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

Biomacromolecules 2001, 2, 844-850

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β-Casein Adsorption at the Silicon Oxide-Aqueous Solution Interface F. Tiberg,*,† T. Nylander,‡ T. J. Su,§ J. R. Lu,§ and R. K. Thomas| Institute for Surface Chemistry, Box 5607, S-114 86 Stockholm, Sweden; Physical Chemistry, Center for Chemistry and Chemical Engineering, University of Lund, Box 124, S-221 00 Lund, Sweden; Department of Chemistry, University of Surrey, Guildford GU2 5XH, U.K.; and Physical and Theoretical Chemistry Laboratory, Parks Road, Oxford OX1 3PJ, U.K. Received February 16, 2001; Revised Manuscript Received April 24, 2001

Neutron reflectometry was used to investigate the time-dependent β-casein adsorption at the silica-aqueous solution interface. The transient and steady-state structural characteristics of the adsorbed layer were determined from reflectivity curves, fitted to three-layer and two-layer models. The results show that the β-casein adsorption to silica is very slow. The adsorption process involves the formation of an inner dense protein layer with a mean thickness of about 30 Å onto which a more hydrated outer layer is self-associated. The surface excess and the total layer thickness of the asymmetric bilayer were, after 5 h adsorption time, estimated to be about 6.5 mg/m2 and 105 Å, respectively. The adsorption behavior observed on silica contrasts with that previously reported for hydrophobic substrates, where β-casein adsorption is much more rapid and the final surface excess is less than half of that observed for silica. Rinsing the silica surface with protein-free buffer resulted in a substantial desorption; much more pronounced than observed for hydrophobic substrates. This behavior suggests a weak adsorption affinity for a fraction of the adsorbed casein molecules; most likely the outer self-associated casein molecules in the adsorbed bilayer. The comparative desorption from hydrophobic surfaces was shown to be marginal. The difference between the layer structures adopted on hydrophobic and hydrophilic surfaces is also mirrored in the effects that the addition of a specific proteolytic enzyme (endoproteinase Asp-N) has on the adsorbed layer properties. The rinsing and endoproteinase cleavage processes result together in more than 80% reduction of the originally adsorbed mass at the silica surface. Only a thin but dense adsorbed layer remains after these treatments. The corresponding reduction reported for the hydrophobic adsorbent system was only about 20%. It is concluded that β-casein adsorption on silica results in the formation of an asymmetric surface bound bilayer that stands in strong contrast to the monolayer structure formed at hydrophobic surfaces. This finding support the previous results obtained by using ellipsometry. The study also shows that neutron reflection, despite its limitations in time resolution, can be used for studying dynamic interfacial phenomena in protein systems. Introduction Among four caseins found in milk, β-casein is the most amphiphilic.1 β-casein, like many other amphiphilic molecules, forms micellar-like aggregates (cmc ∼ 0.5 mg/mL) in aqueous solutions.2,3 The biological role of casein is to stabilize the colloidal form of calcium phosphate in milk and thereby inhibit crystal growth in the secretory cells (glands).4-8 The stabilizing ability of casein is also the basis for its frequent use as additive in food, paint, glue and coating colors for paper.4 The nature of the surfaces of the colloidal material varies greatly between these different applications, where the primary function of the added β-casein is to modify the interfacial characteristics. Being the most abundant and surface active protein, β-casein is here bound to play a crucial role.1 * To whom correspondence should be addressed. † Institute for Surface Chemistry. ‡ University of Lund. § University of Surrey. | Physical and Theoretical Chemistry Laboratory.

Studies of β-casein adsorption have mainly been performed at hydrophobic surfaces. A variety of different experimental techniques have been used, including ellipsometry, surface force measurements, dynamic light scattering, neutron reflectivity, and wetting force measurements.9-19 The common picture of these studies is that the protein forms monolayers at hydrophobic surfaces, with the hydrophobic part of the protein sticking to the surface and the highly charged N-terminal portion extending into the solution. This picture is confirmed by our recent neutron reflection study of casein adsorption on hydrophobised silica.20 The main driving force for adsorption at hydrophobic surfaces is the hydrophobic interaction between apolar protein segments and the hydrophobic surface. This picture of adsorption at hydrophobic surfaces was also experimentally verified in studies where a specific proteolytic enzyme (endoproteinase Asp-N) was used to interact with the adsorbed layer of β-casein.16,17,20 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

10.1021/bm0155221 CCC: $20.00 © 2001 American Chemical Society Published on Web 07/25/2001

β-Casein Adsorption

hydrophobic region (residues 129 and 184).21,22 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 brushlike structure when adsorbed onto hydrophobic surfaces with its hydrophilic fragments exposed to the solution. Despite the importance of β-casein as stabilizer of dispersions of colloidal calcium phosphate and other hydrophilic colloids, surprisingly few studies of the adsorbed layer properties at hydrophilic surfaces have been presented. β-Casein adsorption at such surfaces may also be of interest with respect to hard surfaces of glass exposed to, e.g., milk. In this work, we have therefore investigated the adsorption behavior of β-casein molecules at the hydrophilic silicon oxide surface. This includes characterizing the kinetics of adsorption and the nature of the transient adsorbed layer structures thus adopted. Emphasis was also put on the effects of rinsing and added proteolytic enzymes. Prior to the neutron reflection work, we have examined β-casein adsorption on the hydrophilic surface using ellipsometry.16,17 One advantage of ellipsometry is that it is effective in studying fast adsorption processes. However, the output data ordinarily only permit the determination of mean layer thickness, which is obtained in the frame of a homogeneous slab model of the adsorbed layer. When the adsorbed layer density varies strongly in the normal direction, the meaning of such parameters as the layer extension becomes indistinct. In comparison with ellipsometry, neutron reflection is a rather slow technique. This limits its application for studying fast dynamic processes. Fortunately, however, the adsorption of β-casein onto bare silicon oxide surface occurs over a period of hours. Neutron reflection proved to be a very useful tool under such circumstance. Like ellipsometry, neutron reflection also measures the change of the refractive index profile, but in the form of scattering length density.23-25 The main benefit here is the difference of scattering lengths between hydrogen and deuterium. By substitution of H2O by D2O, the interfacial layer is highlighted. 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. Experimental Details 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 (catalog no. 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 endopro-

Biomacromolecules, Vol. 2, No. 3, 2001 845

teinase Asp-N (0.04 µg/mL) was dissolved in 50 mL of 0.02 M imidazole-HCl buffers (pH 7.0). 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 neutron reflection measurements were made on the “white beam” time-of-flight reflectometer CRISP at the Rutherford Appleton Laboratory, ISIS, Didcot, U.K. 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 cm3.26 The collimated beam enters the end of the silicon block at a fixed angle, is then reflected at a glancing angle from the solidliquid interface, and exits from the opposite end of the silicon block. Each reflectivity profile was measured at three different glancing angles of 0.35, 0.8, and 1.8°, 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 (wave vector) transfer κ (κ ) (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 κ > 0.2 Å-1, although the limiting signal at this point was dependent on the H2O/D2O ratio. The background for the D2O runs was typically 2 × 10-6 and for H2O was 3.5 × 10-6, given in terms of reflectivity (see next section). The procedure for polishing the large face (111) of the silicon block has been described earlier.26 Before use, the surface was cleaned in 5% Decon 90 solution, and the whole block was then thoroughly rinsed in UHQ water. Following this, the silicon block was then immersed in a mixture of 25% NH4OH (pro analyse, Merck), 30% H2O2 (pro analyse, Merck), and H2O (1:1:5, by volume) at 80 °C for 5 min, followed by rinsing and another cleaning step in a mixture of 32% HCl (pro analyse, Merck), 30% H2O2 (pro analyse, Merck), and H2O (1:1:5, by volume) at 80 °C for another 5 min. 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 CMSiO2 (H2O/D2O weight ratio of 0.401/0.599), water CMSi (H2O/D2O weight ratio of 0.595/0.405), and finally pure H2O. The combined fitting gave the oxide layer thickness of 15 ( 3 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 experiment, both incoming and reflected beam intensities are monitored, and the ratio

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Tiberg et al.

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 obtained 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 24) F)

∑nibi

(1)

where ni is the number density of element i and bi its scattering amplitude (scattering length). In comparison with conventional techniques such as ellipsometry and X-ray reflection, the main advantage of neutron reflection is that the scattering length or scattering amplitude varies from element to element. Thus, different neutron reflectivity profiles can be produced by using 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 neutron reflectivity profiles is usually done by means of the optical matrix formalism, which has been described in detail elsewhere.27 A typical modeling procedure usually starts with an assumption 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, thickness (τ), and the corresponding scattering length density (F) for 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 interface, however, in general the minimum number of layers that will successfully fit the data is chosen. Results and Discussion The time-dependent adsorption of β-casein at the silicon oxide-water interface was studied by neutron reflection using a buffered aqueous solution containing 0.1 mg/mL β-casein. The effects on the adsorbed layer properties of flushing the sample cell with protein-free buffer and subsequently adding a proteolytic enzyme (endoproteinase AspN) to the buffer solution was also investigated and shown to provide valuable information on the nature of the interfacial adsorption process. Endoproteinase AspN has four potential cleavage sites along the β-casein polypeptide chain, which are located at residues 43 and 47 in the hydrophilic part of the protein and 129 and 184 in the hydrophobic part. By analyzing the effects of the protease, information about the availability of these segments to enzymatic attack was obtained. The reflectivity profiles obtained after different addition and rinsing sequences are discussed in terms of the transient structural properties of the adsorbed layer. Whenever possible, the neutron reflection data are compared with previously reported data on β-casein adsorption obtained by ellipsometry and wetting force techniques.17,19 Figure 1 shows reflectivity vs the momentum transfer curves measured in buffered D2O solution before and after injection of β-casein solution into the measurement cell. The reflectivity profiles change considerably with time after the addition of β-casein into the sample cell. The reflectivity in the low momentum transfer region decreases significantly, and the destructive interference moves in the direction of lower momentum transfer values with increasing adsorption time. This suggests a thickening of the adsorbed β-casein layer. The best three-layer fits to the reflectivity curves are also shown in Figure 1. The reasons for using a three-layer model for the adsorption measurements were the following: First the three-layer fits were found to be somewhat better than fittings with two- or one-layer models. Second, we had strong indications from our previous investigations using

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β-Casein Adsorption

Figure 1. Reflectivity vs momentum transfer curves measured in D2O-imidazole buffer solution before and after injection of the β-casein solution into the measurement cell. The different reflectivity curves represent measurements performed: (b) prior to injection of the β-casein adsorbent and after ([) 0.25 h, (]) 1.6 h, and (×) 4.9 h adsorption time in a 0.1 mg/mL buffered β-casein solution (i.e., runs 1, 3, and 8 in Table 1, respectively). The solid lines are the best-fit curves using a three-layer model of the adsorbed layer.

Figure 2. Time evolution of the total surface excess of β-casein (Γ) and the cumulative layer thickness (τ) of layers 1-3 as determined from best fits to the experimental reflectivity curves. The surface was first exposed to a 0.1 mg/mL β-casein in D2O-imidazole buffer solution at t ∼ 0. The time where rinsing with protein-free buffer was done is indicated by the open arrow, while the time when 0.04 mg/ mL endoproteinase Asp-N was added is indicated by the closed arrow.

Table 1. Adsorption of β-Casein on Silicaa run

1

2

3

4

5

6

7

8

t τ1 ( 3Å τ2 ( 3Å τ3 ( 10 Å τT ( 10 Å F1 ( 5% F2 ( 5% F3 ( 20% φ1 ( 5% φ2 ( 5% φ3 ( 20% Γ1 ( 5% Γ2 ( 5% Γ3 ( 20% ΓT ( 5%

0.25 20 20 15 55 4.7 5.9 6.1 0.48 0.13 0.07 1.3 0.4 0.14 1.8

0.92 29 21 20 70 4.4 5.7 6.0 0.57 0.19 0.1 2.3 0.5 0.3 3.1

1.6 30 28 25 83 4.2 5.1 6.0 0.63 0.37 0.1 2.6 1.4 0.3 4.3

2.3 30 32 31 93 4.2 4.9 5.8 0.63 0.42 0.16 2.6 1.8 0.7 5.1

2.9 30 35 35 100 4.2 4.8 5.7 0.63 0.45 0.19 2.6 2.2 0.9 5.7

3.6 30 35 40 105 4.2 4.7 5.6 0.63 0.48 0.22 2.6 2.3 1.2 6.1

4.3 30 35 40 105 4.2 4.6 5.4 0.63 0.51 0.28 2.6 2.4 1.5 6.5

4.9 30 35 40 105 4.2 4.6 5.4 0.63 0.51 0.28 2.6 2.4 1.53 6.5

a Time evolution of the properties of adsorbed β-casein layers from three-layer fits to consecutive neutron reflectivity measurements in D2O solution (with 0.02 M imidazole set to pH 7 with HCl) during exposure to 0.1 mg/L β-casein solution. t Is time (h), τi the layer thickness (Å), Fi the scattering length density, φi the volume fraction of protein, and Γi the surface excess (mg/m2) of layer i. Subscript T stands for the sum over all layers.

ellipsometry that β-casein adsorption on silica results either in the formation of an asymmetric bilayer structure or, less likely, a surface micellar structure.17 In both cases, a threelayer optical model seems a rational choice. The layer thickness, volume fraction, and surface excess values obtained from the fits to the reflectivity curves are shown in Table 1. The time dependence of the total surface excess and the cumulative thickness values are also displayed in Figure 2. These results illustrate nicely how the interfacial layer properties develop with time during successive adsorption, rinsing, and proteolytic action (induced by the addition of endoproteinase Asp-N). Overall, the results obtained in the neutron reflectivity experiment agree very well with the previously obtained results by ellipsometry; see Figure 3.

Figure 3. Time evolution of the surface excess of β-casein and the mean layer thickness on silica as determined by ellipsometry. The measurement was done in H2O-Imidazol buffer solution. The time where rinsing with protein-free buffer was started is indicated by the open arrow, while the time when 0.04 mg/mL endoproteinase Asp-N was added is indicated by the closed arrow.

Note that the ellipsometric measurement was performed under continuous stirring, while the neutron reflection study was executed without stirring. Because of these different measurement conditions, some dissimilarity in the time dependence is expected. Furthermore, the ellipsometric results were analyzed in the framework of a one-layer optical model, thus explaining the relatively small thickness obtained. Adsorption of β-Casein on Silica. The results presented in Table 1 and Figures 1 and 2 show that the adsorption of β-casein is a very slow process, which probably involves extensive molecular rearrangements in the interfacial region. The layer properties are not stabilized until the end of the 5-h period during which the adsorption was followed. During

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this time, a large surface excess of protein is built up. The adsorbed amount after 5 h adsorption time is almost 6.5 mg/ m-2, which is more than twice the amount corresponding to monolayer coverage at hydrophobic surfaces under identical experimental conditions as measured by neutron reflection (Γ ) 2.46-2.82 mg/m2),20 and ellipsometry (Γ ) 2.75 mg/ m2).17 This indicates that the adsorbed layer structure is not a monolayer. In our earlier study, it was suggested that an asymmetric bilayer is formed on silica under such conditions. This picture is supported if the reflectivity profiles are fitted to a three-layer optical model. The adsorbed layer has an inner region with a high protein volume fraction and two outer zones in which the volume fraction tails off. The volume fractions in these different regions were estimated to be 0.63, 0.51, and 0.28, respectively, at the end of the adsorption measurement. The results suggest that the protein initially anchors to the silica surface to form a rather thin and dense layer onto which a second layer self-associates. The thickness was estimated to 105 Å, which is much larger than the mean layer thickness determined by means of ellipsometry which was estimated to be 60 Å 17(see also Figure 3). As noted before, the mean thickness from ellispometry is not representative of the actual extension of the outer dilute fraction of protein molecules when the density varies strongly normal to the surface plane. A more representative picture of the adsorbed layer structure is obtained from the neutron reflectivity profiles fitted by a multilayer model. The driving force for adsorption on silica is not clear. The β-casein molecule is overall negatively charged and so is the silica surface. There exists, however, in the β-casein molecule a positively charged domain with a charge surplus of +5 in the more hydrophobic portion of the protein, i.e., between amino acid residues 134 and 183.28 The electrostatic attraction between this domain and charged groups at the silica surface may well facilitate adsorption when the electrostatic screening of negatively charged protein segments is sufficient. Hydrophobic interactions and hydrogen bonding may also contribute to the surface affinity. Although the time-resolution in the neutron measurements is not very good, it is sufficient to follow the slow buildup of the adsorbed layer noticed in the present study (see Figure 2). The long-term changes are readily accessible by this technique, but the initial adsorption regime observed in the ellipsometric study could not be identified. Table 1 and Figure 2 show nevertheless that the thickness and density in the inner part of the adsorbed layer are stabilized rather quickly with time, while much slower changes occur in the outer regions of the adsorbed layer. The slow buildup of the layer may be related to the fact that extensive rearrangements of protein molecules are needed in the adsorbed layer region to facilitate further adsorption from the bulk. A low affinity to preadsorbed casein molecules may also inhibit diffusive mass transfer from the bulk solution. Substantiation of the latter hypothesis requires the determination of the adsorption isotherms, which has not yet been done. In our previous ellipsometry study, it was concluded that the rate of the very first stages of adsorption process was similar for hydrophilic and hydrophobic surfaces, but adsorption at a hydrophobic surface was found to equilibrate much faster than that at a relatively

Tiberg et al.

Figure 4. Surface excess of β-casein vs the square-root of time during adsorption. The data are taken from Figures 2 and 3.

hydrophilic silica surface. The adsorption kinetics measured at the silica/aqueous solution interface by the neutron reflectivity and the ellipsometry technique are shown in Figures 3 and 4, respectively. The results obtained in the neutron reflection experiment show that adsorption initially increases proportional to the square-root of time. The curves from both experiments are, however, qualitatively similar. The initial adsorption in the ellipsometric experiment is somewhat faster than in the neutron reflection experiment, presumably because of stirring. Since the adsorption isotherms have not been measured, it is not possible to conclude directly whether the adsorption is diffusion-controlled or not. Taking into account the small influence of the different hydrodynamic conditions in the two experiments and the much lower adsorption rates on silica compared to on hydrophobised silica, it is nevertheless reasonable to propose a kinetic mechanism where structural rearrangements in the adsorbed layer are rate determining. This notion is supported by the fact that the adsorption measured in the ellipsometry experiment initially is faster than that in the corresponding neutron experiment, whereas the situation is reversed at higher surface coverage values. Thus, the faster initial adsorption detected in the ellipsometric experiment may not allow proper ordering in the inner layer, which may then result in the slower adsorption observed at higher surface coverages. An alternative explanation of the pattern of the adsorption kinetics is as follows. The buildup of a second layer is effectively driven by the protein-protein interaction, similar in nature to the interactions leading to the formation of β-casein micelles, which should be rather weak considering the high critical micellar concentration of β-casein. That this is also the case at the interface can be deduced from the reversible character of the adsorption for a large fraction of the β-casein adsorbed at the silica surface (see Figure 2). A straightforward description of the adsorption kinetics in terms just of adsorption and desorption rate constants has been suggested by Corsel et al.29 D(kon(Γmax - Γ(t))Cb - koffΓ(t)) d Γ(t) ) dt D + δkon(Γmax - Γ(t))

(6)

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β-Casein Adsorption Table 2. Rinsing and Exposure to Protein-Free Buffera run

1

2

3

time t1 t2 t3 τT F1 F2 F3 φ1 φ2 φ3 Γ1 Γ2 Γ3 ΓT

5.8 30 32 33 95 4.2 4.8 5.8 0.63 0.45 0.16 2.6 2 0.7 5.3

6.3 30 29 30 89 4.2 5.1 6 0.63 0.37 0.1 2.6 1.5 0.4 4.5

7.1 30 29 30 89 4.2 5.2 6 0.63 0.34 0.1 2.6 1.3 0.4 4.3

4 in CM4

5 in CM2.1

30 29 30 89 3.05 3.65 3.85 0.65 0.24 0.1 2.7 1.0 0.4 4.1

30 29 30 89 2.18 2.14 2.1 0.63 0.34 0.1 2.6 1.3 0.4 4.3

a Time evolution of the properties of adsorbed β-casein layers from three-layer fits to consecutive neutron reflectivity measurements in D2O (with 0.02 M imidazole set to pH 7 with HCl) after rinsing with protein solutions. Runs 4 and 5 are subsequent contrast variation measurements in CM4 and CM2.1. Symbols and errors are specified in Table 1.

Figure 5. Reflectivity vs momentum transfer curves measured in buffered D2O solution after (b) 4.9 h adsorption time of β-casein (run 8 in Table 2), then (]) 2 h of exposure to protein-free buffer (run 3 in Table 3), and finally ([) after about 1.2 h exposure to buffered solution containing 0.04 mg/mL endoproteinase Asp-N (run 3 in Table 4).

where D is the diffusion coefficient of the protein, δ is the thickness of the diffusion zone, Γ(t) is the adsorbed amount of protein at time t, Γmax is the maximal adsorbed amount, and kon and koff are the intrinsic adsorption and desorption rate constants. This simple equation can be used to discuss the adsorption data for the buildup of the second layer. If adsorption is diffusion-controlled, then the adsorption rate will decrease when the thickness of the unstirred layer, δ, increases. This is in agreement with the results seen in Figure 4. However, if δkonΓmax, D, then the adsorption process is controlled by the adsorption step. If we now return to Figure 4 and compare the adsorption process beyond the formation of the monolayer, it is obvious that the adsorption rate after about 1 h of previous adsorption is faster in the neutron experiment. This is not consistent with eq 6, which predicts a lower adsorption rate in the absence of agitation, provided that the adsorption is diffusion limited. The reason for the higher adsorption rate could be that the proteins adsorbed early on in the process have a longer time to rearrange to present binding sites for newly arriving casein molecules in the adsorbed layer. In other words, kon or koff varies with time, allowing for rearrangements within the interfacial zone, and the overall process is thus not diffusion-controlled. Effect of Rinsing with Protein-Free Buffer. The effect on the adsorbed layer properties of rinsing with protein-free buffer is shown in Table 2 and Figure 2. Figure 5 shows the change in the reflectivity curves caused by rinsing. A significant fraction of adsorbed β-casein is removed from the surface by rinsing the sample cell with protein-free buffer solution. This contrasts with the behavior at hydrophobic surfaces where adsorption of β-casein is essentially irreversible.20.17 The adsorption on the silica appears more complex involving both low and high affinity binding of casein molecules in the interfacial region. The inner layer appears not to be affected at all by the rinsing process, while more than half of the measured surface excess molecules in the outer regions (as defined by the optical model) is desorbed within 2 h after flushing the cell with protein-free buffer solution. Correspondingly, the layer thickness decreases from 105 to about 90 Å.

Table 3. Exposure to Endoproteinase Asp-N and Second Rinsinga run

1

2

3

4 rinse D2O

5 rinse D2O

6 in CM4

7 in CM2.1

t τ1 ( 4 Å τ2 (8 Å τT ( 12Å F1 ( 10% F2 ( 30% φ1 ( 10% φ2 ( 30% Γ1 ( 10% Γ2 ( 30% ΓT ( 15%

7.7 20 29 49 5 5.7 0.39 0.19 1.07 0.75 1.8

8.4 20 29 49 5.5 6 0.25 0.1 0.68 0.4 1.1

9.0 20 29 49 5.5 6 0.25 0.1 0.68 0.4 1.1

10 20 29 49 5.5 6 0.25 0.1 0.68 0.4 1.1

10.6 20 29 49 5.5 6 0.25 0.1 0.68 0.4 1.1

20 29 49 3.64 3.85 0.25 0.1 0.68 0.4 1.1

20 29 49 2.1 2.1 .25 0.1 0.68 0.4 1.1

a Time evolution of the properties of adsorbed β-casein layers from three-layer fits to consecutive neutron reflectivity measurements in D2O (with 0.02 M imidazole set to pH 7 with HCl) after addition of an endoproteinase Asp-N solution, followed by rinsing with buffered D2O. Runs 6 and 7 are subsequent contrast variation measurements in CM4 and CM2.1 with added imidazole buffer. The measurement time scale is stopped during contrast variation runs during which no changes were observed in the layer properties over this period of measurements. The symbols used are specified in Table 1.

Cleavage of Adsorbed β-Casein by Endoproteinase Asp-N. The effects on the adsorbed layer properties of adding the proteolytic enzyme endoproteinase Asp-N into the sample cell are shown in Table 3 and Figure 3. The reflectivity profiles could in this case be fitted well using only a twolayer profile. The surface excess was reduced by about 75% on the addition of endoproteinase and only a thin layer with a total thickness of about 49 Å remained. The polypeptide segments remaining after the enzymatic attack most probably include the amino acid residues 134-183 with a charge surplus of +5, which are located in the more hydrophobic portion of the protein. The small residual adsorption indicates that cleavage also occurs at residue 129 and perhaps also 184 in the hydrophobic part. These residues are not available for enzymatic attack when β-casein is adsorbed at the hydrophobic surface. Assuming that cleavage occurs at all potential cleavage sites and that the positively charged hydrophobic domain between residues 129 and 184 remains adsorbed after cleavage, then the predicted surface excess

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Table 4. New Addition of β-Casein and Third Rinsinga run

1

2

3

t t1 t2 t3 τT F1 F2 F3 φ1 φ2 φ3 Γ1 Γ2 Γ3 ΓT

11.4 20 30 15 65 4.7 5.9 6.1 0.48 0.13 0.07 1.31 0.53 0.14 1.98

12.1 30 30 40 100 4.6 5.8 6.05 0.51 0.16 0.09 2.09 0.66 0.49 3.24

12.7 30 30 40 100 4.4 5.6 6.0 0.57 0.22 0.1 2.34 0.9 0.55 3.79

4 rinse D2O

5 rinse D2O

6 CM4

13.5 30 30 30 90 4.4 5.6 6.0 0.57 0.22 0.1 2.34 0.9 0.41 3.65

14.1 30 30 30 90 4.4 5.6 6.05 0.57 0.22 0.09 2.34 0.9 0.37 3.61

30 30 30 90 3.17 3.68 3.87 0.57 0.22 0.09 2.34 0.9 0.37 3.61

a Time evolution of the properties of adsorbed β-casein layers from three-layer fits to consecutive neutron reflectivity measurements in D2O solutions (with 0.02 M imidazole set to pH 7 with HCl) after new exposure to a 0.1 mg/L β-casein solution, followed by rinsing with buffered D2O. Run 6 represents a contrast variation measurement performed in CM4. Symbols and errors are specified in Table 1.

should be about 1.1 mg/m2. This agrees well with the measured plateau value obtained after the addition of endoproteinase Asp-N (see Figure 2 and Table 3) thus supporting the idea that β-casein adsorption is facilitated by the surface affinity of this polypeptide segment. The calculated value is based on the fact that the adsorbed amount prior to cleavage is 4.3 mg/m2 as seen in Table 2 and that residues 129-184 constitute about 30% (w/w) of the 209 amino residues in the β-casein molecule. Readsorption of β-Casein on Silica with Residual β-Casein Polypeptide Segments. The effect of re-exposing the substrate with residual polypeptide segments to β-casein is shown in Table 4. The total surface excess increases from 1.1 mg/m2 prior to the exposure to the β-casein solution to about 3.8 mg/m2 after about 2 h adsorption. This corresponds to an increase of about 2.7 mg/m2, consistent with the surface excess obtained at the hydrophobic surface in the previous ellipsometric study.17 The thickness values measured after readsorption as well as limited desorption caused by rinsing are also consistent with those observed on a hydrophobic surface. The layer of β-casein left after exposure to endoproteinase Asp-N appears to be quite hydrophobic, at least judging from readsorption measurements of β-casein onto this layer. Final Remark. The insight gained from this study has some relevance to the self-association of β-casein in its solution micellar state. Thus, at the hydrophilic silica surface, β-casein self-assembles to form an asymmetric bilayer structure, where the inner surface bound β-casein layer functions as a substrate for further adsorption. It has been suggested that the calcium phosphate nanoclusters of casein micelles are stabilized by interaction with the phosphorylated sequences of the calcium sensitive caseins.5,7,8 Our results suggest that this interaction could be a β-casein bilayer type interaction, which effectively cross-links the calcium phos-

phate nanoclusters. This suggestion for cross-linking is in addition to the possibility for cross-linking provided by the Rs1- and Rs2-caseins, which unlike β-caseins contain two or more sequences rich in phosphorylated residues and hence could directly link the nanoclusters through a single peptide chain. Acknowledgment. This work was sponsored by the Swedish Foundation for Strategic Research (SSF) and the Institute for Research and Competence Holding (IRECO). References and Notes (1) Dickinson, E. Int. Dairy J. 1999, 9, 305- -312. (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-. (6) Holt, C.; Wahlgren, M. N.; Drakenberg, T. Biochem. J. 1996, 314, 1935-1039. (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) Dalgleish, D. G.; Leaver, J. J. Colloid Interface Sci. 1991, 141, 288294. (10) Leaver, J.; Dagleish, D. G. J. Colloid Interface Sci. 1992, 149, 4955. (11) Brooksbank, D. V.; Davidson, C. M.; Horne, D. S.; Leaver, J. J. Chem. Soc., Faraday Trans. 1993, 89, 3419-3425. (12) Mackie, A. R.; Mingins, J.; North, A. N. J. Chem. Soc., Faraday Trans. 1991, 87, 3043-3049. (13) Dickinson, E.; Horne, D. S.; Phipps, J. S.; Richardson, R. M. Langmuir 1993, 9, 242-248. (14) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 2847-2854. (15) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657-660. (16) Nylander, T.; Wahlgren, N. M. J. Colloid Interface Sci. 1994, 162, 151-162. (17) Kull, T.; Nylander, T.; Tiberg, F.; Wahlgren, M. Langmuir 1997, 13, 5141-5147. (18) Nylander, T.; Wahlgren, N. M. Langmuir 1997, 13, 6219-6225. (19) Nylander, T.; Tiberg, F. Colloids Surf. B: Biointerfaces 1999, 15, 253-261. (20) Nylander, T.; Tiberg, F.; Su, T. J.; Lu, J. R.; Thomas, R. K. Biomacromolecules 2001, 2, 278-287. (21) Drapeau, G. R. J. Biol. Chem. 1980, 255, 839-840. (22) Wahlgren, N. M. A Nuclear Magnetic Resonance Approach to the Milk System; University of Lund: Lund, Sweden, 1992. (23) 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.; Burgess, A. N.; Simister, E. A.; White, J. W. J. Chem. Soc., Faraday Trans. 1997, 93, 3899-3917. (24) Lu, J. R.; Thomas, R. K. J. Chem. Soc., Faraday Trans. 1998, 94, 995-1018. (25) Lu, J. R.; Thomas, R. K. The application of neutron and X-ray specular reflection to proteins at interfaces. In Physical chemistry of biological interfaces; Baszkin, A., Norde, W., Eds.; Marcel Dekker: New York, 2000; pp 609-650. (26) Fragneto, G.; Lu, J. R.; McDermott, D. C.; Thomas, R. K. Langmuir 1996, 12, 477-486. (27) Born, M.; Wolf, E. Principles of optics; Pergamon: Oxford, England, 1970. (28) Eigel, W. N.; Butler, J. E.; Ernstrom, C. A.; Farrell, H. M., Jr.; Harwalkar, V. R.; Whitney, R. M. J. Dairy Sci. 1984, 67, 1599. (29) Corsel, J. W.; Willems, G. M.; Kop, J. M. M.; Cuypers, P. A.; Hermens, W. T. J. Colloid Interface Sci. 1986, 111, 544-554.

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