Salt Concentration and pH-Dependent Adsorption of Two

Feb 3, 2007 - C. M. Pradier*, V. Humblot, L. Stievano, C. Méthivier, and J. F. Lambert. Laboratoire de Réactivité de Surface, CNRS UMR 7609, Univer...
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Langmuir 2007, 23, 2463-2471

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Salt Concentration and pH-Dependent Adsorption of Two Polypeptides on Planar and Divided Alumina Surfaces. In Situ IR Investigations C. M. Pradier,* V. Humblot, L. Stievano, C. Me´thivier, and J. F. Lambert Laboratoire de Re´ actiVite´ de Surface, CNRS UMR 7609, UniVersite´ Pierre et Marie Curie, 4 place Jussieu, Case 178, 75252 Paris Cedex 05, France ReceiVed July 27, 2006. In Final Form: December 12, 2006 The adsorption of proteins is the first process to take place when a solid is immersed in a biological fluid; though not yet thoroughly understood at a molecular level, this process is also known to be strongly influenced by the presence of salt in solution or by pH changes. In the present work, poly-L-glutamic acid (PG) and poly-L-lysine (PL) were selected to mimic the behavior of some protein fragments. Their adsorption was investigated by infrared spectroscopy in various modes, both on planar and on divided (powder) surfaces of aluminum oxide. These two peptides were shown to have different behaviors when adsorbed from solutions with or without CaCl2 and at various pH values. Polarization modulation-reflection absorption infrared spectroscopy, applied in a special cell designed to characterize the solid surface in contact with the liquid, enabled the observation of the influence of pH and salts upon polypeptide adsorption. At pH values higher than 5 and in the presence of CaCl2 in solution, a net increase of the PG adsorbed amount is observed, whereas no such effect could be detected for PL. Specific interactions between the COO- groups on the side chains and the surface, or between those of two different molecules, was inferred. Interestingly, similar conclusions could be drawn for the surface of alumina powders contacted with solutions of PG and PL and characterized by attenuated total reflectance IR. This work demonstrates the potential for IR investigations of solid oxide-liquid interfaces combining the study of planar and finely divided surfaces.

1. Introduction The mechanism of protein aggregation in a biological medium has been widely studied for its importance in various domains like separation, blood coagulation, biofilm growth, food, or in vivo applications. In particular, poly-L-Lysine (PLL or PL) and poly-L-glutamic acid (PLGA or PG) have often been used as protein models because their molecular weights are close to that of several common proteins. They have contrasting chemical structures: PL has basic amine groups on its side chain, whereas PG has carboxylic acid groups (see Scheme 1). They have been previously used to illustrate and study attractive or repulsive long-range interactions in colloidal solutions as a function of pH,1 but they have been used also for their prospective applications as the layer-by-layer deposition of PL and PG is a well-explored route to the obtention of nanostructured films.2-7 Studies of the topic, however, usually focus on the global properties of multilayer films, while the initial step of polyelectrolyte adsorption on the substrate is less well understood. It has been known for a long time that divalent cations, and in particular calcium ions, favor protein aggregation in solution8 but the reason for that remains somewhat controversial with the direct bridging between negatively charged sites in protein molecules and Ca2+ cations being often invoked9 but never demonstrated at a molecular level. Caseinate solutions were * Corresponding author. E-mail: [email protected]. (1) Watson, G. S.; Blach, J. A.; Cahill, C.; Nicolau, D. V.; Pham, D. K.; Wright, J.; Myhra, S. Biosens. Bioelectron. 2004, 19, 1355. (2) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422. (3) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355. (4) (4) Zhi, Z. L.; Haynie, D. T. Macromolecules 2004, 37, 8668. (5) Bouldemais, F.; Frisch, B.; Etienne, O.; Lavalle, P.; Picart, C.; Ogier, J.; Voegel, J. C.; Schaaf, P.; Egles, C. Biomaterials 2004, 25, 2003. (6) Haynie, D. T.; Zhang, L.; Rudra, J. S.; Zhao, W. H.; Zhong, Y.; Palath, N. Biomacromolecules 2005, 6, 2895. (7) Haynie, D. T. J. Biomed. Mater. Res. B 2006, 78, 243. (8) Barbut, S.; Foegeding, E. A. J. Food Sci. 1993, 5, 867. (9) Roefs, S. P. F. M.; Peppelman, H. A. Food Colloids: Fundamentals of Formulation; Royal Society of Chemistry: Cambridge, UK, 2001; Chapter 12.

Scheme 1. (a) Schematic Representation of PG; (b) Schematic Representation of PL

studied in the presence of monovalent or divalent cations; in addition to an effect on the solution viscosity, divalent ions rapidly induced protein aggregation.10 Several studies reported comparative investigations of the respective roles of monovalent and divalent cations. Although monovalent ions, such as Na+ or K+, do increase the viscosity of a protein solution, the influence on aggregation is always significantly stronger with divalent ions. The ion strength is highly important in calcium binding to proteins; there is a competition between Ca2+ and Mg2+, both having similar affinity for the albumin binding sites regardless of their different sizes.11 On the other hand, the turbidity of a soy protein isolate was higher with Ca2+ than with Mg2+ at similar concentrations indicating Ca2+ binding selectivity.12 The interaction between proteins in solution and solid surfaces shows phenomena that are similar to those observed in protein aggregation. For instance, the adsorption of proteins on hydrophobic media has been investigated in the presence of salts at various concentrations to understand the operation of gradient elution chromatography.13 In NaCl solutions, the amount of adsorbed BSA on a polymeric membrane decreased when the salt concentration increased (50-150 mM), which was attributed to shielded electrostatic interactions with the solid phase.14 A model of this elution behavior of proteins on a hydrophobic medium, based on a two-state protein (hydrated or not), was recently proposed by Chen and Sun, leading to protein adsorption (10) Carr, A. J.; Munro, P. A.; Campanella, O. H. Int. Dairy J. 2002, 12, 487. (11) Pedersen, K. O. Scan. J. Clin. Lab. InVest. 1972, 29, 427. (12) Molina, M. I.; Wagner, J. R. Food Res. Int. 1999, 32, 135. (13) Oscarsson, S.; Karsnas, P. J. Chromatogr., A 1998, 803, 83. (14) Hunter, A. K.; Carta, G. J. Chromatogr., A 2001, 930, 79.

10.1021/la062208p CCC: $37.00 © 2007 American Chemical Society Published on Web 02/03/2007

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isotherms that are highly dependent on the nature and concentration of salt in the medium.15 These few examples from the literature reveal that strong effects of divalent cation salts on protein behavior exist, but controversial arguments are given for their interpretation. The main controversy is between purely electrostatic effects (shielding of the repulsive interactions by counterions, which should only depend on the charge of the ions and not on their chemical nature) and specific, coordinative binding implying the divalent cations. On the one hand, de Jongh et al. used β-lactoglobulin as a model protein and by changing the number of its negative charges concluded that increasing the number of carboxyl groups does not enhance aggregation; according to these authors, calcium plays a determining role in the screening of electrostatic repulsions between proteins rather than in specific intraprotein bridging.16 On the other hand, Bhosle et al. studied the adsorption of a bacterial polysaccharide on a Ge-oxide film; they evidenced a specific influence of divalent cations at constant ionic strength, and attributed this influence to the formation of “divalent cationic bridges” between functional groups of the polysaccharide and the oxide layer17 (i.e., to coordinative binding between specific groups of the polymer and the Ca2+ ions). Some model experiments, using simpler molecules like peptides or amino acids, would help to understand the effect of salts on protein or peptide aggregation and/or adsorption at a molecular level. In this frame, the purpose of this paper is twofold: first, to investigate the effect of a divalent cation, Ca2+, on the adsorption of two types of peptides on an oxide surface and second, to draw a parallel between results on a model planar surface and on divided solids. Thus, it constitutes an attempt to fill the materials gap between studies on model surfaces and on real ones on which roughness and defects are uncontrollable. The long-term goal of this work is the control of biofilm growth in a marine environment. Adsorption of these peptides on Al2O3 thin films or powder surfaces was monitored ex situ and in situ thanks to the use of the Fourier transform infrared (FT-IR) spectroscopy in different modes: attenuated total reflection (ATR) was used to characterize alumina powders in solutions while polarization modulationreflection absorption infrared spectroscopy (PM-RAIRS) was performed both under air and in a dedicated cell to monitor adsorption processes at planar alumina surfaces ex and in situ, respectively. 2. Experimental 2.1. Adsorption on Alumina Thin Films. 2.1.1. Materials. Alumina surfaces were obtained by evaporation of aluminum on glass substrates covered with an intermediate Cr layer.1 Samples were cleaned by successive batches in pure ethanol and H20 Millipore prior to immersions in peptide solutions. Poly-L-glutamic acid sodium salt (Sigma Aldrich, noted as PG) with 50 000-100 000 g mol-1 molecular weight corresponding to an average chain length of ca. 350-650 units and poly-L-lysine hydrobromide (Sigma Aldrich, noted as PL below) 90 000-100 000 g mol-1 molecular weight corresponding to an average chain length of ca. 450-500 units solutions were prepared at 15 mg/L in pure water at pH ) 7 for PG and pH ) 5.6 for PL, corresponding to the anionic and cationic forms of the peptides, respectively. To investigate the effect of salt in solution, CaCl2 (PROLABO, 99%) was added to reach a concentration of 2.5 × 10-2 M. This value is ten times (15) Chen, J.; Sun, Y. J. Chromatog., A 2003, 992, 29. (16) Simons, J.-W. F. A.; Kosters, H. A.; Visschers, R. W.; de Jongh, H. H. J. Arch. Biochem. Biophys. 2002, 406, 143. (17) N. Bhosle; Suci, P. A.; Baty, A. M.; Weiner, R. M.; Geesey, G. G. J. Colloid Interface Sci. 1998, 205, 89.

Pradier et al. higher than in human body fluids and is close to the concentration in natural seawater. All solutions were submitted to a continuous bubbling of N2 to avoid CO2 enrichment in solution. The pH/pD of the H2O/D2O (Sigma Aldrich, 99.9%) solutions were adjusted by adding the necessary amount of 1M solutions of NaOH/NaOD or HCl/DCl. 2.1.2. PM-RAIRS ex situ Surface Characterizations. In the PMRAIRS module, the sample is placed in the external beam of a FT-IR instrument (Nicolet 5700 spectrometer) and the reflected light is focused on a nitrogen-cooled mercury cadmium telluride (MCT) detector. A ZnSe grid polarizer and a ZnSe photoelastic modulator to modulate the incident beam between p and s polarizations (HINDS Instruments, PEM90, modulation frequency ) 36 kHz) are placed prior to the sample. The detector output is sent to a two-channel electronic device that generates the sum and difference interferograms. Those are processed and Fouriertransformed to lead to the PM-RAIRS signal ∆R/R0 ) (Rp - Rs)/ (Rp + Rs). All reported spectra are recorded at an 8 cm-1 resolution by the coaddition of 128 scans; the use of polarization modulation enabled us to perform rapid analyses of the samples after immersion without purging the atmosphere nor requiring a reference spectrum. 2.1.3. PM-RAIRS in situ Surface Characterizations. In situ PMIRRAS measurements were performed using the same FT-IR spectrometer as above and a cell especially built for letting a thin (ca. 1 µm) layer of liquid between a CaF2 half-spherical window and the sample during analysis. The detail of the experimental setup has been presented in a previous paper.18 In the time between two analyses, the peptide solutions were circulated at 200 mL/min, passing through a peristaltic pump, with nitrogen bubbling in the solution. A small reservoir prior to the pump enabled a change to the pH, salt concentration, or to pass to the rinsing solution so that the sample is continuously in contact with the solution. All reported spectra are recorded at an 8 cm-1 resolution by the coaddition of 32 scans. 2.2. Adsorption on Alumina Powders. 2.2.1. Solutions. Aqueous solutions of sodium poly-L-glutamate (Sigma-Aldrich, MW ) 97 800 Da) or poly-L-lysine hydrobromide (Sigma-Aldrich, MW ) 93 800 Da) with a concentration of poly(amino acid) of 1.5 g/L, corresponding to about 10 mmol of peptide bonds per L, were used in this study. The influence of the concentration of Ca2+ on the adsorption properties of these peptides was investigated by using in parallel solutions containing no CaCl2 and solutions with a CaCl2 concentration of 0.02 mol/L (corresponding to an ion strength of 0.12). The pH of all studied solutions was adjusted to the desired value by adding appropriate amounts of 1M solutions of NaOH or HCl. 2.2.2. In situ Adsorption on Alumina Powders. FT-IR in the ATR mode was used to monitor in situ the adsorption of polypeptides on Al2O3 powders suspended in solutions. Ten milliliters of a solution of sodium poly-L-glutamate or poly-L-lysine hydrobromide (15 mg/ mL) of known pH and CaCl2 concentration were contacted with 100 mg γ-Al2O3 powder (Procatalyse (Axens) EC-1285, specific surface 160 m2/g), 1 mL of the obtained suspension was rapidly transferred in a cell fitted with a ZnSe ATR crystal at the bottom (t ) 0), and ATR-IR measurements were repeated at increasing contact times. Specific solutions of pure water adjusted at the same pH and CaCl2 concentration of each suspension were previously measured to provide background spectra. 2.2.3. Ex situ Adsorption on Alumina Powders. FT-IR in the transmission mode was used to monitor the adsorption of polypeptides on Al2O3 powders after contacting with solutions of polypeptides. Ten milliliters of an aqueous solution of sodium poly-L-glutamate and poly-L-lysine hydrobromide of known pH and CaCl2 concentration were contacted in a closed vessel with 100 mg of γ-Al2O3 for 4 h. The solid was separated by centrifugation and was washed by resuspending it for 30 min in 10 mL of water previously adjusted to the same pH and CaCl2 concentration as the former poly(amino acid) solution. The solid then was recovered by centrifugation and vacuum-dried overnight at 30 °C. Each sample was pressed into a self-supported pellet (27 mg/cm2) for the FT-IR transmission (18) Me´thivier, C.; Beccard, B.; Pradier, C. M. Langmuir 2003, 19, 8807.

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Figure 1. PM-IRRAS spectra of the alumina surface after immersion in PG aqueous solutions at various pH. Lower spectra: PG in pure water. Upper spectra: PG in CaCl2 solutions. All surfaces have been rinsed in pure water and dried under nitrogen. measurement. The absorption of a self-supported pellet of pure alumina previously treated in water at the same pH and CaCl2 concentration as the studied sample was used as the background of the FT-IR spectra. 2.2.4. Infrared Spectroscopy. FT-IR spectra were recorded on a Nicolet-Magna 5700 FT-IR spectrometer equipped with a liquid nitrogen-cooled MCT detector. ATR-IR measurements on solutions and suspensions were obtained by adding 256 scans recorded at 8 cm-1 resolution, whereas FT-IR transmission spectra of selfsupported pellets were recorded by adding 128 scans at 4 cm-1 resolution.

3. Results 3.1. Adsorption of Poly-L-Glutamic Acid on Al2O3, Thin Films. 3.1.1. Ex situ Analyses. The PM-RAIRS spectra obtained in the air after immersion of the Al thin film in PG solutions at pH 2, 7, or 11, copious rinsing, and drying, are shown in Figure 1. The lower spectra correspond to a 30 min adsorption in PG solutions in pure water; the upper spectra were obtained after a 75 min immersion in a PG and CaCl2 solution after rinsing in pure water. After immersion in nonsalted solutions, all spectra exhibit bands characteristic of the polypeptide with their intensities slightly varying with the pH value. At neutral pH, two bands at 1659 and 1566 cm-1 are easily ascribed to the amide I and II bands of the polymer; a weaker signal at ca. 1410 cm-1 can be attributed to the symmetric stretch of the carboxylate groups while that at 1464 cm-1 is the δCH of CH2 groups; at pH ) 2, noticeable spectral changes are observed with the appearance of an intense and expected νCdO band at 1718 cm-1 due to the COOH groups and an increase of all other signals indicating that a larger amount of PG was adsorbed. At pH ) 11, bands are very similar to those observed at pH ) 7, although slightly more intense. On all spectra, the intense band at 953 cm-1 can be attributed to the δOH vibrations of bulk hydroxyls of bohemite.19 Its intensity was stable regardless of the time of immersion. After adsorption in CaCl2-containing solutions, the IR signals do not change in position but significantly increase indicating much larger amounts of adsorbed polypeptide; the effect of the presence of salts in the peptide solutions is strong at basic or neutral pH and almost zero at acidic pH. (19) Mikkelsen, K.; Nielsen, S.O. J. Phys. Chem. 1960, 64, 632.

Some adsorption tests then were performed with PL. Only two pH values were tested: 5.5 and 9.2. At pH ) 5.5, all amine groups are expected to be protonated while at pH ) 9.5 the polymer likely bears a large fraction of NH2 groups. Immersions were performed in solutions without and with CaCl2 (2.5 mM). After rinsing the samples in pure water, the surface spectra are reported in Figure 2. In the absence of salt, one observes IR absorption bands showing that like PG, this peptide adsorbs in a weak amount on alumina surfaces. Considering the intensities of the peptide bands, the amount of PL is about twice as low than that of PG; moreover, at pH ) 5.5 or 9.5, there is no effect, or a very weak effect, of CaCl2 in solution upon the amount of adsorbed PL. 3.1.2. In situ Analyses. Immersions in PG and PG and CaCl2 solutions were repeated in D2O instead of H2O solution to perform an in situ analysis of the surface by PM-RAIRS. In fact, although the PM acquisition procedure is expected to cancel out all contributions from the bulk solution, when the adsorption experiments were conducted in light water, a huge absorption signal was observed in the 1600 cm-1 range that prevented any detection of the amide signals. This was because of some uncontrolled loss of polarization at a certain distance of the surface. Using D2O instead of H2O produces instead an intense absorption in the 1200 cm-1 region that does not prevent the observation of the peptide signals.19 The spectra recorded in the presence of the PG solutions at natural pD value, and corrected for the absorption at time 0, are shown in Figure 3b. When this procedure of data treatment is followed, the observed signals are strictly those from the adsorbed molecules. No absorption could be detected during the first 30 min (i.e., as long as the D2O solution contained no salt); as soon as the solution was changed to a D2O, CaCl2, and PG solution, one observed a rapid increase of the amide and carboxylic bands of the peptide. The saturation was attained after ca. 70 min. This experiment was repeated in the same PG solutions but modifying the pD by adding small amounts of DCl or NaOD as described in Experimental. The pD (where pD is related to pH via PD ) pH - 0.420) values tested were 2 and 11, so as to have the fully protonated and fully deprotonated forms of the peptide, (20) Kiss, A. B.; Keresztury, G.; Farkas, L. Spectrochim. Acta, Part A 1980, 36, 653.

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Figure 2. PM-IRRAS spectra of the alumina surface after immersion in PL solutions, at various pH. Lower spectra: PL in pure water. Upper spectra: PL in CaCl2 solutions. All surfaces have been rinsed in pure water and dried under nitrogen.

respectively. The in situ spectra recorded in the presence of the PG solutions are shown in Figure 3a, c. At pD 2, no PG adsorption was detected from solutions prepared in pure water; nevertheless, the spectrum recorded in the air shows that a weak amount of PG was indeed adsorbed (spectrum not shown). We carried out the adsorption at pH ) 2 in a manner slightly different from the experiments at higher pH: the in situ measurements (Figure 3, lower spectra) were started in the presence of CaCl2 without previous exposure to salt-free solutions, so that the effect of the salt could be studied before corrosion of the alumina surface makes the spectra unexploitable. Weak CdO stretch bands were observed from amide and COOH groups (1650 and 1710 cm-1), indicating the adsorption of a small amount of PG; they did not increase with contact time. At pD ) 11, again no amide bands could be seen in the absence of salt but the addition of CaCl2 strongly favored PG adsorption; the amide I and II bands increase with time in solution. Moreover, at that basic pH the band at ca. 1550 cm-1 is more intense than that at ca. 1650 cm-1, which is in contrast to what happened at neutral pD. The increase of the former signal, together with that at 1410 cm-1, can be explained by the contributions of the COOgroups plus that of the NH2 group of the peptide (δNH expected at ca. 1560 cm-1 21) accompanying the speciation change of the molecule. Adsorption of PL in D2O and in D2O and CaCl2 solutions was characterized in situ at neutral pH. A weak adsorption of the polypeptide was detected with no change upon addition of CaCl2 (see Supporting Information). 3.2. Adsorption of Poly-L-Glutamic Acid on Al2O3 Powders. 3.2.1. Ex situ Analyses. The FT-IR spectra of the sample obtained after contacting the alumina powder with the solutions of PG in the presence and absence of CaCl2 are shown in Figure 4. At neutral pH, no absorption attributable to the polypeptides was observed when the samples were prepared in the absence of CaCl2, while bands around 1650 and 1560 cm-1, corresponding (21) Pearson, J. F.; Slifkin, M. A. Spectrochim. Acta, Part A 1972, 28, 2403.

to the Amide I and II bands of the adsorbed polypeptide, respectively, are clearly visible in the spectra of the samples prepared with CaCl2. Thus, the behavior of this system is basically similar to that of the Al-coated planar surface. The same observations can be made at basic pH, except that in addition a strong band at 1418 cm-1 is present, which is probably mainly because of the formation of calcium carbonate by adsorption of CO2 from the surrounding atmosphere during the preparation. Note that the spectrum of pure carbonates directly formed in a CaCl2 solution in the absence of peptides and alumina also was recorded and revealed bands at exactly the same positions as those of the upper spectrum of Figure 4 (spectrum not shown). In the case of the adsorption at pH ) 2, the solution became opalescent already during the acidification before the addition of Al2O3 whether CaCl2 was present or not. This indicates that PG precipitates at acidic pHs, and thus the peaks appearing between 1720 and 1550 cm-1 are due to the solid precipitated polypeptide rather than to the adsorbed species. This interpretation is corroborated by the ATR spectrum of the solution obtained after microfiltration of the precipitate (not shown), which indicates that PG is not present in the solution anymore. Adsorption experiments also were performed in poly-L-lysine solutions at acidic, neutral, and basic pH, one without and one with CaCl2 (Figure 5). In this case, no adsorption could be detected whether in the presence or absence of calcium chloride at acidic or neutral pHs. At basic pHs, very small contributions at 1658 and 1540 cm-1 may correspond to the amide I and II bands, because they are quite close to the bands observed by Roddick-Lanzilotta and McQuillan for polylysine/ TiO2;22 in the presence of CaCl2, strong calcium carbonate bands also are present, indicating the precipitation of calcium carbonate due to the adsorption of CO2 from the atmosphere during the preparation. (22) Roddick-Lanzilotta, A. D.; McQuillan, A. J. J. Colloid Interface Sci. 1999, 217, 194.

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Figure 4. Transmission FT-IR spectra of Al2O3 powder immersed in PG aqueous solutions at different pH and in the presence or in the absence of CaCl2.

of calcium carbonate upon exposure to the ambient atmosphere (see Supporting Information). Figure 3. In situ PM-IRRAS spectra of the alumina surface in contact with PG D2O solution; CaCl2 was added to the solutions at pD ) 7 or 11; CaCl2 was present from the beginning at pD ) 2.

3.2.2. In situ Analyses. The ATR-IR spectra of a suspension of Al2O3 powder in PG aqueous solutions at neutral pH in the absence or in the presence of CaCl2 are shown in Figure 6a and b. In the absence of CaCl2, the peaks at 1652 and 1550 cm-1, attributed to the amide bands of PG, do not change in intensity with time. When CaCl2 is present in the solution, the initial intensity of these bands is much higher; moreover, they significantly increase with the contact time. This shows an enrichment of the concentration of PG on the surface of alumina, deposited on the ZnSe ATR crystal, compared to the starting solution. These observations confirm that, at neutral pH, PG is preferentially adsorbed on alumina when calcium chloride is present in the solution, whereas only little adsorption is detected in the absence of CaCl2. The above experiments were repeated with the poly-L-lysine peptide. Irrespective of whether CaCl2 was present in solution or not, the spectrum did not change with time with very weak bands at ca. 1400, 1547, and 1644 cm-1; no noticeable adsorption of the peptide on the surface of alumina is detected in agreement with the data obtained ex situ. One may only note that in the presence of CaCl2, a slight increase of a band at 1458 cm-1, indicating the slow precipitation

4. Discussion One of the aims of this paper is to clarify the role of ions in solutions and of the pH on PL and PG adsorption. The first step in evaluating likely adsorption mechanisms is to assess the speciation of the two partners involved in the adsorption, namely, the alumina surface and the polyelectrolyte. The alumina surface is covered with hydroxyls (aluminols) that are expected to constitute reactive sites for organic molecule interaction,23,24 but also to determine the surface charge through their amphoteric behavior. The nature of these hydroxyl groups is still the object of much speculation25 but at an elementary level the surface can be described in the “1-site, 2-pK” model.26,27 In this frame, the speciation of the alumina surface mostly depends on its point of zero charge (PZC). For the γ-alumina powder we used, this is known to be 8.2. For the planar alumina thin film, the situation is less clear. According to van den Brand et al., passivation of aluminum in the air, followed by treatment in an acidic or basic solution, should result in a hydrated aluminum oxide layer (pseudobohemite oxide) with an increase of the OH and global surface charge when going from an acidic to a basic pretreatment.28 The PZC of boehmite and pseudoboehmite has (23) Kasprzyk-Hordern, B. AdV. Colloid Interface Sci. 2004, 110, 19. (24) Fowkes, F. M. J. Adhes. Sci. Technol. 1990, 4 (8), 669. (25) Digne, M.; Sautet, P.; Raybaud, P.; Euzen, P.; Toulhoat, H. J. Catal. 2002, 211, 1. (26) Hiemstra, T.; VanRiemsdijk, W. H. J. Colloid Interface Sci. 1999, 210, 182. (27) Piasecki, W. Langmuir 2002, 18, 4809.

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Figure 5. Transmission FT-IR spectra of Al2O3 powder immersed in PL aqueous solutions at different pH and in the presence or in the absence of CaCl2.

been variously reported in the 7.2-9.2 range according to the sample studied.29-31 In the glutamic acid molecule, the pKavalues of the terminal (side chain) COOH is 2.2 whereas that of the internal COOH is 4.25. Polyglutamate in solution is a weak polyacid with a pKa for the side chains carboxylic acid group that also is close to 4.2. In comparison, the effective pKa of PG has been reported to lie around 2.5 in an immobilized film.4,32 As for PL, its pKa value in solution is said to be around 933 and to increase in the immobilized film; in summary, polyglutamic acid becomes a stronger acid and polylysine a stronger base when deposited in films.4 These values have direct implications toward the possible ligand behavior of these polymers; at intermediate pH values, PG bears many carboxylate groups, which are specific ligands for calcium,34 whereas PL bears protonated ammonium groups, which are unable to act as ligands toward a Lewis acid such as Ca2+ (and a single, terminal carboxylate). The speciations of the surface and the polypeptides, estimated on this basis, are summarized in Table 1. Let us first consider polylysine adsorption. All experimental data converge to show that (i) only a limited amount of PL is (28) van den Brand, J.; Snijders, P. C.; Sloof, W. G.; Terryn, H.; de Wit, J. H. W. J. Phys. Chem. 2004, 108, 6017. (29) Alwitt, R. S. J. Colloid Interface Sci. 1972, 40, 195. (30) Sidorova, M. P.; Ermakova, L. E.; Savina, I. A.; Kavokina, I. A. Colloid J. 1997, 59, 495. (31) Vucina-Vujovica, A. J.; Jankovic, I. A.; Milonjic, S. K.; Nedelijkovic, J. M. Colloids Surf., A 2003, 223, 295. (32) Richert, L.; Arntz, Y.; Schaaf, P.; Voegel, J. C.; Picart, C. Surf. Sci. 2004, 570, 13. (33) Fasman, G. D. Handbook of Biochemistry and Molecular Biology; Boca Raton, FL, 1976. (34) Frau´sto da Silva, J. J. R.; Williams, R. J. P. The Biological Chemistry of the Elements; Oxford, 2001.

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adsorbed at low or intermediate pH with a somewhat higher amount at pH ) 11 (the latter data is only available for powders ex situ) and (ii) the effect of CaCl2 in the adsorbing solution is negligible. We may look at the electric charges of the surface and polylysine in Table 1 and ask whether these trends are compatible with an electrostatic adsorption mechanism for the polyelectrolytes. The only clear conclusion is that a strong repulsive electrostatic interaction must be present at pH ) 2. However, the hypothesis of a purely electrostatic adsorption does not explain the more extensive adsorption at pH ) 11. Moreover, the addition of CaCl2 would be expected to screen the repulsive electrostatic interaction at pH ) 2, and therefore to favor adsorption, which is not observed. Let us now consider the adsorption of polyglutamate. In the absence of CaCl2, both in situ and ex situ data on the planar surface indicate significant adsorption at pH (or pD) ) 2 (the ex situ data on powder alumina, however, suggest that the presence of polyglutamate in the solid phase may be because of bulk precipitation35 followed by nonspecific deposition). At neutral pH, the adsorption is less significant and it might be more important again at pH ) 11, according to the ex situ data on the planar surface, which are not confirmed by the in situ data in this case. Also in this case, as shown by Table 1, electrostatic interaction alone does not provide a rationale for the observed trends. The experimentally observed effect of CaCl2 is to significantly increase PG adsorption at neutral and high pH (indicated by all four procedures) but probably not at low pH (according to ex situ powder and in situ planar surface data; ex situ planar surface data, however, indicate a small increase in the adsorbed amount). If the reason for this effect was a nonspecific electrostatic screening, one would expect it to be manifest only when there is a strong net repulsion between the surface and the polyelectrolyte (i.e., at pH ) 11) but not at pH ) 2 or 7. This does not correspond to the experimental observations. More generally, when comparing the two electrolytes the electrostatic hypothesis does not explain why the promoting effect of CaCl2 is manifest on polyglutamate but not on polylysine adsorption. Thus, this comparison speaks against a primarily electrostatic mechanism in polyelectrolytes adsorption. The low PL adsorption also tends to show that the often invoked hydrophobic interactions between peptides chains in certain conformations36,37 are not sufficient to enable the formation of peptide multilayers in our samples. Conversely, a specific interaction by the formation of “calcium bridges” between the side chain groups of the polyelectrolyte and the surface groups of alumina is in line with experimental evidence. These bridges would correspond to a partly covalent, localized binding comparable to the well-defined coordination complexes known in biochemistry for calcium-binding proteins.38 In this model (see Scheme 2a), calcium ions would be coordinatively bound to negatively charged surface aluminolates, and two or more ligands from the polyelectrolyte. Only negatively charged carboxylates are good ligands for calcium ions, conversely to carboxylic acid, and especially to protonated ammonium groups, explaining why the effect is only manifest at moderate and high pHs for PG and not at all for PL. Regarding (35) Richert, L.; Arntz, Y.; Schaaf, P.; Voegel, J. C.; Picart, C. Surface Sci. 2004, 570, 13. (36) Ball, V. Collloids Surf., A 2004, 33, 129. (37) Zhao, W.; Zheng, B.; Haynie, D. T. Langmuir 2006, 22, 6668. (38) Dudev, T.; Lim, C. Acc. Chem. Res. [Online early access.] DOI: 10.1021/ ar068181i. Published online: Oct. 14, 2006. http://pubs.acs.org/cgi-bin/asap.cgi/ acr/asap/html/ar068181i.html.

In Situ IR InVestigation of Two Polypeptides on Alumina Surfaces

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Figure 6. ATR-IR spectra of a suspension of Al2O3 powder in a PG aqueous solution at neutral pH; (a) in the absence of CaCl2 and (b) in the presence of CaCl2. Table 1. Speciation Data of Alumina, Polylysine, and Polyglutamic Acid as a Function of Solution pH approximate pH

alumina surface charge

PL net charge

2 7 to 9 11

positive close to neutral negative

positive positive close to neutral

PG net charge 2 neutral negative negative

PL, each polyelectrolyte molecule still has a single terminal carboxylate, and although this group has been proposed to play a role in the specific, coordinative adsorption of oligolysine on TiO2,22 this remained a minor effect in comparison to electrostatic interactions for PL. On the other hand, the condition on the surface state for this model to be operative is simply the presence of a large enough number of (AlO-) groups, which are predominant at pH > 8 and still present to a large extent at pH ) 7 (even though they may already be outnumbered by (AlOH2+) groups) but almost absent

at pH ) 2; this is compatible with the effect being observed at high and neutral but not at low pH. Therefore this coordinative binding, or calcium bridge hypothesis, accounts satisfactorily for the available data on polyglutamate adsorption enhancement. Of course not all of the PG carboxylate groups must be involved in calcium bridges with the surface; actually, free carboxylates may later form additional calcium bridges with another PG chain (see Scheme 2b), resulting in incipient multilayer formation. The slow (on a time scale of a few hours) increase in adsorbed PG bands, following the strong initial adsorption that is observed for in situ measurements on powders at pH 7, may be due to this phenomenon although alternative growth models based on hydrophobic interactions might be invoked.39 In this particular case, it could also be argued that the rather high polyelectrolyte concentration (1.5 g L-1) could cause flocculation in the solution, followed by nonspecific (39) Haynie, D. T.; Zhang, L.; Zhao, W.; Smith, J. M. Biomacromolecules 2006, 7, 2264-2268.

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Scheme 2. (a) Formation of a Calcium Bridge between the Polyelectrolyte Chain and the Surface. The Coordination of Calcium May Be Different from That Shown Here; the Cation May Be Coordinated to Water Molecules and/or Other Carboxylate Groups. (b) Incipient Multilayer Adsorption by Formation of Calcium Bridges

precipitation. However, the Ca-containing solutions remained translucent in the absence of alumina powders. Moreover, the slow building up of PG bands intensity is even more pronounced for in situ planar surface experiments (Figure 3) in which the PG concentration in solution is lower by a factor of 100 excluding bulk flocculation. Actually, the observed band intensity is impressive if one remembers that in this case the signal is attenuated by a 1 µm water layer; in these conditions, monolayer adsorption is often hardly distinguishable. The only likely explanation is that we are witnessing the formation of multilayers in a kind of “surface-induced aggregation”; although the PG concentration is much too low to induce polyelectrolyte chains aggregation, the increased concentration in the surface region due to adsorption is sufficient to start this phenomenon. One additional argument for surface-induced precipitation is provided by ex situ planar surface data: even after copious rinsing, a significant amount of peptides remains bound to the surface regardless of the pH of the solution. Since nonspecifically precipitated PG chains would be washed away by this treatment, it follows that the chains are strongly bound to the surface. Our hypotheses are equivalent to models that have been put forward in previous works and sometimes in lesser detail and/or using a different scientific language. Specific ion effects were already invoked in complement of DLVO theory to rationalize protein-salt interactions and understand bioseparation processes.40 The salt specificity, which may highly affect protein adsorption on a charged surface, also is related to the often cited and tentatively explained Hofmeister effect.41 In a recent study by Richert et al., dealing with the pH dependence of PL/PG films, these authors evidenced a shrinking of the films elaborated in pure water when submitted to a medium containing both Ca2+ and Cl-.18 They interpret their data in (40) Curtis, R. A.; Lue, L. Chem. Eng. Sci. 2006, 61, 907. (41) Moreira, L. A.; Bostro¨m, M.; Ninham, B. W.; Biscaia, E. C.; Tavares, F. W. Colloids Surf., A 2006, 282-283, 457.

terms of a “decreased hydration” of the films in the presence of salts. The formation of coordination compounds linking the polyelectrolyte chains would indeed cause a complete disruption of their hydration layer. Strong interactions between hydroxyls and the positively charged side chains of the PL in solution have already been shown to prevail over surface deposition of a PL complex hybrid on a present interface; this experiment was conducted in presence of calcium phosphate.42 More generally, our results are relevant to understand the effect of salts on protein adsorption as compared to aggregation from homogeneous solutions. They confirm the role of Ca2+ when carboxylate groups are present in a high amount on the protein side chains; similar to aggregation, adsorption of proteins is strongly dependent on the presence of carboxylates, and consequently on the pH, as well as on the presence of salts in the medium. For instance, this effect may account for the strong adsorption of bovine serum albumin and of some of its fragments on stainless steel surfaces at pH ) 3,43 or the effect of divalent cations, Ca2+ and Mg2+, on the adsorption of the same protein on stainless steel surfaces.44 To summarize the discussion, the observed enhanced adsorption of PG on alumina surfaces in the presence of salts and at low or neutral pH is ascribed to a specific, coordinative binding of Ca2+ ions with both the side chain of the PG molecules and the surface and/or with side chains of two different molecules; in the latter case the huge IR intensities are due to peptide multilayers. These molecular events manifest at the macroscopic level as enhanced monolayer adsorption and as surface-induced aggregation, respectively. It seems reasonable to assume that both effects exist and are likely to play a cooperative role. (42) Spoerke, E. D.; Stupp, S. I. Biomaterials 2005, 26, 5120. (43) Sakiyama, T.; Tomura, J.; Imamura, K.; Nakanishi. Colloids Surf., B 2004, 33, 77. (44) Pradier, C. M.; Costa, D.; Rubio, C.; Compere, C.; Marcus, P. Surf. Interface Anal. 2002, 34, 50.

In Situ IR InVestigation of Two Polypeptides on Alumina Surfaces

Conclusion This work has shown an in situ characterization of a metal oxide-liquid interface by IR operating in two different modes, PM-RAIRS and ATR, depending on the type of sample. The results demonstrate the promoting effect of Ca2+ cations upon adsorption of PG when the polymer bears carboxylate groups on its side chains. Adsorption of PG is strongly dependent on the pH, being maximal at pH above the COOH pKa value. The combined role of Ca2+ and COO- groups is confirmed by the experiments performed at low pH on PG, or on PL in which no enhancement of the adsorption was seen in the presence of salts. This paper also presents an original comparison of results obtained under similar conditions on planar model surfaces and on divided materials. Thanks to the two IR setups, ATR or PMRAIRS modes, one could analyze both types of materials in the same solutions and come to very similar results. This type of

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experiments validates the use of small area surfaces, like single crystals, that are often easier to control and characterize at a molecular level by surface science techniques. Acknowledgment. C. Poleunis from the “Unite´ de PhysicoChimie et Physique des Mate´riaux” (Louvain-la Neuve, Belgium) is gratefully acknowledged for the sample elaboration (evaporation of Al on a thin layer of Cr on silica wafers, in UHV conditions). The authors thank the CNRS, DGA, and IFREMER for financial help in the frame of a collaborative programme (GdR 2614 “Biosalissures Marines”). Supporting Information Available: In situ PM-IRRAS spectra of the alumina surface and ATR-IR spectra of a suspension of Al2O3 powder. This material is available free of charge via the Internet at http://pubs.acs.org. LA062208P