, 17, 878-882
Protein Adsorption onto Auto-Assembled Polyelectrolyte Films Guy Ladam,†,‡ Pierre Schaaf,*,†,§ Fre´de´ric J. G. Cuisinier,‡ Gero Decher,† and Jean-Claude Voegel‡ Institut Charles Sadron (CNRS-ULP), 6, rue Boussingault, 67083 Strasbourg Cedex, France, Fe´ de´ ration de Recherche “Odontologie” U424 INSERM-ULP, Universite´ Louis Pasteur, 11, rue Humann, 67085 Strasbourg Cedex, France, and E Ä cole Europe´ enne de Chimie, Polyme` res et Mate´ riaux de Strasbourg, 25, rue Becquerel, 67087 Strasbourg Cedex 2, France Received September 12, 2000. In Final Form: November 20, 2000 We investigate the adsorption processes of a series of positively and negatively charged proteins onto the surface of polyelectrolyte multilayers. We find that proteins strongly interact with the polyelectrolyte film whatever the sign of the charge of both the multilayer and the protein. When the charges of the multilayer and the protein are similar, one usually observes the formation of protein monolayers which can become dense. We also show that when the protein and the multilayer become oppositely charged, the adsorbed amounts are usually larger and the formation of thick protein layers extending up to several times the largest dimension of the protein can be observed. Finally, we find that proteins are mainly adsorbed in a strong way on polyelectrolyte multilayers and protein surface diffusion is strongly suggested. Our results confirm that electrostatic interactions play an important role in polyelectrolyte multilayer/ protein interactions.
1. Introduction The buildup of intentionally ordered protein systems constitutes one of the major objectives of biorelated chemistry and biotechnology. Such a nanoconstruction also constitutes the frontier domain between materials and life science. Thus, spatially organized protein layers within supramolecular aggregates can play major roles in biological systems by inducing for example time-delayed specific responses at the cellular level. In this respect, a concept has recently been reported aimed at fabricating multilayers by the consecutive adsorption of positively and negatively charged polyelectrolytes.1,2 Proteins,3-6 some examples of which are immunoglobulin G, antiimmunoglobulin G, cytochrome C, lysozyme, myoglobin, hemoglobin, peroxidase, and glucose oxidase, can be inserted within such architectures, and films can be constructed in which the same or different proteins are embedded at different depths in the film architecture. There are examples in which two oppositely charged proteins separated by at least one polyelectrolyte layer pair were inserted in the film.3,4 In addition, in the case of antibodies inserted in the polyelectrolyte film, it was shown that the embedded proteins kept their reactivity with respect to their antigenic reaction when no more than four polyelectrolyte layers were deposited over them.5 * To whom correspondence should be addressed. Phone: (33) 3 88 41 40 12. Fax: (33) 3 88 41 40 99. E-mail:
[email protected]. † Institut Charles Sadron (CNRS-ULP). ‡ Fe ´ de´ration de Recherche “Odontologie” U424 INSERM-ULP, Universite´ Louis Pasteur. § E Ä cole Europe´enne de Chimie, Polyme`res et Mate´riaux de Strasbourg. (1) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831. (2) Decher, G. Science 1997, 277, 1232. (3) Lvov, Y.; Ariga, K.; Kunitake, T. Chem. Lett. 1994, 6117. (4) Lvov, Y.; Ariga, K.; Ichinoze, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (5) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427. (6) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinoze, I.; Kunitake, T. Langmuir 1998, 14, 4559.
The same holds for the interaction of embedded streptavidin with biotin.7 Moreover, the biological activity was found to be additive when the antibodies were inserted between different outer polyelectrolyte layers. This property offers, for example, large potentiality in the design of bioactive biomaterials aimed at inducing different biological responses at the cellular level. One also has to consider that such materials are developed with the perspective of being introduced on the surface of human repair devices or at least to be put in close contact with various biofluids which also implies interactions and adsorption of proteins on the terminating polyelectrolyte layers. However, only few quantitative data are available for protein adsorption onto such films.3-8 In a previous publication,9 we investigated the adsorption of negatively charged human serum albumin (HSA) onto a positively charged PEI-(PSS-PAH)3 or a negatively charged PEI(PSS-PAH)3-PSS polyelectrolyte film. We observed that HSA adsorbs on both types of surfaces and that on PSSterminating films which are similarly charged as albumin only monolayer adsorption of HSA was found whereas on the oppositely charged PAH-terminating films the adsorbed protein layers extended over thicknesses larger than 4 times the largest dimension of the HSA molecule. We also demonstrated that the intermolecular interactions involved in these processes are mostly of electrostatic origin. The aim of the present report is to investigate the protein coverage in the classical adsorption plateau domain at pH 7.35 for a series of negatively and positively charged proteins on polyelectrolyte films terminating with both negatively and positively charged polyelectrolytes. The goal was also to determine whether the charges of the proteins and the multilayer surface constitute an essential (7) Cassier, T.; Lowack, K.; Decher, G. Supramol. Sci. 1998, 5, 309. (8) Keller, S. W.; Kim, H.-N.; Mallouk, T. E. J. Am. Chem. Soc. 1994, 116, 8817. (9) Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G. Biomacromolecules 2000, 1, 674.
10.1021/la0013087 CCC: $20.00 © 2001 American Chemical Society Published on Web 01/12/2001
Protein Adsorption onto Polyelectrolyte Films
parameter in protein adsorption onto polyelectrolyte multilayers and if some results previously reported for albumin adsorption can be generalized. 2. Materials and Methods All the adsorption experiments were performed at pH 7.35. We thus used as negatively charged proteins R-lactalbumin (RLA, MW 14 200 Da, pHi 4.3, Sigma, Type III, cat. no. L-6010), human serum albumin (HSA, MW 69 000 Da, pHi 4.6), myoglobin (MGB, MW 17 800 Da, pHi 7.0, Sigma, cat. no. M-0630), and R1-acid glycoprotein (RAGly, MW 44 100 Da, pI 2.7, Sigma, cat. no. G-9885) and as positively charged proteins ribonuclease A (RNase, MW 13 680 Da, pHi 9.4, Sigma, cat. no. R-5000) and lysozyme (LSZ, MW 14 600 Da, pHi 11.1, Sigma, cat. no. L-6876). The HSA was provided in concentrated solutions (20 g/100 mL) for intravenous injection (prepared and purified by Cohn’s method;10,11 its purity was controlled by gel electrophoresis). All proteins were used without further purification. Anionic poly(sodium 4-styrenesulfonate) (PSS, MW 70 000, cat. no. 24,305-1), cationic poly(allylamine hydrochloride) (PAH, Mn 50000-65000, cat. no. 28,322-3), and cationic poly(ethyleneimine) (PEI, MW 750 000, cat. no. 18,197-8) were purchased from Aldrich (France). Sodium chloride was purchased from Fluka (France), and trishydroxyaminomethane (Tris) was also purchased from Sigma. All the chemicals of commercial origin were used without further purification. The buffer and protein solutions were prepared, and the cleaning steps were realized with ultrapure water (Milli-Q-plus system, Millipore, Bedford, MA). The resistivity of the water was approximately 18.2 MΩ. cm. The technique used to follow the adsorption experiments, scanning angle reflectometry (SAR), and the experimental approach (cleaning and polyelectrolyte film buildup) are similar to those reported in former publications.9,12,13 Briefly, all experiments were carried out in 10-2 M Tris buffer adjusted to pH 7.35 with HCl and 0.15 M NaCl. All the buffer solutions were degassed under vacuum before use. The polyelectrolyte multilayers were built up from polyelectrolyte solutions prepared by dissolving 0.5 g of polyelectrolyte (PEI, PSS, PAH) in 0.1 L of Tris buffer. Similarly, the protein solutions were obtained by dissolving the proteins in the Tris buffer, with the protein concentrations ranging from 20 to 25 mg/100 mL. The concentrations were precisely determined by UV spectroscopy absorbance at 280 nm. The polyelectrolyte multilayer and protein films were built up as follows: The silica surface of the prism of the scanning angle reflectometry cell was first brought in contact with the buffer solution in order to determine the reflectivity curve in the absence of any deposited film. This curve was used to calibrate the reflectometer. The buffer solution was then replaced by the PEI solution which was left in contact with the silica surface for more than 30 min before being replaced again by pure buffer. After the adsorption of this first PEI layer, a similar procedure was repeated by using instead of a PEI solution alternatively a PSS or a PAH solution. The buildup of the film was stopped after the deposition of either a positively charged PEI-(PSS-PAH)3 or a negatively charged PEI-(PSS-PAH)3-PSS polyelectrolyte multilayer. At this stage, the reflectivity curves were determined for about 16 h, allowing the film to reach full equilibrium. The reflectivity curves corresponding to these polyelectrolyte multilayer films before protein adsorption were analyzed by assuming that they behave as homogeneous and isotropic monolayers of thickness L1 and of refractive index n1 (∆n1 ) n1 - nsol, where nsol is the refractive index of the solution in contact with the film). The typical thickness and refractive index of the PEI(PSS-PAH)3 films were 17.4 ( 0.8 nm and 1.4865 ( 0.002, respectively, and those of the PEI-(PSS-PAH)3-PSS films were 20.4 ( 0.7 nm and 1.492 ( 0.002, respectively.9 (10) Cohn, E. J.; Strong, L. E.; Hughes, W. L.; Mulford, D. J.; Ashworth, J. N.; Melin, M.; Taylor, H. L. J. Am. Chem. Soc. 1946, 68, 459. (11) Cohn, E. J.; Hughes, W. L.; Weare, J. H. J. Am. Chem. Soc. 1947, 69, 1753. (12) Schaaf, P.; De´jardin, P.; Schmitt, A. Langmuir 1987, 3, 1131. (13) Ladam, G.; Schaad, P.; Voegel, J. C.; Schaaf, P.; Decher, G.; Cuisinier, F. J. G. Langmuir 2000, 16, 1249.
Langmuir, Vol. 17, No. 3, 2001 879 We then replaced the buffer solution that was in contact with the film by the protein solution. During the contact between the protein solution and the film, reflectivity curves were determined continuously. When the adsorption process leveled off, we replaced the protein solution by pure buffer and again determined the reflectivity curve. The optical data concerning the multilayer/ protein films were analyzed within the framework of a double layer, characterized by two thicknesses L1 and L2 and by two refractive index excesses ∆n1 and ∆n2. The whole procedure has been validated, and the approximations have been justified by an analysis of the data within the framework of the optical invariants which has been extensively discussed in ref 9. It was shown, in particular, that the monolayer model could not correctly account for the optical data of an architecture constituted by albumin adsorbed on a polyelectrolyte multilayer and the bilayer model constitutes the most simple approximation for the profiles of polyelectrolyte multilayer/protein films. However, because scanning angle reflectometry usually allows access to only two optical parameters we had to maintain as constants two parameters of the bilayer. We chose, as a reasonable procedure, to maintain the thickness L1 and the refractive index excess ∆n1 identical to the values found for the polyelectrolyte film before protein adsorption and to use L2 and ∆n2 as adjusting parameters by the standard method of Abe´le`s.14 The comparison between the experimental reflectivity curves and the theoretical ones was realized by means of a minimization procedure of the quality fit parameter χ2 using the Simplex nonlinear least-squares fitting procedure.15 The quality fit parameter is defined as
χ2 )
1
∑
N
i
(
)
fexp(θi) - fmod(θi) 0.01fexp(θi) + σ0
2
(1)
where fexp(θi) and fmod(θi) represent the measured reflected intensity and the reflected intensity calculated by assuming the bilayer model, respectively. The sum runs over all the N incidence angles, and σ0 corresponds to the baseline noise or roundoff error.
3. Results and Discussion Adsorption kinetics on both surface types was investigated by injecting first (20 min) a protein solution at a concentration of 20-25 mg/100 mL, followed by a second step during which the solution was maintained at rest in contact with the surface. During this second step, the adsorption process was thus governed by the multilayer/ protein interactions and by the diffusion process in the solution. Figure 1 represents typical adsorption kinetics of different proteins adsorbing onto multilayer surfaces terminating with PSS and PAH. Because of the resolution time of the technique (the determination of a reflectivity curve takes about 3 min) and also because of the initial protein injection step, the very early stages of the adsorption process cannot be precisely followed. One can first notice that both positively and negatively charged proteins adsorb on both negatively and positively charged multilayer surfaces. This constitutes a general result as can be seen in Figure 2 which represents the adsorbed amounts and the optical thicknesses of the adsorbed layers on both types of films as a function of the isoelectric point (pHi) of the different proteins. The presented data correspond to the end of the adsorption kinetics. From Figure 2, it can be concluded that the adsorbed amounts and the protein layer thicknesses are larger when the proteins and the terminating polyelectrolyte layer are oppositely charged compared to protein adsorption onto films terminating with a similarly charged polyelectrolyte layer. (14) Abe´le`s, F. Ann. Phys. 1950, 5, 596. (15) Press, W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. Numerical Recipes in Pascal; Cambridge University Press: New York, 1990.
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Figure 1. Adsorption kinetics of RLA (b), MGB (O), LSZ (1), and Rnase (3) onto polyelectrolyte multilayers: adsorbed protein amounts Γ (µg cm-2) on (a) PEI-(PSS-PAH)3 and (b) PEI-(PSS-PAH)3-PSS films. The adsorption processes were performed with protein solutions at concentrations of 20-25 mg/100 mL at pH 7.35 in the presence of 0.15 M NaCl. Arrows indicate the beginning of the rinsing process by means of the buffer solution at pH 7.35 in the presence of 0.15 M NaCl.
This result becomes even clearer in Figure 3 where (ΓPAH - ΓPSS)/max(ΓPAH, ΓPSS) is plotted versus the pHi. ΓPAH and ΓPSS represent the highest adsorbed amounts on the PAHor PSS-terminating multilayers, respectively. This shows that polyelectrolyte/protein electrostatic interactions play an important role in these adsorption processes. This result confirms the conclusions relative to the importance of electrostatic interactions drawn from the study of the HSA adsorption on similar polyelectrolyte multilayer films as a function of the ionic strength of the protein solution in contact with the polyelectrolyte film during adsorption.9 We found that on PAH-ending multilayers the adsorbed HSA amount passes through a maximum for an NaCl concentration of 10-1 M at pH 7.4, whereas it increases slightly with the NaCl concentration on PSS-ending multilayers, as is expected from a charge screening effect. The fact that proteins still adsorb onto similarly charged polyelectrolyte films is not unexpected because proteins bear on their surfaces domains with both positive and negative surface excess charges. Moreover, it is not excluded that on PSS-terminating multilayers some PAH chains can emerge at the outer surface and are thus also able to interact with the proteins; the same is true for PAH-terminating multilayers and PSS chains. The amounts of adsorbed proteins as well as the largest and smallest surface coverages estimated from the largest and smallest projected dimensions of the protein are gathered in Table 1. On cationic PAH-terminating films, the adsorption of negatively charged RLA, HSA, and MGB are not compatible with a monolayer coverage and point toward multilayer formation. In the case of positively charged LSZ adsorbing onto a film terminating with PSS, the adsorbed amounts are larger than those compatible
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Figure 2. (a) Protein layer thickness L (nm) and (b) adsorbed protein amounts Γ (pmol cm-2) onto polyelectrolyte multilayers as a function of pHi: (b) PEI-(PSS-PAH)3 or (O) PEI-(PSSPAH)3-PSS films. The adsorption processes were performed with protein solutions at concentrations ranging between 20 and 25 mg/100 mL at pH 7.35 in the presence of 0.15 M NaCl. Γ was calculated from (∆n)L by using a dn/dc value of 0.18 cm3/g.
with a monolayer. This is particularly striking for HSA and RLA which correspond to protein layers whose extension represents more than 3 times the largest dimension of the proteins (Figure 4). HSA is thus not the only protein showing such a somewhat unusual behavior.9 The formation of such thick protein layers implies attractive protein/protein interactions. However, proteins usually repel each other in solution. Two explanations were proposed for HSA which may also hold for other proteins. The formation of thick protein layers can be due to cooperative attractive interactions consecutive to a protein ordering on the surface of a polyelectrolyte multilayer. This could then lead to a first protein layer adsorbed in such a way onto an oppositely charged polyelectrolyte film as to exhibit itself a net surface charge which again acts as an attractive surface for further proteins from the solution. Subsequent protein layers can thus be similarly attached. However, as the process evolves the protein layer becomes less structured so that the surface excess charge decreases and the process stops. This adsorption process would thus be the result of the interplay between transport and reorganization mechanisms which lead to a structuration of the adsorbed protein layer. In a second explanation, one can also assume that proteins are somehow entangled in the polyelectrolytes of the outer polyelectrolyte layer so that some polyelectrolytes of this layer are also present at the outer part of the
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Langmuir, Vol. 17, No. 3, 2001 881
Table 1. Experimental Data for Various Proteins Adsorbing onto PEI-(PSS-PAH)3 or PEI-(PSS-PAH)3-PSS Films at pH 7.35a protein concentration (mg/100 mL)
L (nm)
n
Γ (µg cm-2)
θmin, θmax (%)
Lrinse (nm)
nrinse
Γrinse (µg cm-2)
% released
1.4693 1.4685 1.4551 1.4677
0.80 0.81 0.10 0.16
5.9 7.0 45.5 22.2
20.1b 21.4b 18.4c 19.5c
11.59 11.81 2.72 2.90
RLA: 3.7 × 3.2 × 3.5 nm3/14 200 g mol-1/pI ) 4.3 1.4661 0.85 404-467 10.66 1.4666 0.87 414-479 10.85 1.4547 0.18 87-101 1.50 1.4595 0.20 95-110 2.13
25.6b 24.3b 24.4c 24.9c
3.98 3.48 5.42 5.48
LSZ: 4.6 × 3.0 × 3.0 nm3/14 600 g mol-1/pI )11.1 1.4523 0.26 97-149 2.85 1.4497 0.22 82-126 2.38 1.4868 0.46 171-262 3.74 1.4866 0.47 173-266 3.95
1.4566 1.4523 1.4888 1.4839
0.19 0.16 0.32 0.33
25.5 30.0 30.1 29.8
∼25b ∼25b ∼25b ∼25c ∼25c
2.78 2.80 2.26 3.99 3.90
RNase: 3.8 × 2.8 × 2.2 nm3/13 680 g mol-1/pI ) 9.4 1.4642 0.20 54-94 1.89 1.4643 0.21 56-97 2.25 1.4693 0.17 45-78 1.74 1.4719 0.31 83-143 2.53 1.4718 0.30 81-140 2.54
1.4670 1.4707 1.4742 1.4780 1.4777
0.14 0.17 0.13 0.20 0.21
30.6 16.2 20.0 34.5 31.5
∼25b ∼25b ∼25c ∼25c ∼25c
5.78 4.74 3.94 3.84 4.05
MGB: 4.5 × 3.5 × 2.5 nm3/17 800 g mol-1/pI ) 7.0 1.4768 0.46 136-246 5.53 1.4802 0.38 113-204 4.52 1.4596 0.28 82-148 1.4537 0.26 76-136 3.37 1.4566 0.28 82-148 3.77
1.4769 1.4800
0.44 0.37
4.8 4.3
1.4518 1.4545
0.22 0.26
13.0 8.0
23.89b 22.74c
44.79 8.95
HSA: 12 × 2.7 × 2.7 nm3/69 000 g mol-1/pI ) 4.6 1.4498 2.91 184-821 43.00 1.4488 1.4359 0.51 33-145 8.52 1.4344
2.77 0.48
4.8 6.5
22.97b 23.02c 22.18d
6.40 1.94 8.09
RAGly: ellipsoid, a ) 6 nm, b ) 4 nm/44 100 g mol-1/pI ) 2.7 1.4347 0.36 79-118 5.17 1.4305 1.4269 0.10 22-33 1.17 1.4197 1.4454 0.50 109-164 7.25 1.4453
0.28 0.06 0.45
23.1 44.4 10.0
a L, n, and Γ and L rinse, nrinse, and Γrinse correspond to the protein layer thickness, the protein layer refractive index, and the adsorbed protein amount at the end of the adsorption kinetics and after rinsing with the buffer solution, respectively. θmin and θmax are the lowest and highest surface coverage percentage estimated from the smallest or the largest projected dimensions of the protein molecule. The last column corresponds to the percentage of proteins released after a buffer rinse of the adsorbed layer. b Adsorption onto PEI-(PSS-PAH)3 films at pH 7.35. c Adsorption onto PEI-(PSS-PAH)3-PSS films at pH 7.35. d Adsorption onto PEI-(PSS-PAH)3-PSS films at pH 5.3.
Figure 3. Influence of the protein charges on the differences between the protein amounts adsorbed onto PEI-(PSS-PAH)3 and PEI-(PSS-PAH)3-PSS films. (ΓPAH - ΓPSS)/max(ΓPAH, ΓPSS) was plotted as a function of the isoelectric point pHi of the protein. ΓPAH - ΓPSS is the difference between the protein amounts uptaken onto PAH- and PSS-terminating films, and max(ΓPAH, ΓPSS) corresponds for each protein to the highest amount adsorbed on either the PSS- or the PAH-terminating film.
protein layer allowing further polyelectrolyte/protein interactions. It is not expected that the proteins penetrate more deeply in the multilayer. If this was the case, one should also observe thick protein layers for systems in which both the protein and the multilayer are of similar charge, but this is never observed. The fact that only the outer polyelectrolyte layer should extend into the protein
Figure 4. L/Ldim versus pHi for the different proteins. L is the layer thickness, and Ldim is the largest dimension of the protein molecule. Adsorption was onto (b) PEI-(PSS-PAH)3 or (O) PEI-(PSS-PAH)3-PSS films.
film still allows the use of the bilayer model for the analysis of the optical data by keeping the thickness and the refractive index of the polyelectrolyte layer fixed and equal to their values determined before the addition of the proteins. One expects only a slight change in the polyelectrolyte layer thickness, and its refractive index should remain unchanged. If this second explanation for the observation of thick protein films was correct, one would, for example, expect a dependence on the molecular weight of the polyelectrolyte fixed on the surface of the film.
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Further studies are thus necessary, in particular by changing the molecular weight of the polyelectrolytes to get a deeper insight into this unexpected adsorption behavior. When proteins are adsorbed onto multilayer surfaces terminating with a polyion of equal charge (i.e., RLA, HSA, or MGB adsorbing onto films terminating with PSS and Rnase or LSZ adsorbing onto films terminating with PAH), one usually forms a protein layer whose thickness is compatible with or very close to a monolayer dimension. However, one often reaches surface coverages of 80% or 90% of the available area even when the smallest dimensions of the molecules are considered, that is, when the proteins adsorb in “end-on” configurations. Such high values can be obtained only when proteins are reversibly bound to the surface and/or can diffuse along it. Indeed, in the case of irreversible adsorption without surface diffusion one knows that the random sequential adsorption model correctly describes the adsorption process and in particular the jamming limit coverage. For noninteracting spheres, the jamming limit coverage is equal to 53%,16 and it is expected that protein/protein electrostatic repulsion would lead to even smaller coverages. To investigate the reversibility of protein adsorption, we rinsed the cell at the end of the adsorption process with pure buffer (thus having a similar ionic strength (0.15 M NaCl) as the adsorption solution) and followed the evolution of the adsorbed layer. Typical protein release processes are seen in Figure 1. Only a fraction varying between 5% and 30% of the initially adsorbed proteins are desorbable (see also Table 1), and for certain proteins (RLA, LSZ, Rnase) a slight densification of the adsorbed layer is observed. This is shown by a slight increase of the layer refractive index (Table 1). A rinse with a buffer solution of similar ionic strength as the one employed for the protein layer buildup does thus not significantly modify the structure of the adsorbed protein layers. The adsorbed proteins appear thus to interact very strongly with the terminating polyelectrolyte layer or with the polyelectrolyte/protein layer complex whatever the sign of the charge of the film. This also implies that the large coverages observed even when proteins adsorb on polyelectrolyte films of same surface charge must be due to protein diffusion along the film. The existence of such surface diffusion is indeed confirmed by recent diffusion studies of albumin on both PSS- and PAH-ending multilayers.17 One can point out that the existence of such a diffusion process would indicate that the first rather than the second explanation for the buildup of thick protein layers on oppositely charged polyelectrolyte films compared to the protein charge is valid. Indeed, a polyelectrolyte/protein layer complex in which the proteins are entangled within the polyelectrolyte film should rather inhibit protein surface diffusion. Finally, on films terminating with PAH RAGly was suspected to form very thick layers because of the important gap between its pHi value (2.7) and the experimental pH value, and experimentally we only found L/largest dimension ∼ 1.1. Two reasons can be proposed (16) Hinrichsen, E. L.; Feder, J.; Jøssang, T. J. Stat. Phys. 1986, 44, 793. (17) Szyk, L. Personnal communication.
Ladam et al.
to explain this observation. First, if the formation of thick protein layers is explained by the first proposed mechanism one can assume that the presence of numerous glycosylated groups on RAGly could hinder a structural reorganization. Second, the strong acidic pHi renders the protein highly negatively charged and salt screening is not sufficient to screen intermolecular repulsion. This is confirmed by the results with RAGly adsorption experiments at pH 5.3 in which one observes an increase in the adsorbed amount by 50% (Table 1). Adsorption kinetics of negatively (RLa, MGB) and of positively (Rnase, LSZ) charged proteins at pH 7.35 are given for adsorption on a positive (Figure 1a) or on a negative film (Figure 1b), respectively. It is observed that for the most stable proteins (LSZ, characterized by a free energy of denaturation ∆GN-D ) 4.0 J g-1, and Rnase, ∆GN-D ) 4.0 J g-1)18 the adsorbed amounts become rapidly constant and remain stable over a long time period (more than 2 h). RLA (∆GN-D ) 1.9 J g-1) behaves differently because the uptaken amount increases continuously during several hours and finally becomes constant. An intermediate behavior is found for MGB (∆GN-D ) 3.1 J g-1). The differences cannot be attributed to diffusionlimited reactions because the proteins have comparable molecular weights and thus also possess similar diffusion coefficients. A possible explanation is that the less stable proteins are able to undergo important conformational changes leading to structural reorganizations which lead to further protein adsorption. Such structural reorganizations could affect the biological activity of the adsorbed proteins. This point would deserve a study in itself. 4. Conclusion We found that the polyelectrolyte multilayers strongly interact with proteins whatever the sign of the charge of both the multilayer and the protein. When the charges of both the multilayer and the protein are the same, one usually observes within the limits of interpretation imposed by the optical model the formation of monolayers which can become dense. When the charges of the protein and the multilayer are opposite, the adsorbed amounts are usually much larger than the ones observed when proteins and polyelectrolyte films are similarly charged, and the formation of thick protein layers extending up to several times the largest dimension of the protein can be observed. Finally, proteins are mainly adsorbed in a strong way on polyelectrolyte multilayers, but protein surface diffusion is suggested. Our results confirm that electrostatic interactions play an important role in protein polyelectrolyte multilayer interactions. Acknowledgment. This work was supported by the program Adhe´sion Cellules-Mate´riaux and by the program CNRS Physique et Chimie du Vivant. It was performed within the framework of the CNRS/INSERM Research Network Me´ canismes physico-chimiques d’adhe´ sion cellulaire: forces d’adhe´ sion entre ligands et re´ cepteurs biologiques. LA0013087 (18) Privalov, P. L. Adv. Protein Chem. 1979, 33, 167.
