β-Casein Adsorption at the Silicon Oxide-Aqueous Solution Interface

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β-Casein Adsorption at the Silicon Oxide-Aqueous Solution Interface: Calcium Ion Effects David Follows,† Carl Holt,‡ Tommy Nylander,*,§ Robert K. Thomas,† and Fredrik Tiberg†,§ Physical and Theoretical Chemistry Laboratory, Parks Road, Oxford OX1 3PJ, United Kingdom, Hannah Research Institute, Hannah Research Park, Ayr, Scotland KA6 5HL, United Kingdom, and Physical Chemistry 1, Center for Chemistry and Chemical Engineering, University of Lund, Box 124, S-221 00 Lund, Sweden Received August 15, 2003; Revised Manuscript Received December 2, 2003

Neutron reflectometry was used to investigate effects of calcium ions on the interfacial behavior of β-casein at the silicon oxide-aqueous solution interface. The structural characteristics of the adsorbed layer were determined from reflectivity curves fitted to three- and two-layer optical models. The results showed that the presence of divalent calcium ions decreased the specific electrostatic adsorption affinity of the protein to silica compared with the calcium-free buffer system studied in an earlier work. In addition, it speeded up the adsorption suggesting that the slow kinetics seen in the calcium-free system are related to conformational adjustments of the β-casein structure driven by the maximization of the number of positive charges on the polypeptide interacting with negative surface charges. In the calcium-free system, a dense inner layer resulted from this process, with cationic segments firmly bound to the negative surface, whereas in the presence of calcium, a less dense inner layer was formed. The difference in binding is also mirrored by the effects on the interfacial layer of a specific proteolytic enzyme, i.e., endoproteinase Asp-N. In the calcium-free case, an inner dense layer remained at the surface after the proteolytic cleavage of the polypeptide, whereas virtually nothing was left after enzymatic action in the presence of calcium ions. Introduction β-Casein is the most abundant and amphiphilic protein among the four caseins present in milk.1 It readily forms micellar-like aggregates, analogous to ionic surfactant micelle formation, in aqueous solution starting at a concentration of ∼0.5 mg/mL.2,3 Apart from the nutritional value, the biological role of casein molecules is to stabilize the colloidal form of calcium phosphate in milk and thereby inhibit crystal growth in the secretory cells (glands).4-9 Caseins are also frequently used as additives in food, paint, glue, and coating colors for paper.4 Knowledge of the mechanisms by which caseins adsorb and interact at interfaces is fundamental for applications as well as to understanding the structure of the casein micelle. Studies of β-casein adsorption have mainly been performed at hydrophobic surfaces by a number of experimental techniques such as ellipsometry, surface force measurements, dynamic light scattering, neutron reflectivity, and wetting force measurements.10-21 The common picture emerging from these studies is that the protein forms a monolayer at hydrophobic surfaces, with the hydrophobic part of the protein sticking to the surface and the highly charged N-terminal portion protruding into the solution. The main driving force for adsorption at hydrophobic surfaces is the * To whom correspondence should be addressed. [email protected]. † Physical and Theoretical Chemistry Laboratory. ‡ Hannah Research Institute. § University of Lund.

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hydrophobic interaction between apolar protein segments and the hydrophobic surface. This picture of adsorption at hydrophobic surfaces was also experimentally verified by sensibly studying the effects of a specific proteolytic enzyme (endoproteinase Asp-N) on the interfacial β-casein layer.16,18,21 Endoproteinase Asp-N can potentially cleave the β-casein molecule at four different sites where two are located in the hydrophilic region (residues 43 and 47) and two in the hydrophobic region (residues 129 and 184) (see Figure 1).22,23 The results obtained at hydrophobic surfaces indicated that the enzyme could access the sites in the hydrophilic part of the protein and the rest of the adsorbed protein layer seemed to remain intact. This confirms that the protein adopts a brush-like structure when adsorbed onto hydrophobic surfaces with its hydrophilic fragments exposed to the solution. Despite the importance of β-casein as a stabilizer of dispersions of colloidal calcium phosphate and other hydrophilic colloids, relatively few studies of the adsorbed layer properties at hydrophilic surfaces have been conducted. Aside from the colloidal stabilization issue, understanding the surface interactions of β-casein at such surfaces is also relevant for cleaning and biofouling processes. The hydrophilic moiety of β-casein contains five phosphorylated serine amino acid residues, which binds divalent cations, like calcium, with high affinity.5-9 The present study deals with the effects of divalent calcium ions on the interfacial structure formation and adsorption kinetics of β-casein at the silicaaqueous solution interface. Thus, it is a natural extension of our previous ellipsometry and neutron reflection (NR) studies

10.1021/bm034301n CCC: $27.50 © 2004 American Chemical Society Published on Web 01/16/2004

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wetting force techniques.18,20 Further information about the interfacial availability of protein segments to Endoproteinase Asp-N attack as well as their interaction with the surface was obtained from observed effects that the enzyme has on β-casein surface coverage and interfacial structure. Experimental Details

Figure 1. Schematic representation of the charge distribution in β-casein. As illustrated in the figure, β-casein has a marked N-terminal charged hydrophilic region and a hydrophobic regime with almost zero net charge. The calcium binding sites, the serine-phosphate amino acid residues have a charge of -2 in the absence of calcium. The arrows indicate the potential cleavage sites of Endoproteinase AspN.

performed in calcium-free buffer,18,24 which showed that β-casein adsorbed extremely slowly at the silica-aqueous solution interface; a process ultimately leading to the formation of an asymmetric bilayer structure with an inner dense layer and an outer layer having similar characteristics as on the hydrophobic surface. To investigate the effects of divalent calcium ions on the interfacial structure formation, we choose in this study to exploit the NR technique. Prior to this investigation, we have also used ellipsometry to study β-casein adsorption on hydrophilic surfaces.18 One advantage of ellipsometry is that it is effective in studying fast adsorption processes. However, the output data ordinarily only permits the determination of mean layer thickness, which is obtained in the frame of a homogeneous slab model of the adsorbed layer. Because we were here mainly interested in effects of structure, neutron reflection was considered a more attractive choice with is higher structural sensitivity. Although the time-resolution of NR is not great, it turned out to be sufficient in our earlier investigation in which the adsorption of β-casein onto bare silicon oxide surface was found to occur over a period of several hours. Like ellipsometry, neutron reflection also measures the change of density profiles at an interface, but in the form of scattering length density instead of the refractive index profile.25-28 The main benefit here is the difference of scattering lengths between hydrogen and deuterium, which allows for contrast variation/optimization by substituting H2O by D2O. Thereby, the thickness resolution is greatly improved. Another factor leading to a greater depth resolution of the neutron reflection technique is the shorter wavelength of the beam. With the better resolution and the possibility of measuring at different contrasts, more suitable multilayer models can be used for interpreting the adsorption data. The combined effect is such that the inhomogeniety of the protein layer in the normal direction can be revealed. The reflectivity profiles obtained after different addition and rinsing sequences are discussed in terms of the structural properties of the adsorbed layer. When appropriate, the neutron reflection data are compared with previously reported data on β-casein adsorption obtained by ellipsometry and

The β-casein (genetic variant A1, Mw ) 24 000 g/mol) was extracted from bovine milk and purified according to the procedure described by Nylander and Wahlgren.16 Endoproteinase Asp-N was purchased from Boehringen Mannheim Biochemica (Cat. 1054589, Lot 14184025). The water used was passed through an Elgastat ultrapure water system (UHQ). Deuterated water (99.9%, deuterated) was obtained from Fluorochem. All other chemicals used were of analytical grade. β-Casein (0.1 mg/mL) or endoproteinase Asp-N (0.04 µg/mL) was dissolved in 50 mL of 0.02 M imidazol-HCl buffers (pH 7.0) containing 0.017 M calcium chloride. All aqueous H2O and D2O solutions featured in this study were buffered similarly. Fresh solutions were always prepared immediately before the adsorption measurement was started. The adsorption behavior of β-casein at the silicon oxideaqueous solution interface was studied by neutron reflection. As in previous investigations, the effects on the adsorbed layer properties of flushing the sample cell with proteinfree solution and subsequently adding a proteolytic enzyme (endoproteinase Asp-N) to the buffer solution was also investigated and shown to provide valuable information on the nature of the interfacial adsorption process. The neutron reflection measurements were made on the “white beam” time-of-flight reflectometer, D17, at the Institut LaueLangevin, Grenoble, France. Neutron wavelengths from 1 to 6 Å were used in these experiments. The sample cell consisted of a Teflon trough clamped against a silicon block of dimensions 12.5 × 5 × 2.5 cm3 and was maintained at a constant temperature of 298 ( 2 K by circulating water through the outer part of the cell.29 The collimated beam enters the end of the silicon block at a fixed angle, is then reflected at a glancing angle from the solid-liquid interface, and exits from the opposite end of the silicon block. Each reflectivity profile was measured at two different glancing angles of 0.7° and 4°, respectively. The results of these measurements were then combined. The beam intensity was calibrated in D2O with respect to the intensity below the critical angle for total reflection. A flat background determined by extrapolation to high values of momentum transfer Q (Q ) (4π sin θ)/λ, where λ is the wavelength and θ is the glancing angle of incidence) was further subtracted. The reflectivity profiles were always essentially flat for Q > 0.2 Å-1. The background for the D2O runs was typically 2 × 10-6 given in terms of reflectivity (see the next section). The procedure for polishing the large face (111) of the silicon block has been described earlier.29 The silicon blocks used were single crystals measuring approximately 2 × 5 × 12.5 cm. The large face was polished using an Engis polishing machine. The block was polished on a mat using 1 micron silca suspension followed by colloidal alumina.

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Thorough cleaning of the mat between grades of polishing fluid using demineralized water and detergent is necessary. The blocks were then cleaned in a three-step process. The blocks are first rinsed in UHQ water supplied by an Elga UHQ PS machine. Then they are soaked in a solution consisting of water, sulfuric acid (98%), and hydrogen peroxide (27.5% solution in water) mixed in a volume ratio of 5:4:1 respectively at 80 °C for 40 min. The blocks are then removed from the cleaning solution and allowed to cool for a few minutes before being quenched by immersion in UHQ water. Next, the blocks were placed in a stream of oxygen and exposed to ultraviolet light for 30 min. The surface of each block is then clean and very hydrophilic. This procedure rendered the surfaces hydrophilic and highly reproducible with respect both to the silicon oxide layer thickness and the β-casein adsorption. Before the adsorption measurements were started, the oxide layer at the surface of the silicon block was characterized in terms of structural parameters. The characterization was done in different isotopic compositions of water in order to reveal different features of the bare substrate such as roughness and oxide layer thickness. The water contrasts used were pure D2O, water CMSi (H2O/D2O weight ratio of 0.595/0.405), and finally pure H2O. The combined fitting gave the oxide layer thickness of 9 ( 2 Å and the scattering length density of 3.4 × 10-6 Å-2. The scattering length density is the same value as expected for amorphous silica, suggesting little penetration of water into the layer and hence a surface free of defects. Neutron Reflection. In a typical neutron reflection (NR) experiment, both incoming and reflected beam intensities are monitored, and the ratio of the two is the reflectivity, R. The neutron reflectivity is determined as a function of momentum transfer, κ, where κ ) 4π sin θ/λ (θ is the incidence angle and λ is the wavelength length). The reflectivity profile is determined by the variation of the scattering length density, F, along the normal to the surface. In turn, the scattering length density depends on the chemical composition of the sample as (cf. ref 26) F)

∑nibi

(1)

where ni is the number density of element i and bi is its scattering amplitude (scattering length). Different reflectivity profiles can be generated by exploiting isotopic substitution. This feature has been proven to be very effective in revealing the structure of complicated interfaces. Most relevant to this work is the substitution of hydrogen by deuterium. However, in contrast to polymers, it is practically impossible to do partial or full substitution of hydrogen in proteins. Isotopic contrast variation can be achieved by varying the ratio of H2O and D2O. Measurements of more than one reflectivity profile, for the same interfacial system, substantially improve the reliability of the derived structural information. The reflectivity profiles of the adsorbed layer in the present study were therefore recorded both in H2O and D2O. The derivation of structural information from reflectivity profiles is usually done by means of the optical matrix formalism, which has been described in detail elsewhere.30 A typical modeling procedure usually starts with an assump-

tion of a structural model for the adsorbed layer, followed by calculation of the reflectivity based on the optical matrix formula. The calculated reflectivity is then compared with the measured data, and the structural parameters are then varied in a least-squares iteration until a best fit is found. The structural parameters used in the fitting are the number of layers and the thickness (τ) and the corresponding scattering length density (F) of each layer. The area per molecule, A, can be deduced directly from the derived scattering length density and thickness of the layer using A)

∑mibi + nwbw Fτ

(2)

where ∑mibi denotes the total scattering length of the protein molecule, nw is the number of water molecules associated with each protein molecule, and bw is the scattering length of water. The total scattering length of the protein depends on its chemical composition, where each mi number of component i has a scattering length bi. The value of nw can be estimated from the following equation: nw )

Aτ - Vp Vw

(3)

where Vp and Vw are the molecular volumes for protein and water, respectively. From A the surface excess, Γ, can be obtained as Γ)

1 NaA

(4)

where Na is Avogadro’s number. The volume fraction of protein, φp, can be obtained from F ) φpFp + (1 - φp)Fw

(5)

where Fp and Fw are the scattering length densities of protein and water, respectively. Although eqs 2-5 have been developed under the condition of uniform layer distribution, they are directly applicable to each of the sublayers when more than one layer is required to model the density distribution profiles. The total adsorbed amounts are obtained by summing over the sublayers used in the fitting procedure. The choice of the number of sublayers is dependent upon the extent of inhomogeneity across the interfacial region. We do in general use the simplest model by which the experimental data can be successfully fitted, i.e., using a minimum number of sublayers. Although there may be arguments to suggest that a given sublayer is inhomogeneous in the surface plane, neutron reflection is not generally sensitive to such inhomogeneities and, except in unusual circumstances, determines only the average composition of a sublayer. Results and Discussion Figure 2 shows reflectivity versus the momentum transfer curves measured in buffered D2O solution prior to and after

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Figure 2. Reflectivity versus momentum transfer curves measured at different times after adding β-casein (0.1 g/L) into the measurement cell containing the 0.02 M imidazol-D2O buffer (pH7). Also shown are the measured profiles following 2 h exposure to protein-free buffer, and that measured subsequent exposure to the same buffer containing 0.4 mg/L endoproteinase Asp-N. The solid line shows the reference profile measured prior to any adsorption of protein.

injection of β-casein solution into the measurement cell. The calcium concentration in the buffer, 0.017 M, is similar to the total calcium content in milk (16-27 mM).4 Considering the large binding constants for Ca2+ as well as the large molar electrolyte-β-casein ratio used in our experiments of about 800:1, the five high affinity calcium binding sites of the protein are expected to be saturated. Also shown in Figure 2 is the curve measured after thorough rinsing of the sample cell with buffer solution (to a constant coverage of β-casein) and that following treatment with endoproteinase Asp-N. In contrast to previous measurements in calcium-free imidazol buffer, reflectivity profiles did not change significantly with time after the first measured run of β-casein adsorption. This suggests that plateau adsorption is established rather quickly, i.e., well within the first 30 min during which the first reflectivity profile is measured. This contrasts the earlier measurements in the calcium-free buffer for which plateau adsorption conditions were not attained even after a total measurement period of more than 7 h. Before going into the detail of the interfacial layer structure, we briefly discuss the changes in the reflectivity curves following rinsing with the protein free buffer and the addition of endoproteinase Asp-N. Following rinsing, the reflectivity in the low momentum transfer region increased significantly and the destructive interference moved in the direction of higher momentum transfer values. This indicates desorption of β-casein and a thinning of the interfacial layer. It should be noted that after each change of conditions reflectivity versus momentum transfer was recorded several times until no changes in the reflectivity profile could be detected. The given data are therefore expected to represent the equilibrium situation unless stated otherwise. Following the addition of the protease, the reflectivity profile became more or less identical to that obtained for the original bare surface; that is, the β-casein was completely removed. This is in contrast to the calcium-free system studied earlier, for

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Figure 3. Best -fit reflectivity versus momentum transfer curves to “β-casein 2.5 h” and “after rinse” curves in Figure 2 using a two-layer optical model for the adsorbed layer. Table 1. Time Evolution of the Properties of Adsorbed β-Casein Layers from Two-Layer Fits to Consecutive Neutron Reflectivity Measurements in D2O Solution (with 0.02 M Imidazol Set to pH 7 with HCl) during Exposure to 0.1 g/L β-Casein Solution run

t, h τ1 ( 3, Å τ2 ( 5, Å τT ( 8, Å F1 ( 5% F2 ( 5% φ1 ( 5% φ2 ( 10% Γ1 ( 5% Γ2 ( 10% ΓT ( 5%

1 2 3 4 5 β-casein β-casein β-casein after rinse after Asp-N 0.5 66 46 112 5 6 0.43 0.11 3.8 0.7 4.5

1.5 65 50 115 5 6 0.45 0.13 3.9 0.9 4.8

2.5 63 40 103 5 5.8 0.43 0.19 3.7 1.0 4.7

39 39 82 5.2 5.7 0.39 0.2 2.1 1.0 3.1

0 0 0 6.4 6.4 0 0 0 0 0

a t is time (h), τ is the layer thickness (Å), F is the scattering length ι i density (10-6 Å-2), φi is the volume fraction of protein, and Γi is the surface excess (mg/m2) of layer i. Subscript T stands for the sum over all layers.

which a residual thin and strongly adsorbed polypeptide layer remained after the action of the proteolytic enzyme. We now turn to the fits of interfacial structures to the experimental reflectivity curves. In our previous investigation of β-casein adsorption on silica from the calcium-free buffer,24 it was found that an optical model made of three sublayers was required for successful fitting of the experimental data. The same approach was therefore adopted in the initial fitting of the present reflectivity curves. However, it was quickly realized that for the adsorbed layers formed in the presence of calcium ions equally good fits could be obtained using a two-layer model. Typical fits to the experimental curves are shown in Figure 3 and are sufficiently close to the observations that they are not easily distinguished. Table 1 summarizes the best-fit results in terms of layer thickness, scattering length density, protein volume fraction, and surface excess values. To highlight effects of adding calcium ions to the system, thickness and volume fraction values are also presented in Figure 4 together with

β-Casein Adsorption

Figure 4. Comparison of β-casein adsorbed layer properties at silica from the 0.02 M imidazol-D2O buffer at pH7 with and without 0.017 M added CaCl2. The first and second stacked bar shows the adsorption, without and with calcium, respectively. The following two show the corresponding data obtained after rinsing with protein-free buffer solution, whereas the last two show the results after treating the remaining layer with Endoproteinase Asp-N. The structural information was obtained by fitting theoretical reflectivity profiles to experimental reflectivity versus momentum transfer curves. In the presence of calcium ions, the adsorbed layer could be well described using a simple two-layer optical model. However, the calcium-free system was best fit when dividing the first layer into two slabs with different protein volume fractions, L11 and L12, respectively. The stack-bars show the best-fit thickness values of each slab and the inserted values show the corresponding protein volume fraction values. Values for the calcium-free systems are taken from ref 24.

data obtained in the previously mentioned NR study.24 As noted earlier, neutron reflection does not distinguish between a sublayer of uniform lateral composition and one that is heterogeneous and so we cannot eliminate the possibility that β-casein adsorption may become patchy when the coverage becomes low. The total amount of β-casein adsorbed from the calcium containing imidazole buffer is about 4.7 ( 0.2 mg/m2 and the layer thickness is 110 ( 8 Å. This can be compared to an adsorption of 3.5 ( 0.4 mg/m2 and a layer thickness of 63 ( 10 Å measured by neutron reflection on hydrophobized silica in the presence of the same concentration of calcium chloride.21 At hydrophobic silica, β-casein is known to form a monolayer with hydrophobic segments anchoring and the highly charged N-terminal segments forming a polypeptide brush. Considering the larger surface excess and thickness values measured on hydrophilic silica, it seems sensible to propose a “bilayer-type” structure at this surface, where the build-up of the second layer is controlled by protein-protein interactions. Neutron reflection would not be directly sensitive to such lateral inhomogeneity; it would merely give the average composition, but the thickness of the layer is strongly suggestive of such structures. Such a mode of self-assembly of protein at the interface would be expected to parallel the mode of self-assembly of surfactants on such surfaces. The

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latter nearly always form more or less discrete aggregates even to the extent of being discrete surface micelles.31,32 In the case of β-casein, the formation of micelles in bulk solution is well established, although it is not clear that similar structures persist in the presence of calcium. It should also be noted that a large excess of calcium leads to precipitation of β-casein at 40 °C.23 However, under the experimental conditions used in the present study, we could not detect any signs of precipitation and β-casein solutions with calcium were not turbid. There is a large difference in effect of divalent calcium ions on the monolayer structure at hydrophobic surfaces and “bilayer” structure at silica. Adding electrolyte in the former case increases the density of the monolayer due to the screening of intra- and intermolecular repulsions between charged groups along the peptide chain. At the hydrophobic surface, we previously have observed clear indications of specific ion effects on the adsorption of β-casein.18,21 Thus, the amount of β-casein adsorbed from calcium-containing buffer was 20% higher than in a corresponding divalent magnesium ion-containing buffer, and 30% higher than that found using a solution of the same ionic strength but containing monovalent sodium ions instead of divalent calcium. Whereas the adsorbed amount and monolayer density at hydrophobic surfaces were found to be much higher in the presence of divalent ions than in their absence, the opposite effect is observed in the present investigation on the hydrophilic silica surface. It is evident the structure of the adsorbed layer structure is a balance between proteinprotein interaction at the interface (and/or in bulk) and protein surface interactions. As can be seen in Figure 4, the adsorbed β-casein layer density profile differs strongly depending on whether calcium ions are present in the solution. Thus, the “bilayer” structure formed in the presence of calcium ions is overall less dense in protein and more homogeneous than that formed in its absence. The adsorption process at silica is comparatively rapid in the presence of calcium ions indicating less conformational rearrangement than in the absence of calcium ions. In the latter case, the adsorption process was found to continue for more than 7 h and resulted in an asymmetric “bilayer“ structure, in which the sublayer closest to the surface was of higher protein density, with a protein volume fraction of 0.63 compared with 0.43 in the presence of calcium. The β-casein molecule is overall negatively charged and so is the silica surface and still we observed significant adsorption of the protein. However, in the β-casein molecule, there is a positively charged domain with a charge surlus of +5 in the more hydrophobic portion of the protein, i.e., between amino acid residues 134-183 as illustrated in Figure 1.33 Conformational adjustments to optimize the electrostatic interactions, e.g., between this domain and opposite charges on the silica substrate, may explain the slow adsorption kinetics observed in the absence of calcium ions. In the presence of calcium ions, the free energy to be gained by changing conformation at the surface seems to be much smaller, leading to a much more rapid stabilization at the interface as is evident from the repeated neutron reflection runs shown in Figure 2. This difference in structure and

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adsorption rate may originate in the competition between the divalent ions and positive protein charges for the negative surface groups on the silica surface. Although calcium binding to silica increases with pH at alkaline pH, the calcium binding is expected to be negligible at pH 7 at the low calcium concentration used in the present study.34 At least partly, the calcium effect is therefore likely to be a consequence of the protein-protein interactions, which are likely to be more attractive in the presence of calcium. We know for sure that no β-casein micelles are present in the bulk solution in absence of calcium at the low protein concentration used. In analogy with the formation of surfactant micelles, the “cmc” of BCN is expected to be lowered in the presence of calcium, although the self-assembly of β-casein in the presence of calcium is not fully explored. One can argue that the fact that we observe faster adsorption kinetics, indicating less rearrangements, as well as looser adsorbed layer suggest adsorption of micellar-like aggregates in the presence of calcium. Based on the present experimental data, it is impossible to say anything about the mode of interaction between such aggregates and the surface. The effect on the adsorbed layer properties of rinsing with protein free buffer is shown in Table 1 and Figure 4. Figure 3 shows the change in the reflectivity curves caused by rinsing. A significant amount of adsorbed β-casein is removed from the surface by rinsing the sample cell with the calcium containing buffer solution. About one-third of the originally adsorbed amount is desorbed from the silica surface, which although less in terms of adsorbed amount compared with the calcium-free system represents about the same ratio compared to the originally adsorbed mass. In both cases, however, the inner regions of the adsorbed layer seem unaffected by the rinsing. The adsorption on the silica thus appears to involve both low and high affinity binding of casein molecules, in line with the notion of a “bilayer” type structure. This contrasts with the behavior at hydrophobic surfaces where adsorption of β-casein is essentially irreversible.18,21 The effect on the adsorbed layer properties of adding the proteolytic enzyme endoproteinase Asp-N into the sample cell is shown in Table 1 and Figures 2 and 4. Here it should be noted that the enzyme activity was not affected by the presence of 0.017 M CaCl2 as observed in connection with our earlier studies on the effect of Endoproteinase Asp-N on β-casein layers on hydrophobic surfaces.18,21 Enzyme addition to the calcium-containing buffer resulted in complete removal of adsorbed β-casein segments from the silica surface (see Figures 2 and 3). This is in contrast to the situation in the absence of calcium ions, when the surface excess was reduced by about 75%, leaving behind a thin layer with a total thickness of about 49 Å. The enzyme can penetrate into the adsorbed layer and access a significant portion of the aspartic acid residues. This is in agreement with the less dense structure formed in the presence of calcium. Furthermore, the segments that remain after the enzymatic proteolysis have no affinity for the surface. This can be facilitated by strong intermolecular interactions in the calcium containing bulk solution, which help solubilize the hydrolysis products.

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Concluding Remarks In this work, we have confirmed the importance of electrostatic interactions as the driving force for the adsorption of proteins on charged surfaces, even in situations where the net-charge of the protein has the same sign as the surface. The presence of calcium has been found to have a profound effect on the structure of the adsorbed layer of β-casein on hydrophilic surfaces. It shows explicitly the importance of specific ion effects for the adsorption behavior of proteins. The present study is in contrast to our earlier observation of the adsorbed layer structure of β-casein on hydrophobic surfaces, where the presence of calcium merely leads to an increase of the packing of the brush-like structure.18,21 The large amount adsorbed on a hydrophilic surface, more than expected for a β-casein monolayer, shows that the adsorption is controlled by the protein-surface interactions as well as protein-protein interactions, where both are expected to be influence by the presence of calcium. The calcium concentration in milk is far above the saturation limit, where the excess of calcium is present as calcium phosphate nanoclusters.6,7 These are stabilized by calcium sensitive caseins in the casein micelle. One of the steps in forming the casein micelle may be regarded as adsorption of β-casein on a hydrophilic surface in the presence of calcium, where β-casein both ensures colloidal stability as well as limit the growth of the nanoclusters. It has been pointed out that the serine phosphate residues of the protein play an important role in the interaction between the colloidal calcium phosphate and β-casein.6-9,35 The casein micelles can be considered as a relatively uniform matrix that contains a disordered array of calcium phosphate ion clusters.9 Such a model implies that both the interaction between casein, e.g., β-casein, and the surface of the colloidal calcium phosphate particles and protein-protein interactions are important as our study suggests. The implications of interactions between the different casein have been discussed by Horne, who suggests that β-casein on the calcium phosphate nanoclusters can act as a linker to other caseins.36 Acknowledgment. This work was supported financially by the Engineering and Physical Sciences Research Council (EPSRC). References and Notes (1) Dickinson, E. Colloids Surf. B: Biointerfaces 1999, 15, 161-176. (2) Schmidt, D. G.; Payens, T. A. J. J. Colloid Interface Sci. 1972, 39, 655-662. (3) Leclerc, E.; Calmettes, P. Phys. ReV. Lett. 1997, 78, 150-153. (4) Walstra, P.; Jenness, R. Dairy Chemistry and Physics; WileyInterscience: New York, 1984. (5) Holt, C.; Sawyer, L. Protein Eng. 1988, 2, 251-259. (6) Holt, C.; Wahlgren, M.; Drakenberg, T. Biochem. J. 1996, 314, 1035-1039. (7) Holt, C.; Timmins, P. A.; Errington, N.; Leaver, J. Eur. J. Biochem. 1998, 252, 73-78. (8) Holt, C. J. Dairy Sci. 1998, 81, 2994-3003. (9) Holt, C.; de Kruif, C. G.; Tuinier, R.; Timmins, P. A. Colloids Surf. A: Physicochem. Eng. Aspects 2003, 213, 275-284. (10) Dalgleish, D. G.; Leaver, J. J. Colloid Interface Sci. 1991, 141, 288294. (11) Leaver, J.; Dalgleish, D. G. J. Colloid Interface Sci. 1992, 149, 4955. (12) Brooksbank, D. V.; Davidson, C. M.; Horne, D. S.; Leaver, J. J. Chem. Soc., Faraday Trans. 1993, 89, 3419-3425.

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β-Casein Adsorption (13) Mackie, A. R.; Mingins, J.; North, A. N. J. Chem. Soc., Faraday Trans. 1991, 87, 3043-3049. (14) Dickinson, E.; Horne, D. S.; Phipps, J. S.; Richardson, R. M. Langmuir 1993, 9, 242-248. (15) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 2847-2854. (16) Nylander, T.; Wahlgren, N. M. J. Colloid Interface Sci. 1994, 162, 151-162. (17) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657-660. (18) Kull, T.; Nylander, T.; Tiberg, F.; Wahlgren, M. Langmuir 1997, 13, 5141-5147. (19) Nylander, T.; Wahlgren, N. M. Langmuir 1997, 13, 6219-6225. (20) Nylander, T.; Tiberg, F. Colloids Surf. B: Biointerfaces 1999, 15, 253-261. (21) Nylander, T.; Tiberg, F.; Su, T.-J.; Lu, J. R.; Thomas, R. K. Biomacromolecules 2001, 2, 278-287. (22) Drapeau, G. R. J. Biol. Chem. 1980, 255, 839-840. (23) Wahlgren, N. M. A Nuclear Magnetic Resonance Approach to the Milk System; University of Lund: Lund, Sweden, 1992. (24) Tiberg, F.; Nylander, T.; Su, T. J.; Lu, J. R.; Thomas, R. K. Biomacromolecules 2001, 2, 844-850. (25) Penfold, J.; Richardson, R. M.; Zarbakhsh, A.; Webster, J. R. P.; Bucknall, D. G.; Rennie, A. R.; Jones, R. A. L.; Cosgrove, T.; Thomas, R. K.; Higgins, J. S.; Fletcher, P. D. I.; Dickinson, E.; Roser, S. J.; McLure, I. A.; Hillman, A. R.; Richards, R. W.; Staples, E. J.;

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