11906
J. Phys. Chem. B 2001, 105, 11906-11916
Stabilizing Effects of Various Polyelectrolyte Multilayer Films on the Structure of Adsorbed/Embedded Fibrinogen Molecules: An ATR-FTIR Study† P. Schwinte´ ,‡ J.-C. Voegel,‡ C. Picart,‡ Y. Haikel,‡ P. Schaaf,*,§ and B. Szalontai| Contribution from the Unite´ de Formation et de Recherches “Odontologie” U424 INSERM, UniVersite´ Louis Pasteur (ULP), 11 rue Humann, 67085 Strasbourg Cedex, France, Institut Charles Sadron (CNRS-ULP), 6 rue Boussingault, 67083 Strasbourg Cedex, France, and Institute of Biophysics, Biological Research Center (BRC), Hungarian Academy of Sciences, H-6701 Szeged, TemesVa´ ri krt. 62, P.O.B. 521, Hungary ReceiVed: June 18, 2001; In Final Form: August 13, 2001
The structural changes in fibrinogen as a consequence of its adsorption onto the surface of or its embedding into the interior of poly(allylamine hydrochloride) (PAH) or poly(styrenesulfonate) (PSS) multilayers are investigated by means of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. It is found that both adsorption and embedding preserve the secondary structure of the fibrinogen molecules. Furthermore, the interactions of the polyelectrolytes with the protein molecules prevent their aggregation, especially in the embedded state, at room temperature. Thus, it seems that the structure and the biological activity of proteins adsorbed on or embedded in polyelectrolyte multilayers could largely be preserved, which opens up great perspectives in the design of new bioactive surfaces. The nature and the extent of the polyelectrolyte-protein interactions are further studied via analysis of the thermotropic responses of the different architectures. It is found that both PAH- and PSS-terminated polyelectrolyte multilayers can elevate the onset temperature of the structural changes in adsorbed/embedded fibrinogen molecules by about 5 °C as compared with that for fibrinogen in solution. These polyelectrolytes also broaden the thermally induced structural transitions in the adsorbed/embedded fibrinogen molecules. The magnitude of these thermally induced structural changes is polyelectrolyte- and architecture-dependent. Whereas multilayer PAH-fibrinogen and multilayer PSS-fibrinogen constructions exhibit roughly the same large-scale thermally induced structural changes, in all architectures where fibrinogen is embedded the scale of these structural changes is restricted. The restriction becomes stronger as the presence of PSS at the polyelectrolyte-fibrinogen interfaces increases (PAH-fibPAH < PAH-fib-PSS ≈ PSS-fib-PAH < PSS-fib-PSS). In the PSS-fib-PSS arrangement, the secondary structure of fibrinogen as determined from its infrared spectrum changes only slightly up to 90 °C. The underlying processes of the thermally induced structural changes is, in addition, different for fibrinogen molecules adsorbed onto or embedded into PAH-terminated polyelectrolyte multilayers. A tentative model based on “encapsulation” of the embedded protein by the polyelectrolytes is proposed to explain the observed features.
Introduction During the past decade great efforts have been devoted to the development of new classes of biomaterials. Bioactive materials constitute such a class.1 These materials mainly exhibit bioactivity through biological recognition. Recognition can be obtained by incorporating active molecules in the bulk of the materials or by immobilizing them on their surfaces. This is usually achieved by covalently binding or simply adsorbing the molecules onto the surfaces. A promising new method based on the alternate adsorption of polycations and polyanions, leading to polyelectrolyte multilayers, was developed recently.2-4 The buildup of such multilayers offers great advantages over simple adsorption or covalent coupling: for instance, proteins can be easily adsorbed onto multilayers or incorporated into polyelectrolyte structures.5-8 This allows the construction of †
Part of the special issue “Howard Reiss Festschrift”. * Corresponding author. E-mail:
[email protected]. Telephone: (33) 3 88 41 40 00. Fax: (33) 3 88 41 40 99. ‡ U424 INSERM. § Institut Charles Sadron. | BRC.
complex architectures with targeted properties.9 As examples, fibronectin, fibrinogen, or vitronectin can be adsorbed onto or incorporated into these structures in order to promote cell adhesion. For these films to be bioactive, however, the secondary structure must be preserved, at least partially, when the proteins are embedded, and they must be able to diffuse laterally in/on the polyelectrolyte films. It has been demonstrated that the degree of ligand mobility along the surface strongly influences cell adhesion.10 The extent of cell spreading, for example, may depend on both the overall amount of the ligands immobilized on the surface and on their ability to cluster into spatial microdomains. The lateral mobility of proteins adsorbed on or embedded in polyelectrolyte multilayers has recently been demonstrated, the diffusion coefficient of albumin in polyelectrolyte films lying in the range 10-10 to 10-11 cm2‚s-1.11 Several studies support the idea that proteins embedded in polyelectrolyte multilayers can preserve some of their biological activity: Anti-IgG molecules embedded in polyelectrolyte films but covered by fewer than four polyelectrolyte layers retained their biological activity as measured via their binding capacity
10.1021/jp0123031 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/25/2001
Adsorbed/Embedded Fibrinogen Molecules with respect to their antigens.12 Additive activity was observed when anti-IgG molecules were inserted simultaneously between several consecutive polyelectrolyte layers. Since the antigenantibody binding requires a definite structure of the antibodies, this result suggests that the conformation of protein molecules is not fundamentally altered when they are adsorbed on or embedded in polyelectrolyte multilayers. It has also been shown that polyelectrolyte multilayers preserve enzymatic activity of different incorporated proteins.13,14 However, as yet no study has directly addressed the question of the conformational changes in proteins inserted into polyelectrolyte multilayers, and the aim of the present study is to provide an answer to this question. We therefore followed the structural changes in fibrinogen molecules in consequence of their adsorption onto polyelectrolyte surfaces or their insertion into different polyelectrolyte film architectures formed by the successive deposition of poly(styrenesulfonate) and poly(allylamine hydrochloride). Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode15 was applied to follow the structural changes in the protein and we focused mainly on the analysis of the amide I band (for a review see ref 16). The changes in the secondary structure of fibrinogen upon its adsorption/embedding were followed at room temperature and its thermal stability was also tested under these conditions. As will be shown in this paper, the secondary fibrinogen structure is little affected either by adsorption onto the surface or by embedding into the interior of polyelectrolyte multilayers. The tendency of fibrinogen to aggregate, however, is strongly reduced when it interacts with these polyelectrolytes. Moreover, the stability of fibrinogen, even against higher temperatures, can be enhanced when the proteins are embedded in polyelectrolyte multilayers. This result confirms the observed enhanced thermostability of enzymes in polyion films.17 Materials and Methods Materials. Fibrinogen (Fraction I) from human plasma was purchased from SIGMA (product number 4883 Lot 117H7602), and used without further purification. The solvent was deuterium oxide (99.9 atom % D) from Aldrich. The buffer medium, prepared form phosphate-buffered saline tablets (Sigma), contained 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride at pD ) 7.0 corresponding to pH ) 7.4. The polyelectrolytes used in this study were the positively charged poly(allylamine hydrochloride) (MW ) 50000-65000, cat. no.: 28322-3) (PAH), polyethylenimine (MW ) 750000, cat. no.: 18179-8) (PEI), and the negatively charged poly(sodium 4-styrenesulfonate) (MW ) 70000, cat no.: 243051) (PSS), all from Aldrich. Sample Preparation. For all ATR-FTIR experiments, lyophilized protein, and polyelectrolytes, were dissolved in 0.01 M deuterated phosphate buffer, at a concentration of 0.05 mg‚mL-1, and 5 mg‚mL-1. The protein concentration was chosen because it should lie in the adsorption plateau and is low enough to avoid protein aggregation in solution. For transmission absorption measurements, the same buffer was used and three types of fibrinogen solutions were prepared: a dilute one with a weighed concentration of 2 mg‚mL-1 , a solution containing 5 mg‚mL-1 protein, and a very concentrated one at 50 mg‚mL-1, obtained by hydrating a weighed amount of lyophilized fibrinogen directly in the FTIR cell with a known volume of D2O added by a syringe. Infrared Measurements. D2O was used as solvent instead of water because the amide I band of the protein is strongly affected by the appreciable absorption band of water at around
J. Phys. Chem. B, Vol. 105, No. 47, 2001 11907 1643 cm-1 (O-H bending), whereas the corresponding vibration in D2O absorbs at around 1209 cm-1.18 Conventional absorption spectra were recorded between two CaF2 windows separated by a 15 µm spacer, mounted in a liquid cell, using the DTGS detector and the sample shuttle device of an Equinox 55 spectrometer (Bruker, France). At the low protein concentration of 2 mg‚mL-1, the weak, broad combination band of the D2O solvent of the buffer solution cannot be neglected. The buffer spectrum therefore had to be fitted and subtracted from the spectrum of the fibrinogen solution. The temperature-induced changes in fibrinogen in solution were followed by recording absorption spectra on a PU9800 FTIR spectrometer (Philips, England) equipped with a computer-controlled sample shuttle and temperature setting. Reference and sample spectra at the same temperatures were taken by collecting 128 interferograms at 2 cm-1 resolution. The temperature was increased in 2-3 °C steps, a period of 7 min being allowed for thermal equilibrium. The uncertainty in the set temperature was smaller than 0.5 °C. ATR infrared spectra were taken with an Equinox 55 FTIR spectrometer, using a liquid nitrogen-cooled MCT detector. For adsorption studies, protein solution (about 3 mL) was flowed into an overhead in-compartment ATR cell (Graseby-Specac, England), equipped with a thermostabilized top-plate (110 µL cell volume) fitted with a 45° trapezoidal germanium IRE (internal reflection element) crystal (six reflections, dimensions 72 mm × 10 mm × 6 mm). Samples were introduced into the cell by a peristaltic pump at a flow rate of approximately 0.25 mL‚min-1. Single-channel spectra from 512 interferograms were calculated between 400 and 4000 cm-1 with 2 cm-1 resolution, using Blackman-Harris three-term apodization and the standard Bruker OPUS/IR software (version 3.0.4). Preparation of Polyelectrolyte Multilayers/Fibrinogen Films. For the adsorption of multilayers, polyelectrolyte solutions of 5 mg‚mL-1 were flowed through the cell (kept at 28 °C), and single-channel spectra were collected. Completion of each adsorption process was controlled by waiting until the difference absorption spectrum -log(Sn/Sn-1) calculated from two successive single-beam spectra recorded from 64 interferograms during the deposition of the layer had become equal to zero. After the deposition of each polyelectrolyte layer, the cell was rinsed with buffer. Multilayers of PEI-(PSS-PAH)3 or PEI-(PSS-PAH)3-PSS were deposited on the Ge crystal surface by successive steps of adsorption and washing of the different polyelectrolytes, with alternation of anionic and cationic species. At this point, a final single-beam spectrum obtained from 512 interferograms, was recorded. Great care was taken to avoid any contact between the multilayers and air. In particular, the multilayers were never dried, which was often not the case in other studies reported in the literature. Fibrinogen was adsorbed onto the polyelectrolyte multilayers from a circulating solution at a concentration of 0.05 mg‚mL-1. The progress of the adsorption was followed by the collection of successive single-beam spectra until the difference between two successive spectra became equal to zero, as described above. Then, after washing with buffer, 512 interferograms were again collected to determine the absorption of the adsorbed fibrinogen. This was done by calculating -log(SUL+fib/SUL), where SUL indicates the single-channel spectrum of the underlying multilayer and SUL+fib the single-channel spectrum of the multilayer with fibrinogen adsorbed on it. Experiments with Fibrinogen Embedded in Polyelectrolyte Multilayers. In another series of experiments, the fibrinogen layer deposited on polyelectrolyte films was covered with five
11908 J. Phys. Chem. B, Vol. 105, No. 47, 2001 or six supplementary polyelectrolyte layers. Four such architectures were investigated: PEI-(PSS-PAH)3-fib-(PSSPAH)3, PEI-(PSS-PAH)3-PSS-fib-(PSS-PAH)3, PEI(PSS-PAH)3-fib-(PAH-PSS)2-PAH, and PEI-(PSSPAH)3-PSS-fib-(PAH-PSS)2-PAH. The contribution of the underlying PEI-(PSS-PAH)3 or PEI-(PSS-PAH)3-PSS multilayers (UL) was subtracted from the total contribution of the “sandwich” buildup by taking -log(SUP/SUL), where SUP is the single channel spectrum of the whole film and SUL is that of the multilayers underlying the protein. Accordingly, the resulting spectrum is not that of fibrinogen alone, but that of fibrinogen together with the covering multilayer film. Fortunately, neither PSS nor PAH has a structured absorption in the range 16001700 cm-1 (the protein amide I region), except for a fine band at 1600 cm-1 characteristic of PSS. Thus, the contribution of the outer polyelectrolyte layer did not interfere substantially with the absorption spectrum of the adsorbed fibrinogen. ATR-FTIR Investigation of Heat-Induced Structural Changes in Fibrinogen. Fibrinogen adsorbed on PEI-(PSSPAH)3, or on PEI-(PSS-PAH)3-PSS, or embedded in each of the four architectures described in the previous section, was subjected to ATR-FTIR experiments as a function of temperature by means of a thermostated bath (Julabo, Germany). Control experiments were performed on naked Ge and with the two underlying multilayer structures. During all temperature change experiments, a continuous buffer flow of 15 mL‚h-1 was circulated through the thermostated cell. The temperature was increased in steps of 4 °C. At each temperature, 512 interferograms were collected and used to calculate singlechannel spectra. A period of 5 min was allowed for new thermal equilibrium to be reached before new interferograms were recorded. The absorption of the adsorbed fibrinogen was calculated as described above, by dividing the sample spectrum at the actual temperature by the appropriate single-channel reference spectrum taken at room temperature. Heating induced a reversible change in the baseline of the spectra: the baseline moved up toward lower frequencies on elevation of the temperature and this baseline alteration had to be eliminated. An interactive baseline correction (Bruker, OPUS software) was therefore performed individually for each spectrum in the temperature runs. Water vapor absorption was also subtracted from the spectra by means of an interactive OPUS function until it could no longer be detected by eye. Evaluation of the Infrared Spectra. All processing of the infrared spectra (except when indicated separately) was carried out by using the SPSERV© software (version 3.20, BCS Software: Dr. Csaba Bagyinka, Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, P.O.B. 521, Hungary). To eliminate water vapor contamination and to reduce noise, Fourier smoothing was performed on the spectra before component band analysis of the amide I band was carried out. This procedure and all details of the protein secondary structure determination are described in the Appendix. Results and Discussion Analysis of the Structure of Fibrinogen Adsorbed on or Embedded in Polyelectrolyte Films at Room Temperature. First, we followed the adsorption of fibrinogen onto PSS and PAH-terminated films. Figure 1 shows the evolution of the infrared spectrum of fibrinogen in the region 1400-1700 cm-1 during its adsorption from a 0.05 mg‚mL-1 solution onto the surface of a PAH-terminated polyelectrolyte multilayer. The intensities increase rapidly during the first 1200 s of adsorp-
Schwinte´ et al.
Figure 1. Evolution of the infrared spectrum of fibrinogen in the region 1400-1700 cm-1, due to its adsorption onto the surface of a PEI(PSS-PAH)3 multilayer. Fibrinogen was adsorbed from a 0.05 mg‚mL-1 solution circulating at a flow rate of 0.25 mL‚min-1 above the polyelectrolyte-coated Ge internal reflection element. The numbers along the amide I band indicate the time (in seconds) elapsed since the beginning of the adsorption. The insert shows the evolutions of the signals at 1450 cm-1 (b), 1580 cm-1 (O), and 1640 cm-1 (1).
tion: in the amide I region 1600-1700 cm-1, at around 1580 cm-1 (glutamic and aspartic acid carboxylate residues)19,20 and in the range 1420-1480 cm-1 (δCH2 and amide II′),21 before leveling off. A similar time evolution is observed on PSSterminated films (data not shown). In a separate control experiment, we determined the multilayer buildup and the amounts of fibrinogen adsorbed on these types of polyelectrolyte multilayers from a 0.05 mg‚mL-1 protein solution by using optical waveguide lightmode spectroscopy (OWLS). Details of this optical technique can be found elsewhere.22,23 The evolutions of both the adsorbed amount and the film thickness during the multilayer buildup process are shown in Figure 2 for a typical experiment. As expected, the film thickness increases linearly with the number of bilayers,3 the thickness per bilayer being of the order of 4 nm. The last step represented in Figure 2 corresponds to the adsorption of fibrinogen on a PSS ending film. We found 0.74 ( 0.06 and 0.96 ( 0.06 µg‚cm-2 adsorbed amounts of fibrinogen on the PSS- and PAH-terminated films (mean of three experiments for each value). The isoelectric point of fibrinogen being at 5.8, fibrinogen is negatively charged at pH 7.4.24 The larger amount of fibrinogen found at the positively charged PAH ending film is thus compatible with the electrostatic nature of the protein/multilayer interaction. A similar behavior has been observed for the adsorption of several proteins on both PSS and PAH ending films.25 With regard to the dimensions of a fibrinogen molecule (5 × 9 × 45 nm3),26 the thickness of 19 nm (and 27 nm) found for the fibrinogen layer adsorbed on a PSS (and PAH) ending film reveals that the proteins are adsorbed both in “end on” and “side on” configurations. These values represent mean optical thicknesses, the largest extent of the film being certainly larger. A surface concentration of 0.7 µg‚cm-2 corresponds to a minimum of 55% and a maximum of 500% surface coverage for proteins respectively adsorbed in “end-on” and “side-on” arrangements. If it is assumed that fibrinogen is distributed within a 45 nm (the largest dimension of fibrinogen not to be confused with the optical thickness that represents a mean thickness) thick layer, a surface concentration of 0.7 µg‚cm-2 would correspond to a local protein concentration of the order of 155 mg‚mL-1. This value is certainly only
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J. Phys. Chem. B, Vol. 105, No. 47, 2001 11909
Figure 3. Normalized infrared absorption spectra of fibrinogen under different conditions. The spectra were Fourier smoothed as described in the Appendix, with the exception of fibrinogen in the 50 mg‚mL-1 solution (for which raw data are shown). Curves: (b) PEI-(PSSPAH)3-fib; (2) PEI-(PSS-PAH)3-fib-(PAH-PSS)2-PAH; (4) PEI-(PSS-PAH)3-PSS-fib-(PSS-PAH)3; ([) fibrinogen in 2 mg‚mL-1 solution; (]) fibrinogen in 50 mg‚mL-1 solution.
Figure 2. Evolution of the multilayer thickness d (A) and of the amount of polyelectrolyte Q (B) during the film buildup determined by OWLS. The multilayer is constructed by first adsorbing PEI on the waveguide followed by alternate deposition of PSS and PAH layers from a 5 mg‚mL-1 polyelectrolyte solution. The film before fibrinogen adsorption corresponds to PEI-(PSS-PAH)3-PSS. The final step corresponds to the fibrinogen adsorption from a 0.05 mg‚mL-1 protein solution.
a crude order of magnitude due to the fact that the multilayer films can create surface roughness so that the surface area available for protein adsorption is underestimated and on the other hand that the proteins are adsorbed in both “end-on” and “side-on” configurations. This high surface concentration should nevertheless be kept in mind when features of the spectra of fibrinogen adsorbed on or embedded in polyelectrolyte multilayers are compared with those of fibrinogen in solution. It should be noted here that in the OWLS experiments the multilayers were deposited on a Si0.8Ti0.2O2 substrate and not on Ge. However, the structure of the multilayer should become substrate independent after the deposition of the first polyelectrolyte layer.27 To extract structural information relative to the adsorbed fibrinogen molecules, we concentrated our analysis on the amide I region (1600-1700 cm-1). As concerns the polyelectrolytes applied in this study, fortunately, only a single band near 1600 cm-1, originating from the stretching vibration of the C-C bonds in the aromatic benzene rings of PSS, overlaps with the amide I protein band. This contribution of the multilayer films could be subtracted from the multilayer/protein films. On the other hand, when fibrinogen was embedded in a polyelectrolyte architecture, only the contribution of those polyelectrolyte layers
could be subtracted which were under the protein; the contribution of the covering layers could not be separated from the protein spectrum. The relative weight of the PSS-related 1600 cm-1 band in the spectra of the different polyelectrolyte architectures varies considerably (see Figure 3), since the multilayers covering the protein have different compositions. Figure 3 reveals that the highly concentrated fibrinogen solution spectrum (50 mg‚mL-1) exhibits a very broad contour in the amide I region as compared with the spectrum of a 2 mg‚mL-1 protein solution. Since the adsorbed protein concentrations on the polyelectrolyte surfaces are very high, one would expect a similar broadening in the spectra of adsorbed fibrinogen. This is not the case, however. Figure 3 reveals that there are only slight variations in the region 1600-1700 cm-1 between the spectra of fibrinogen on or in the multilayers and the spectra of 2 mg‚mL-1 fibrinogen solutions. Thus, the secondary protein structure appears to be essentially unaffected, or even protected by the polyelectrolyte multilayers against concentration-induced structural changes. Decomposition of the complex amide I region of the IR spectra allows us to extract more detailed information concerning the secondary structure of the protein. After the noise had been filtered out by a method involving Fourier smoothing, as described in the Appendix, we were consistently able to decompose the amide I region of fibrinogen into five Gaussianshaped bands, whose frequencies, bandwidths, and intensities are presented in Table 1. It can be seen that the widths of the component bands do not depend on the environment of fibrinogen (solution, adsorbed, or embedded). For fibrinogen in solution at increasing concentrations, the contribution of the band attributed to the intramolecular β-sheets (1633-1635 cm-1) decreases, whereas that of the band attributed to the R-helices (1650-1653 cm-1) remains almost constant. The contributions of the turn structures (1668-1670 cm-1) and the two components assigned to the intermolecular β-sheets (1616-1618 and 1683-1688 cm-1) increase. The two intermolecular β-sheet-related components are situated at the edges of the amide I band, and their relative increase can therefore explain the broadening of the amide I band in concentrated fibrinogen solutions (Figure 3). Such an increase in the intermolecular β-sheet content is to be expected because
11910 J. Phys. Chem. B, Vol. 105, No. 47, 2001
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TABLE 1: Structural Analysis of Fibrinogen at Room Temperature under Different Conditionsa fibrinogen in solution at 2 mg‚mL-1 fibrinogen in solution at 5 mg‚mL-1 fibrinogen in solution at 50 mg‚mL-1 - - -PAH-fib - - -PSS-fib - - -PAH-fib-PAH- - - - -PSS-fib-PSS- - - - -PAH-fib-PSS- - - - -PSS-fib-PAH- - -
intermolecular β-sheet
intramolecular β-sheet
R-helix
turn
intermolecular β-sheet
1616-1618 18-20 4-6 1621 15 4 1617 19 9 1615-1616 20-22 5-7 1611-1613 15 2 1618-1622 9-12 0-3 1621-1624 8-9 1 1618-1623 7-8 0-2 1623 10-12 2-3
1633-1635 25-27 42-50 1635 25 39 1633 23 25 1635 24-26 42-43 1635 27-28 44-46 1636 24-27 46-53 1636 24 39-40 1636 23-25 40-44 1636 23 39-43
1650-1653 24 35-37 1652 23 35 1652 24 32 1652-1653 24-25 36 1652-1653 23-25 34-37 1652-1653 21-22 25-39 1652 23 35-36 1652 22 33-37 1652 22 34-37
1668-1670 19-23 8-11 1668 22 19 1669 21 21 1668-1669 19-20 9-10 1668-1669 20-21 10-12 1668-1669 20-22 12-17 1669 23 20 1669-1670 20-22 19 1668-1669 21-23 18
1683-1688 15-20 3-5 1683 17 4 1684 19 12 1680-1681 19-20 6-7 1681 20-21 6-7 1682-1683 14-20 1-3 1681-1683 16 3-4 1682-1683 12-15 1-4 1680-1682 16 3-4
a Gaussian-shaped component bands were obtained after Fourier smoothing of the infrared spectra as described in the Appendix. The errors in both the frequency and bandwidth determinations in the individual spectra were in the range of 1 cm-1. The errors in the relative intensity determinations for the weak and strong components were lower than 10% and 5%, respectively. For simplicity, only the fibrinogen-contacting polyelectrolytes are given from the architectures described in the Materials and Methods. For each component band, the range of the central frequency (cm-1), the bandwidth (cm-1), and the intensity relative (%) to the total amide I intensity are given successively. The limits represent the variations between different experiments.
these structures originate from protein-protein interactions, which should be favored at high protein concentrations. These interactions seem to take place at the expense of the intramolecular β-sheets. It should be mentioned here that the distribution of the secondary structure elements as shown in Table 1, obtained by our Fourier-smoothing aided IR spectrum evaluation method, is consistent with those published by Yongli et al.,28 i.e., 35% R-helix, 21% intramolecular β-sheet, 31% random, and 12.8% turn as determined by circular dichroism, the protein solution concentration not being specified, or by Azpiazu and Chapman,29 who report 37% R-helix, 30% intramolecular β-sheet, 14% turn, and 16% unassigned structures, determined by IR on a 10 mg‚mL-1 protein solution. As concerns the amide I component bands of fibrinogen adsorbed on PAH- or PSS-terminated multilayers, these are very similar to those found for fibrinogen in dilute solution. This confirms that the secondary protein structure is essentially unaffected by the adsorption and that fibrinogen is even protected by the polyelectrolytes against concentration-induced structural changes. When embedded in multilayers, fibrinogen molecules exhibit similar intramolecular β-sheet and R-helix contents but have a larger amount of turn structures and virtually no intermolecular β-sheet contents as compared with fibrinogen in dilute solution. The integrated relative intensities of the bands attributed to the turn structures and intermolecular β-sheets of the embedded fibrinogen molecules were independent of the multilayer architecture and were similar to those found for fibrinogen adsorbed on multilayers or dissolved at low concentration. Thus, the intermolecular β-sheets observed in solution or in adsorbed fibrinogen appear to be formed at the expense of the turn structures. The proportions of the relative weights of these two
secondary structure elements in surface-related and in solution IR spectra of fibrinogen may imply that interactions with polyelectrolyte multilayers preserve the secondary protein structure and additionally hinder formation of the intermolecular β-sheets otherwise observed because of protein-protein interactions at high concentrations. Temperature-Induced Changes in the Structure of Fibrinogen Interacting with Polyelectrolyte Films. Another way to check the structural stability of proteins incorporated in polyelectrolyte films is to follow their structural changes upon heating. Figure 4 depicts the spectra of a 5 mg‚mL-1 fibrinogen solution (Figure 4A), a highly concentrated (50 mg‚mL-1) fibrinogen solution (Figure 4B), and fibrinogen adsorbed on a PAH-terminated polyelectrolyte multilayer (Figure 4C) or embedded in a polyelectrolyte architecture between two PAH layers (Figure 4D) at different temperatures ranging from 28 to 85 °C. Similar spectra were obtained (data not shown) for fibrinogen adsorbed on a PSS-terminated film or embedded in any of the three other multilayer architectures listed in Table 1. A characteristic change in the fibrinogen amide I band upon heating is the appearance of a strong shoulder at around 16161619 cm-1. Parts C and D of Figure 4 also reveal that the structural changes are irreversible once fibrinogen has been heated above 65 °C. This phenomenon seems to be a general one; it has been likewise observed for soluble29-33 and membrane proteins.34 Early studies on polypeptides, and particularly fibrous systems,35,36 led to the conclusion that this component arises from the formation of intermolecular β-sheets. The work of Susi et al.37 on poly(L-lysine) clearly demonstrated this phenomenon upon temperature increase, the change being so great that the 1616 cm-1 band even became predominant with respect to the amide I profile.
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Figure 4. Deformation of the amide I band of fibrinogen in solution and in different polyelectrolyte constructions as a function of temperature: (b) 28 °C; (0) 60 °C; (2) 85 °C; (A) fibrinogen in 5 mg‚mL-1 solution; (B) fibrinogen in 50 mg‚mL-1 solution; (C) PEI-(PSS-PAH)3-fib; (D) PEI-(PSS-PAH)3-fib-(PAH-PSS)2-PAH. The irreversibility of the changes is revealed by the spectra of the polyelectrolyte/protein structures recooled to the starting temperature (3, 28 °C) in panels C and D.
A detailed analysis of the structural changes in fibrinogen as a function of temperature was carried out by decomposing the amide I region of the IR spectra into five component bands, as shown in Figure 7 in the Appendix. The temperature-related evolutions of the different component bands, for fibrinogen in solution, or adsorbed on or incorporated in different multilayers, are given in Figure 5. It can be seen that fibrinogen molecules undergo a structural change in the indicated temperature range. This change can be characterized by a decrease in the 1635 cm-1 component (intramolecular β-sheet) and the appearance of and/or an increase in the 1616 cm-1 component (intermolecular β-sheet). The onset temperature of this increase lies between 40 and 50 °C. The existence of a transition in fibrinogen in this temperature range has already been reported. It was attributed to the unfolding of the globular distal domains of the protein (mainly β-sheet),29,38 which is in accordance with our findings. Donovan and Mihalyi39 found a similar transition by calorimetric experiments. They measured a maximum of an enthalpic peak at 61 °C but the transition already started at 40 °C. They determined the corresponding denaturation enthalpy, which was of the order of 4.2 cal‚g-1. For fibrinogen molecules in 5 mg‚mL-1 solution (Figure 5A), the evolution of the intermolecular β-sheet contribution characterized by the intensity of the 1616 cm-1 component band starts at 40 °C with an abrupt increase between 40 and 50 °C, whereas the decrease in the intramolecular β-sheet (1635 cm-1 component band) proceeds smoothly throughout the whole temperature range above 40 °C. A continuous, slight increase
in the contribution of the 1670 cm-1 band assigned to turn structures can also be observed. This increase seems to be slightly enhanced at around 70 °C. The 1616 cm-1 intermolecular β-sheet content also shows a second, but slight increase at around 70 °C. This may suggest the existence of a second structural transition, whose existence has been mentioned previously in the literature.29,38 It was attributed to a disorganization of the helical parts (coiled coils) of the fibrinogen molecules. However, this should lead to a decrease in the R-helix content, but this was not observed up to 85 °C in our experiments. On the other hand, Donovan and Mihalvi39 also found a second thermal transition with an enthalpic peak centered at around 100 °C but already starting at around 80 °C. It was attributed to the denaturation of the E nodule. Band evolutions similar to those observed for the 5 mg‚mL-1 fibrinogen solution were also found for the 50 mg‚mL-1 fibrinogen solution (Figure 5B), but the amplitudes of the variations were much smaller. This was particularly true for the decrease in the 1635 cm-1 intramolecular β-sheet contribution. This is not surprising since the relative weight of this band is initially smaller than in the case of the 5 mg‚mL-1 solution, the proportion of the intermolecular β-sheet bands being much larger at room temperature for this more concentrated solution. The fibrinogen in the concentrated solution has already undergone self-aggregation, which induces structural changes, reducing the scope of temperature-induced protein aggregation. It should be noted, however, that the R-helix content is practically identical at the two concentrations, indicating that the “core”
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Figure 5. Changes in the relative intensities of the five component bands of the amide I region of fibrinogen during a temperature elevation experiment. Band decomposition was carried out as described in the Appendix, with Gaussian component band shapes. The errors of the fits are in the range of the size of the symbols; error bars indicate deviations between different experiments: (b) intermolecular β-sheet (1618 cm-1); (.) intermolecular β-sheet (1684 cm-1); (0) intramolecular β-sheet (1635 cm-1); (4) R-helix (1652 cm-1); (]) turn (1668 cm-1); (A) fibrinogen in 5 mg‚mL-1 solution; (B) fibrinogen in 50 mg‚mL-1 solution; (C) PEI-(PSS-PAH)3-fib; (D) PEI-(PSS-PAH)3-PSS-fib; (E) PEI-(PSS-PAH)3fib-(PAH-PSS)2-PAH; (F) PEI-(PSS-PAH)3-PSS-fib-(PSS-PAH)3.
of the fibrinogen structure seems not affected by the concentration-induced protein-protein interactions. For fibrinogen molecules adsorbed on multilayers, the 1616 cm-1 intermolecular β-sheet content is observed to increase to a much larger extent and over a much broader temperature range than in solution, though the onset temperature remains at around 40 °C (Figure 5C,D). The enhanced formation of intermolecular β-sheets can result from the fact that the adsorbed protein molecules are constrained to each other over a much longer time than in solution. This may favor interactions giving rise to intermolecular β-sheets. However, while such protein selfassociation occurs in solution at room temperature at high fibrinogen concentration (see the starting values of the 1618 and 1683 cm-1 component bands in Figure 5B), it does not
take place at room temperature in any of the polyelectrolyteadsorbed fibrinogen molecules, despite their high surface concentration (Figure 5C-F), as reflected by the very low starting intensities of the intermolecular β-sheet-related 1618 and 1683 cm-1 bands. This may mean that the formation of an intermolecular β-sheet requires a minimum of protein mobility for the interacting surfaces of two protein molecules to adapt (probably by successive approximation) to each other, and adsorbed proteins do not have this mobility at room temperature; they may gain it only at elevated temperatures. The embedding of fibrinogen in polyelectrolyte multilayers reveals two additional effects concerning its thermal behavior as compared with being in solution or terminally adsorbed on the surface of the polyelectrolyte multilayer:
Adsorbed/Embedded Fibrinogen Molecules First, the temperature at which intermolecular β-sheets appear in fibrinogen is upshifted from 40 to 45-50 °C for the embedded fibrinogen molecules, irrespective of the polyelectrolyte film architecture. This effect is indicative of the enhanced hindrance to intermolecular fibrinogen interactions when the polyelectrolytes surround the protein relative to the case when the fibrinogen is adsorbed onto the polyelectrolyte surface. Second, the covering polyelectrolytes restrict the magnitude of the thermally induced structural changes in the fibrinogen, this restriction being dependent on the type of the polyelectrolytes forming the contact surface with fibrinogen. The restriction effect increases with the abundance of PSS in contact with the protein (-PAH-fib-PAH- < -PAH-fib-PSS- ≈ -PSSfib-PAH- < -PSS-fib-PSS-). For clarity, only an analysis of the two extremities is given in Figure 5E,F. The magnitude of the temperature-induced increase in the 1616 cm-1 band symptomatic of protein association is strongly reduced for fibrinogen molecules embedded in the interior of polyelectrolyte architectures as compared with that observed for adsorbed fibrinogen molecules. This may indicate that the thermal transition of the structure of fibrinogen must involve a somewhat different route in the encapsulated fibrinogen as compared with that for fibrinogen in solution or adsorbed on multilayers: the constraints imposed by the encapsulating polyelectrolytes mean that the chances for the fibrinogen to build up intermolecular contacts is rather limited, as shown by the low weight of the intermolecular β-sheet component. Thus, the thermally induced structural rearrangements must proceed intramolecularly. Indeed, the changes in fibrinogen inserted between PAH-terminated multilayers (-PAH-fib-PAH-) (Figure 5E) can be better characterized by the appreciable decrease in the 1635 cm-1 intramolecular β-sheet contribution and by the concomitant increase in the turn contribution. In fibrinogen embedded in -PSS-fib-PSS- (Figure 5F) architectures, the temperature-induced structural changes were further decreased: the contributions of the turn structures and of the R-helices remained almost constant as the temperature was increased, while a slight decrease in the intramolecular β-sheet content compensated for the similarly small increase in the intermolecular β-sheet content. The limited scale of the structural changes observed for embedded fibrinogen molecules is revealed by the very similar relative intensities of the R-helical structures and their constancy in any of the polyelectrolyte architectures at any temperature. These results permit the conclusion that protein molecules embedded in polyelectrolyte multilayers are strongly hindered as concerns the formation of intermolecular β-sheets. Moreover, the embedding of fibrinogen molecules in multilayers between PSS layers seems to result in a great stabilization of the protein structure over the whole investigated temperature range, up to 90 °C.
J. Phys. Chem. B, Vol. 105, No. 47, 2001 11913 cases no room-temperature self-association occurs at all, whereas the thermally induced structural changes are enhanced. If adsorbed fibrinogen is covered by additional polyelectrolyte layers, the thermally induced structural transitions become strongly hindered and seem to follow a different path from that in solution. All these features can be explained if it is assumed that the polyelectrolytes somehow “encapsulate” the fibrinogen molecules. This encapsulation is much more complete when the protein is embedded in the multilayer films than when it is adsorbed only on such films. Partial encapsulation can prevent room-temperature selfassociation by immobilizing the adsorbed fibrinogen. In consequence of the same immobilizing effect, the fibrinogen molecules probably stay close to one another over a longer time, which may enhance the chances of formation of intermolecular β-sheets from intramolecular β-structures, as was seen here at elevated temperatures. In the event of complete encapsulation, achieved by covering the adsorbed fibrinogen with additional polyelectrolyte layers, the hindrance to intermolecular protein interactions is more effective. Here, the thermally induced intermolecular protein structural changes are also strongly restricted. Thus, the heatinduced structural transition of the protein must take a different path. Instead of being transformed to intermolecular β-sheets, the intramolecular β-structures decompose into other structural elements, mostly turn structures of the same fibrinogen molecule. All the above data show that the interactions with these polyelectrolytes preserve and stabilize the protein structure. We believe that this preserved protein structure is very close to the native one. Such a hypothesis is strongly supported by the observation of Caruso et al.12 that the biological recognition of antigens by their antibodies was unaffected when the latter were adsorbed onto a polyelectrolyte surface and were covered by fewer than four additional polyelectrolyte layers. The conservation of the protein structure by polyelectrolyte multilayers is a prerequisite for the proteins to keep their biological activity but it is not sufficient. One already knows that enzymes embedded in films14 keep their biological activity. However, the polyelectrolytes interacting with the proteins could hinder some activity and in particular interactions with cells. We are currently investigating the interactions between proteins and cells and in particular cell adhesion on protein-functionalized polyelectrolyte multilayers. A positive cell response would open the possibility to design targeted biofilms, allowing, for example, to control cell adhesion, cell growth and/or present thromboresistant properties. Other applications in the conditioning of proteins by preserving their biological activity can also be anticipated. Appendix
Conclusions In solution, fibrinogen undergoes concentration-dependent self-association at room temperature, and exhibits a tendency to form further aggregates as the temperature is increased. These structural changes occur at the expense of the native intramolecular β-structures, which are transformed into intermolecular β-sheets. The interactions between fibrinogen and the polyelectrolytes investigated in this study cause considerable alterations in the concentration-dependent features of fibrinogen. While extremely high local concentrations can be achieved by adsorbing fibrinogen onto the surfaces of polyelectrolyte multilayers, in these
There are several methods whereby information concerning secondary protein structures can be extracted from the complex amide I band. This band is the sum of overlapping component bands, each related mainly to the CdO stretching of the peptide bonds and affected by characteristic environments dependent on the different kinds of secondary structures (R-helix, β-structures, turns, random coils, etc.). The decomposition of the spectra into Lorentzian or Gaussian component bands usually requires a method by which the central frequency of the component bands can be determined. This can be done by taking second derivatives40 or using resolution enhancement.41 It should not be forgotten, however, that these methods are very sensitive to
11914 J. Phys. Chem. B, Vol. 105, No. 47, 2001
Schwinte´ et al. Figure 6 shows the process of “spectrum cleaning” to eliminate the vapor effects. Curve a in Figure 6A depicts the amide I region of fibrinogen “sandwiched” between PAH polyelectrolyte layers. This spectrum was chosen because it reflects the most difficult experimental conditions discussed in this paper and is heavily contaminated with vapor bands. Curve b shows the same spectrum after subtraction of the water vapor spectrum (using the corresponding function of the Bruker OPUS software in interactive mode). In the processing of the infrared spectra, it is common practice to smooth the curves; such a smoothed spectrum (Savitzky-Golay, nine points, Bruker OPUS software) is depicted as curve c. To improve vapor elimination, we used Fourier smoothing; i.e., we applied a Fourier transformation to the spectrum, multiplied it by an apodization function, and then performed an inverse transformation. The apodization function was as follows:
A(ω) ) 1 for ω < ω0 A(ω) ) exp{-[(ω - ω0)2/2σ2]
Figure 6. Illustration of spectrum “cleaning” from water vapor contamination. A: (a) Raw spectrum; (b) spectrum obtained after interactive vapor subtraction; (c) spectrum obtained after a 9-point Savitzky-Golay smoothing applied to spectrum b; (d) spectrum obtained after Fourier smoothing applied to spectrum b, [b-c] difference between spectrum b and its Savitzky-Golay smoothed derivative represented as curve c; [b-d] difference between spectrum b and its Fourier-smoothed derivative represented as curve d. B: (e) Second derivative of the Savitzky-Golay-smoothed spectrum (curve c) after an additional 5-point Savitzky-Golay smoothing; (f) second derivative, without any further smoothing, of the Fourier-smoothed spectrum (curve d). For details, see Appendix.
noise, and that noise sensitivity is strongly dependent on the spectrum of the given noise. The major source of concern is the presence of water vapor rotational bands in the amide I region. Since the rotational bands are approximately 10 times narrower than the protein component bands, they give very strong signals in the second-derivative spectrum. With the help of simulated spectra (not shown), we concluded that the amplitude of the water vapor bands should be around 3 orders of magnitude lower than that of the protein bands if disturbance of the second derivative (calculated without smoothing) of the amide I band is to be avoided. The contribution of the water vapor can be almost entirely eliminated in conventional transmission experiments in which a shuttle device is used to measure sample and reference spectra under the same conditions. This is not possible in ATR experiments, where there is always a long time interval between the sample and reference measurements. Moreover, the absorption of the adsorbed protein is in the range of only a few times 10-3 optical density units. We have therefore developed a technique with which to handle the water vapor problem under these conditions too.
for ω g ω0
Thus, it had values between 1 and 0, the transition from 1 to 0 following the shape of a Gaussian curve; the cutting frequency (ω0) and the curve width (σ) could be changed; shifting the cutting edge to lower values resulted in stronger smoothing. The Fourier-smoothed curve b is shown as curve d in Figure 6A. Of course, any smoothing causes distortions in the original spectrum. While polynomial smoothing can have only local effects, Fourier smoothing, if too strong, can distort the whole spectrum. At the bottom of Figure 6A, the differences between the raw and Savitzky-Golay or Fourier-smoothed spectra are shown, respectively. The level of the noise is higher in the difference spectrum in the Fourier-smoothed case (b-d), indicating a stronger smoothing. While there is some distortion at around 1600 cm-1 for the strong, narrow band originating from PSS, there is no such distortion in the amide I region, a lack of distortion in this region being the limiting criterion for the applied Fourier smoothing. Throughout this paper, the spectral region 1000-2000 cm-1 was Fourier transformed using 2048 points; the parameters of the apodization function used for smoothing were set to ω0 ) 60 and σ ) 45. Second-derivative spectra are generally used to determine starting values of the band positions for curve decomposition procedures. In Figure 6B, curve e shows the second derivative of the Savitzky-Golay-smoothed spectrum (curve c in Figure 6A). A further five-point Savitzky-Golay smoothing was performed before the second derivative (again a common practice in spectrum evaluations) was taken. It can be seen that the features of this second derivative reflect a strong correlation with the water vapor spectrum (shown at the bottom of Figure 6B), which is generally believed to be eliminated by this procedure. Accordingly, this does not seem to be the case. This might be the reason for the occurrence in the literature of frequencies assigned to components of the secondary protein structure, but very close to those of water vapor bands. Curve f in Figure 6B shows the second derivative of the Fourier-smoothed spectrum (curve d in Figure 6A). No further smoothing was applied when the second derivative was taken. It can be seen that the number of minima is decreased, as is the noise level in the region 1750-1800 cm-1, which is good for testing, since there are no bands here in the original spectrum.
Adsorbed/Embedded Fibrinogen Molecules
J. Phys. Chem. B, Vol. 105, No. 47, 2001 11915 depicted in Figure 7. All amide I band decompositions presented in this paper were carried out with the above method. Acknowledgment. This work was performed within the frame of the French-Hungarian bilateral cooperation project “Balaton” (F-1/97). B.S. expresses his thanks for an INSERM “poste jaune” and for a grant of the Hungarian Science Foundation (OTKA T0 31973). P.S. thanks the Faculte´ de Chirurgie Dentaire of Strasbourg for financial support. This work was also supported by the program “Adhe´sion CellulesMate´riaux” and by the CNRS program “Physique et Chimie du Vivant”. We are grateful to Prof. G. Decher for stimulating discussions about polyelectrolyte multilayers and for his careful reading of the manuscript. We also thank Dr. F. Poumier for her participation in this work. References and Notes
Figure 7. Decomposition of the amide I band of fibrinogen. In this example, fibrinogen was embedded in a PEI-(PSS-PAH)3-fib(PAH-PSS)2-PAH architecture at 61 °C. The uppermost curve is the second derivative of the Fourier-smoothed absorption spectrum (equivalent to curve f in Figure 6); stars indicate the minima used as starting values in the band decomposition. In the middle curve, circles indicate the data points of the spectrum, the solid line through the circles is the result of the fit of the five component bands, which are shown displaced below. At the bottom, the difference between the spectrum and the fit is given, using the same abscissa as for the spectrum.
Moreover, the strong band at around 1600 cm-1 is well reproduced in the second derivative. Hence, we suggest that, with the Fourier-smoothing approach and with the criterion of a nondistorted amide I band, a more realistic number of component bands in the region 1600-1700 cm-1 can be defined. The applicability of these predicted bands for curve fitting is shown in Figure 7. Determination of Secondary Structure. The second-derivative signal allowed an estimation of the positions of the main structural components (β-sheet, R-helix, and turns) (Figure 7). Prior to the fit, linear baseline subtraction was performed in the interval 1600-1700 cm-1, where the secondary-structure determination was carried out. The parameters of the underlying bands of the amide I envelope (band intensity, position, and width) were determined by a least-squares iterative fitting procedure of the SPSERV program. As suggested by the second derivative, we fitted five Gaussian bands into the amide I region. First, the widths and positions of the component bands were fixed and the intensities were fitted. The intensities and the frequencies were then fixed, and the widths were fitted. Next, the intensities remained fixed, and the frequencies together with the widths were refined. In the last step, the intensities were refined by fixing the previously optimized frequencies and widths. By using this procedure for each spectrum, we obtained the following five frequencies for the component bands in the amide I region: 1616 and 1684 cm-1 (intermolecular β-sheet, mainly present at higher temperatures), 1635 cm-1 (intramolecular β-sheet), 1652 cm-1 (R-helix), and 1668 cm-1 (turns and unordered structures).42 These values are averages; the variations in band positions between any of the spectra were rarely larger than (2 cm-1. Similarly, the agreement between the frequencies predicted by the second derivatives and those found by the fitting procedure described above in the spectra was excellent: the difference was always within (1 cm-1. As an example for the quality of the fit, the absorption spectrum of fibrinogen in PEI(PSS-PAH)3-fib-(PAH-PSS)2-PAH, recorded at 61 °C, is
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