β-casein adsorption at the hydrophobized silicon oxide−aqueous solution interface and the effect...

â-Casein Adsorption at the Hydrophobized SiliconOxide -Aqueous Solution Interface and the Effect of Added

Electrolyte

Tommy Nylander,*,† Fredrik Tiberg,‡ Tsueu-Ju Su,§ Jian R. Lu,§ and Robert K. Thomas|

Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, Box 124,S-221 00 Lund, Sweden, Institute for Surface Chemistry, Box 5607, S-114 86 Stockholm, Sweden,

Department of Chemistry, University of Surrey, Guildford GU2 5XH, U.K., and Physical and TheoreticalChemistry Laboratory, Parks Road, Oxford OX1 3PJ, U.K.

Received October 24, 2000

The effect of the presence of NaCl, CaCl2, or MgCl2 at the same ionic strength on the structure ofâ-caseinlayers adsorbed on hydrophobic surfaces has been investigated by neutron reflectivity measurements. Thedata were fitted to a four-layer model. The volume fraction versus distance profiles have a similar shapewhetherâ-casein is adsorbed from NaCl, CaCl2, and MgCl2 of the same ionic strength or whether the proteinconcentration is lowered 10 times. In particular at larger distances from the surface, the volume fractionvalues are low and similar. However, close to the hydrophobic surface the volume fraction of protein decreasesin the order CaCl2 > MgCl2 > NaCl. We have also used a specific proteolytic enzyme, endoproteinaseAsp-N, which cleaves off the hydrophilic part ofâ-casein, as a tool to reveal the interfacial structure of theprotein. For all the different types of added electrolytes, endoproteinase Asp N only affects the outermostâ-casein layer. Subsequent addition ofâ-casein in all cases led to large increases in amounts adsorbed andin the thickness of the outer layers.

Introduction

The milk proteins and casein in particular have, due totheir excellent properties as stabilizers of emulsions, foams,and dispersions, come to be used in a range of applicationsboth in foods and in other applications, such as glue, paint,and putty.1-3 One of the major constituents of milk caseinis â-casein.4 Therefore, quite a number of studies have beendevoted to the behavior ofâ-casein at the air-aqueous andoil-aqueous interfaces and the interfaces between hydro-phobic solid surfaces and an aqueous phase.5-20 These studieshave been conducted under various conditions, using a rangeof different experimental techniques and various types ofhydrophobic surfaces. However, they all seem to be consis-tent in terms of the adsorbed amount, of the order of 3 mg/m2, and in the structure of the adsorbed layer. One reasoncould be that the protein has such a strong amphiphiliccharacter, with the amino acid sequence divided into onehydrophilic and one hydrophobic domain. Thus,â-caseinforms quite well structured monolayers on hydrophobicsurfaces, in which the hydrophobic protein segments areattached to the surface, forming a densely packed inner layer.The highly charged N-terminal portion of this casein extendsinto the aqueous solution to form a brushlike structure. Theconsequence is that the surface becomes more hydrophilic,and this is manifested by a change in the wetting properties.20

There are strong similarities betweenâ-casein and am-phiphilic (di-)block copolymers in terms of their ability toassociate in solution and in their interfacial behavior.â-Casein, for instance, forms micellar-like aggregates (cmc≈ 0.5 mg/mL) in aqueous solutions.21,22In comparison withblock copolymers the structure of interfacial layers ofproteins is hard to model due to the variety of monomers,amino acid residues, present in the proteins. However,proteins have the advantage that they are monodisperse.Thus, we know thatâ-casein contains 209 amino acidresidues in a particular order giving it a molecular mass of24 020 Da,4,23 although different genetic variants, where afew of the amino acid residues are different, exist. Dickinsonet al. have modeled the structure of the adsorbed layer of amodel polymer, resemblingâ-casein, on a hydrophobicsurface,13,14,17 by using the self-consistent field theories ofScheutjens and Fleer.24 Their results qualitatively confirmthe structure based on experimental studies. However, themodel calculations showed no evidence of two distinct layerswith markedly different volume fractions of protein segments.Instead, the volume fraction of protein segments decreasesexponentially with the distance from the surface. They alsofound significant effects of ionic strength and pH on thesegment density profiles. Similar effects have been demon-strated in a number of experimental studies.3,9,11,15,18This isdue to the large number of charged and chargeable residuesin the N-terminal 50 residues of the protein. In fact the netnegative charge of this part is at least-16 at pH 7.4 Thesecharge residues include five phosphorylated serines, whichgives the protein a specific affinity to divalent ions such as

* To whom correspondence should be addressed. E-mail:[email protected].

† Lund University.‡ Institute for Surface Chemistry.§ University of Surrey.| Physical and Theoretical Chemistry Laboratory.

278 Biomacromolecules 2001,2, 278-287

10.1021/bm0056308 CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 02/15/2001

calcium and magnesium.25,26Our earlier study indicated thatthe surface excess increased in the order NaCl< MgCl2 <CaCl2 at constant ionic strength, although no significantchange in the layer thickness was observed.15 However, otherstudies have indicated a decrease in layer thickness oncalcium addition.11,18 To investigate further the effect ofadded electrolyte on the structure ofâ-casein layers onhydrophobic surfaces, the present neutron reflectivity studywas undertaken. This has previously been demonstrated tobe a powerful technique for studying the structure ofâ-caseinlayers at hydrophobic surfaces12 as well as the air-waterinterface.8,11,13 We have also made use of a specific pro-teolytic enzyme, endoproteinase Asp-N,27 as a tool to revealthe interfacial structure ofâ-casein.10,15,20This protein hasfour potential cleavage sites for the enzyme, where two arelocated in the hydrophilic part (residues 43 and 47) and twoin the hydrophobic part (residues 129-184).

Materials and Methods

The â-casein (genetic variant A1, Mw ) 24 000 g/mol)was extracted from bovine milk and purified according tothe procedure described by Nylander and Wahlgren,10 wherealso details of the analyses of the sample are given. Thesample was free from contamination of other proteins asdetermined by FPLC and in addition the protein was appliedon a Chelex-100 (200-400 mesh, BioRad) column forcomplete removal of cations before being lyophilized.Endoproteinase Asp-N was purchased from BoehringenMannheim Biochemica (catalog no. 1054589, lot 14184025).The water used was passed through an Elgastat ultrapurewater system (UHQ). Deuterated water (99.9%, deuterated)was obtained from Fluorochem. All other chemicals usedwere of analytical grade.â-Casein (0.1 or 0.01 mg/mL) orendoproteinase Asp-N (0.04µg/mL) was dissolved in 50 mLof 0.02 M imidazol-HCl buffers (pH 7.0) containing theappropriate amount of sodium chloride, calcium chloride,and magnesium chloride as described in the Results. Freshsolutions were always prepared immediately before theadsorption measurement was started.

The procedure for polishing the large face (111) of thesilicon block and hydrophobizing with deuterated octa-decyltrichlorosilane (d-OTS, C18H37SiCl3) has been describedearlier.28 We will describe only the basic steps. The siliconblock was initially soaked for 10 min in nitric acid followedby etching for 10 min in a pH-buffered mixture of NH4F(40%)/HF (10%) at volume ratio of 7:1. The substrate wasthen immersed in a mixture of 25% NH4OH (pro Analysi,Merck), 30% H2O2 (pro analysi, Merck), and H2O (1:1:5,by volume) at 70°C for 10 min, followed by thorough rinsingby water and finally CH2Cl2. The self-assembled layer ofd-OTS on the silica surface was formed by immersing theblock in a freshly prepared solution of d-OTS in CH2Cl2 ata concentration of 9.4× 10-4 M for 1 h. The block wasthen rinsed in CH2Cl2, ethanol, and water. Before use andbetween each adsorption experiment, the surface was cleanedin 5% Decon 90 solution and the whole block was thenthoroughly rinsed in UHQ water.

The neutron reflection measurements were made on the“white beam” time-of-flight reflectometer CRISP at the

Rutherford Appleton Laboratory, ISIS, Didcot, U.K. Adetailed description of the experimental procedure and dataevaluation employed in this study is given elsewhere.29-32

The sample cell consisted of a Teflon trough clamped againsta silicon block of dimensions 12.5× 5 × 2.5 cm3.28 Thecollimated beam enters the end of the silicon block at a fixedangle, is then reflected at a glancing angle from the solid-liquid interface, and exits from the opposite end of the siliconblock. The neutron reflectivity, that is the ratio of thereflected and incoming beam intensities, is determined as afunction of momentum transfer (wave vector),κ, whereκ

) 4π sin θ/λ (θ is the incidence angle andλ is thewavelength). Neutron wavelengths from 1 to 6 Å were usedin these experiments. Each reflectivity profile was measuredat three different glancing angles of 0.35°, 0.8°, and 1.8°,and the results were combined. The beam intensity wascalibrated using the totally reflected beam below the criticalangle with D2O in the cell. A flat background determinedby extrapolation to high values of momentum transfer,κ,was further subtracted. The reflectivity profiles were alwaysessentially flat forκ > 0.2 Å-1, although the limiting signalat this point was dependent on the H2O/D2O ratio. Thebackground for the D2O runs was typically 2× 10-6 andthe background for H2O was 3.5× 10-6, given in terms ofreflectivity.

The structural parameters of the d-OTS layer on the surfaceof the silicon block were fully characterized before anyadsorption of the protein. To remove entrapped air on thehydrophobic surface, the cell was initially filled with ethanol,which was swept from the cell with excess of water. Thecharacterization was then done in different isotopic composi-tions of water: pure D2O, water CMSiO2 (contrast matchingfor SiO2 with H2O/D2O weight ratio of 0.401/0.599), waterCMSi (contrast matching for Si with H2O/D2O weight ratioof 0.595/0.405), and finally pure H2O. The cell was thenfilled with protein solution (about 40 mL), generally preparedfrom D2O, and the reflectivity profile was recorded versustime until no changes with time could be observed. It tookabout 15 min after the protein was added until the reflectivitymeasurement was started, and each measuring cycle tookabout 40 min (depending on the neutron flux). At least twomeasuring cycles were performed, and the reflectivity profileswere compared. If they were similar, the protein solutionwas removed and the cell was filled with pure buffer preparedfrom D2O (or H2O for the experiment with CaCl2). Thissolution was then removed from the cell, and the cell wasfilled with a fresh aliquot of buffer. The reflectivity profilewas then recorded as for the protein solution. The bufferwas then changed to one prepared from H2O, and thereflectivity profile was recorded. This experimental protocolwas followed for the addition of enzyme and the secondaddition ofâ-casein.

The reflectivity profile,R(κ), is determined by the variationof the scattering length density,F, along the normal to thesurface,z, by

R(κ) ) 16π2

κ2

|F̂(κ)|2 (1)

Casein Adsorption Biomacromolecules, Vol. 2, No. 1, 2001 279

whereF(κ) is the one-dimensional Fourier transform ofF-(z), that is

In turn the scattering length density depends on the chemicalcomposition of the sample as (cf. ref 30)

whereni is the number density of elementi and bi is itsscattering amplitude (scattering length). After assumption ofa structural model for the adsorbed layer, the reflectivityprofile is calculated. The calculated reflectivity is thencompared with the measured data, and the structural param-eters are varied until the fit is optimized. The structuralparameters used in the fitting are the number of layers,thickness (τ), and the corresponding scattering length density(F) for each layer. The surface excess,Γ, for each layer canbe deduced directly from the derived scattering length densityand thickness of the layer using

where∑mibi denotes the total scattering length of the proteinmolecule,Na is Avogadro’s number,nw is the number ofwater molecules associated with each protein molecule, andbw is the scattering length of water. The total scattering lengthof the protein depends on its chemical composition, whereeach numbermi of componenti has a scattering lengthbi.The volume fraction of protein,φp, can for each layer beobtained from

whereFp andFw are the scattering length densities of proteinand water, respectively. The parameters used for the calcula-tions are given in Table 1.

Results

The characteristics of the silicon sample block with theOTS layer are given in Table 2.

As reported previously,12,28 the only model that could fitthe reflectivity profiles recorded for all four of the differentwater contrasts for the OTS layer on its own was a three-layer model. The lower oxide layer thickness was found to

be 18 Å, in agreement with these earlier studies. As discussedby Fragneto et al.,12,28the d-OTS layer could be divided intotwo layers, where the layer next to the silicon oxide surfaceis crystalline. Consequently, this 17 Å thick layer consistsalmost entirely of d-OTS with only 0.01 volume fractionentrapped air. This outer layer, however, is defective andcontains a significant amount of water and air. The thicknessof this layer was found to be about 20 Å, and the volumefraction of d-OTS is about 0.27. This defective layer isthicker than the value, 11 Å, reported earlier by Fragneto etal.28 However, they reported a higher surface coverage of5.9µmol/m2, compared with a d-OTS coverage of 1.7µmol/m2 calculated from the values given in Tables 1 and 2. Theouter d-OTS layer in the present study can therefore beconsidered to be more defective than that obtained in theearlier studies. It is worth commenting at this stage that thedivision of the OTS into two layers is somewhat artificial.A more gradual distribution would probably also fit the data,but the composition at the two limits, the total amount onthe surface, and the overall thickness, would be little changedfrom the simpler two-layer division. Here, and in thediscussion of theâ-casein results, we have opted for usingthe minimum number of distinct layers required to fit thedata. While this may not be entirely realistic, it is the standardpractice unless there are other reasons for describing thesystem in terms of smooth distributions (for example to fita theoretical model). The errors quoted in Table 2 reflectthe maximum uncertainty in the fitting and any possiblecoupling of the fitting parameters. In the fitting we have alsoallowed for the presence of air. We do normally see someair present at the hydrophobic surface unless it has beenspecially treated with ethanol and water. It is displaced whenadsorption of other material occurs. We note that the surfaceswere very hydrophobic and had a water contact angle of>100°. They also gave very reproducible scattering curves.

Since the outer part of the OTS layer was defective,â-casein was likely to penetrate into the outer part of theOTS. Thus when it came to fitting the reflectivity data foradsorbed protein, we used the parameters already given forthe oxide and lower OTS layer but allowed the scatteringlength density of the outer layer to vary according to howmuch protein was in it. It was immediately clear when fittingthe data for the protein that the volume fraction declines awayfrom the surface. This allows the choice of using a functionalform for the decay or of dividing the protein into uniformlayers each containing less protein than the last. Theresolution of the experiment cannot distinguish these two

Table 1. Constants Used for the Fitting of the Reflectivity Profiles(Taken from Reference 12)

materialdensity(g/cm3)

volume(Å3) b (10-4 Å) F (10-6 Å-2)

H2O 0.9975 30 -0.168 -0.56D2O 1.105 30 1.905 6.35water CMSi 1.038 30 0.621 2.07water CMSiO2 1.059 30 1.023 3.41Si 2.32 20 0.415 2.07SiO2 2.16 47 1.585 3.41-C18D37 0.7768 542 36.65 6.76â-casein 1.365 29594 532.6 1.80

F̂(κ) ) ∫-∞

+∞e-iκz F(z) dz

F ) ∑nibi (2)

Γ ) FτNa(∑mibi + nwbw)

(3)

F ) φpFp + (1 - φp)Fw (4)

Table 2. Characterization of the Hydrophobic Interface (d-OTS(95% D)/SiO2)a

layerτ ( 3(Å)b FD2O FH2O φOTS ( 0.1b φw ( 0.1b φair ( 0.1b

oxide 18 4.00 2.62 0.2OTS (inner) 17 6.35 6.35 1.0 0.0 0.0OTS (outer) 20 5.40 1.41 0.3 0.6 0.2

a τ is the layer thickness (Å), FD2O and FH2O are the scattering lengthdensities in D2O and H2O, respectively, and φOTS, φw, and φair are thevolume fraction of OTS, water, and air, respectively b The errors givenreflect the maximum uncertainty in the fitting and any possible couplingof the fitting parameters.

280 Biomacromolecules, Vol. 2, No. 1, 2001 Nylander et al.

cases and would not be able to distinguish at all clearly thefunctional form of the decay. We therefore opted to use adescription in terms of the minimum number of distinct layersrequired to fit the data. The experiment is sensitive to theoverall thickness of the layer until the volume compositiondrops below about 10%; i.e., the uncertainty in each layerthickness increases as there is less protein in it. It is alsosensitive to the total amount adsorbed and to the moredetailed distribution within the OTS layer. On the basis ofthis we have estimated the errors as shown in the tables,and these estimates include contributions that could arisefrom coupling of the fitting parameters. Our data wereanalyzed by implementing a four-layer model, where thelower layer is the outer, defective d-OTS-layer. Typicalscattering curves for the pure d-OTS layer at various stagesof the â-casein adsorption study are given in Figure 1.

The results from the fit to the experimental scatteringcurves are given in Tables 3-5. For the sake of clarity, theevolution of thickness and the surface excess in the fourdifferent layers during the different steps in the adsorptionexperiments are illustrated by bar charts in Figure 2-5. Thethickness of the outer d-OTS layer could in all cases be fittedto 20 Å. However, during the course of adsorption the densityof this layer changed, which indicates penetration of BCN.

The Adsorption of BCN. The adsorption data for BCNin the various electrolytes could all be fitted to the four-layer model (Table 3).

In every case the adsorption of BCN on the OTS surfacereaches a plateau after the first measuring cycle, which iscompleted about 55 min after adding the protein. Here, wenote that the time resolution of neutron reflectivity measure-ments is rather limited and the plateau might have beenreached after a significantly shorter time. The largest totalamount adsorbed is found in the presence of CaCl2 (Figure

4b), while the amounts in the presence of NaCl and MgCl2

(Figures 2b and 5b) are smaller but similar to each other.However the total thickness in the presence of the threeelectrolytes is quite similar (Figures 4a, 2a, and 5a, respec-tively). To see the effect of the protein concentration, oneadditional experiment was performed in the presence of 0.05M NaCl, with a 10-fold lower protein concentration of 0.01mg/mL. This led to a decrease in the adsorbed amount(Figure 3b) as well as in the total thickness of the proteinlayer (Figure 3a). The adsorbed amount in the first two layersis basically the same at this lower protein concentration(Figure 3b) as in the presence of NaCl and MgCl2 (Figures2b and 5b), while it is slightly higher in the presence of CaCl2

Table 3. Adsorption/Desorption of â-Casein on/from Hydrophobized Silicaa

adsorption rinse

0.05 MNaCl

0.05 MNaClb

0.017 MCaCl2c

0.017 MMgCl2

0.05 MNaCl

0.05 MNaClb

0.017 MCaCl2

0.017 MMgCl2

τ0 ( 3d 20 20 20 20 20 20 20 20τ1 ( 3d 8 8 6 8 8 8 6 8τ2 ( 5d 25 15 25 25 25 15 25 25τ3 ( 10d 25 25 30 30 25 30 25F0 4.7 4.7 2.44-3.02 5.1 4.7 4.7 3.9 5.1F1 4.2 4.4 1.47 3.8 4.2 4.4 3.4 3.8F2 5.8 5.8 0.03 5.5 6.0 5.9 5.5 5.7F3 6.1 6.1 -0.39 6.1 6.2 6.1 6.2φ0 ( 0.08d 0.36-0.49 0.36-0.49 0.44-0.72 0.24-0.37 0.36-0.49 0.36-0.49 0.44-0.72 0.24-0.37φ1 ( 0.1d 0.6 0.6 0.9 0.8 0.6 0.6 0.9 0.8φ2 ( 0.05d 0.16 0.16 0.25 0.25 0.1 0.13 0.25 0.19φ3 ( 0.03d 0.07 0.07 0.07 0.07 0.04 0.07 0.04Γ0 ( 0.2d 1.0-1.3 1.0-1.3 1.2-2.0 0.7-1.0 1.0-1.3 1.0-1.3 1.2-2.0 0.7-1.0Γ1 ( 0.1d 0.7 0.6 0.7 0.8 0.7 0.6 0.7 0.8Γ2 ( 0.15d 0.6 0.4 0.9 0.9 0.3 0.3 0.9 0.6Γ3 ( 0.1d 0.2 0.2 0.3 0.3 0.1 0.3 0.2τT 58 48 61 63 33 48 61 58ΓT/mg/m2 2.5-2.8 2.2-2.5 3.1-3.9 2.7-3.0 2.0-2.3 2.0-2.3 3.1-3.9 2.3-2.6

a The properties of adsorbed â-casein layers were obtained from a four-layer model fit to data obtained from neutron reflectivity measurements (in 0.02M imidazole-HCl buffered D2O, pH ) 7, with added electrolyte) after exposure to 0.1 mg/mL â-casein solution unless stated otherwise. τi is the layerthickness (Å), Fi is the scattering length density, φi is the volume fraction of protein, and Γi is the surface excess (mg/m2) of layer i, where i ) 0 is the OTSlayer next to the silica surface. Subscript T stands for the sum over all layers. b Containing 0.01 mg/mL â-casein. c In H2O. dThe errors given reflect themaximum uncertainty in the fitting and any possible coupling of the fitting parameters.

Figure 1. Neutron reflectivity versus momentum transfer curves in0.02 M imidazole-HCl buffered D2O solution, pH 7: the curve for theclean d-OTS block (×); the layer adsorbed from a 0.1 mg/mL â-caseinsolution containing 0.0167 M CaCl2 after changing solution to 0.02M imidazole-HCl buffered D2O solution (+); the layer after treatmentwith endoproteinase Asp-N (0.04 µg/mL) and subsequent rinsing inbuffered D2O solution (4); the layer formed after second addition ofa 0.1 mg/mL â-casein solution and subsequent rinsing in bufferedD2O solution (O) are shown together with the corresponding fits, whichare inserted as solid lines.


(Figure 4b). Here, we also note that the build-up ofâ-caseinlayer in the presence of CaCl2 was time dependent unlike inthe other conditions studied. It took about 2 h until theconstant reflectivity profile was obtained. The slower equili-bration time is related to the fact that a more dense layer isformed, which set demands on interfacial ordering and thusrequires more extensive rearrangements of casein molecules

at the surface. The thicknesses of the three inner layers arebasically the same for the different electrolytes, while thelower protein concentration leads to a substantial decreasein thickness of the outermost layer. However, the amountsadsorbed are different and below we will discuss this furtherin terms of protein volume fraction versus distance. Theadsorbed amount and the thickness of the two outer layers

Table 4. Exposure to Endoproteinase Asp-N and Subsequent Rinsinga

endoproteinase rinse

0.05 MNaCl

0.05 MNaClb

0.017 MCaCl2

0.017 MMgCl2

0.05 MNaCl

0.05 MNaClb

0.017 MCaCl2

0.017 MMgCl2

τ0 ( 3c 20 20 20 20 20 20 20 20τ1 ( 3c 8 8 6 8 8 8 6 8τ2 ( 5c 20 15 25 25 20 15 25 25τ3 ( 10c

F0 4.7 4.7 3.9 4.3 4.7 4.7 3.9 4.3F1 4.2 4.4 4.2 4.6 4.2 4.4 4.2 4.6F2 6.2 6.1 6.0 6.1 6.2 6.1 6.0 6.1F3

φ0 ( 0.08c 0.36-0.49 0.36-0.49 0.44-0.72 0.47-0.6 0.36-0.49 0.36-0.49 0.44-0.72 0.47-0.6φ1 ( 0.1c 0.6 0.6 0.6 0.5 0.6 0.6 0.6 0.5φ2 ( 0.02c 0.04 0.07 0.1 0.07 0.04 0.07 0.1 0.07φ3

Γ0 ( 0.2c 1.0-1.3 1.0-1.3 1.2-2.0 1.3-1.6 1.0-1.3 1.0-1.3 1.2-2.0 1.3-1.6Γ1 ( 0.1c 0.7 0.6 0.5 0.6 0.7 0.6 0.5 0.6Γ2 ( 0.07c 0.14 0.14 0.34 0.24 0.14 0.14 0.34 0.24Γ3

τT 28 23 31 33 28 23 31 33ΓT/(mg/m2) 1.8-2.1 1.7-2.0 2.0-2.8 2.1-2.4 1.8-2.1 1.7-2.0 2.0-2.8 2.1-2.4

a The properties of adsorbed â-casein layers after addition of an endoproteinase Asp-N solution, followed by rinsing with buffered D2O. The resultswere obtained from a four-layer model fit to data obtained from neutron reflectivity measurements in 0.02 M imidazole-HCl buffered D2O, pH ) 7. Theâ-casein layers were adsorbed from buffers containing different electrolytes and a protein concentration of 0.1 mg/mL â-casein unless stated otherwise.τi is the layer thickness (Å), Fi is the scattering length density, φi is the volume fraction of protein, and Γi is the surface excess (mg/m2) of layer i, wherei ) 0 is the OTS layer next to the silica surface. Subscript T stands for the sum over all layers. b Containing 0.01 mg/mL â-casein. cThe errors givenreflect the maximum uncertainty in the fitting and any possible coupling of the fitting parameters.

Table 5. Second Addition of â-Casein and Subsequent Rinsinga

second â-casein addition rinse

0.05 MNaCl

0.05 MNaClb

0.017 MCaCl2

0.017 MMgCl2

0.05 MNaCl

0.05 MNaClb

0.017 MCaCl2

0.017 MMgCl2

τ0 ( 3c 20 20 20 20 20 20 20 20τ1 ( 3c 8 8 6 8 8 8 6 8τ2 ( 5c 30 23 25 34 30 23 25 34τ3 ( 10c 35 30 35 37 25 25 35 30F0 4.2 4.1 3.8 4.3 4.2 4.1 3.8 4.3F1 3.2 3.8 3.2 3.1 3.2 3.8 3.2 3.1F2 5.0 5.3 5.2 4.8 5.7 5.7 5.2 4.8F3 5.9 6.1 6.1 5.6 6.2 6.2 6.1 5.8φ0 ( 0.08c 0.5-0.63 0.53-0.66 0.47-0.71 0.47-0.6 0.5-0.63 0.53-0.66 0.47-0.71 0.47-0.6φ1 ( 0.1c 0.9 0.8 0.9 1.0 0.9 0.8 0.9 1.0φ2 ( 0.05c 0.39 0.31 0.34 0.45 0.19 0.19 0.34 0.45φ3 ( 0.03c 0.13 0.07 0.07 0.22 0.04 0.04 0.07 0.16Γ0 ( 0.2c 1.4-1.7 1.4-1.8 1.3-1.9 1.3-1.6 1.4-1.7 1.4-1.8 1.3-1.9 1.3-1.6Γ1 ( 0.1c 1.0 0.8 0.8 1.0 1.0 0.8 0.8 1.0Γ2 ( 0.15c 1.6 1.0 1.2 2.1 0.8 0.6 1.2 2.1Γ3 ( 0.1c 0.6 0.3 0.3 1.1 0.1 0.1 0.3 0.7τT 73 61 66 79 63 56 66 72ΓT/(mg/m2) 4.6-4.9 3.5-3.9 3.6-4.2 5.5-5.8 3.3-3.6 2.9-3.3 3.6-4.2 5.1-5.4

a The properties of endoproteinase Asp-N treated â-casein layers after (second) addition of 0.1 mg/mL â-casein solution, followed by rinsing withbuffered D2O. The results were obtained from a four-layer model fit to data obtained from neutron reflectivity measurements in 0.02 M imidazole-HClbuffered D2O, pH ) 7. The â-casein layers were adsorbed from buffers containing different electrolytes and a protein concentration of 0.1 mg/mL â-caseinunless stated otherwise. τi is the layer thickness (Å), Fi is the scattering length density, φi is the volume fraction of protein, and Γi is the surface excess(mg/m2) of layer i, where i ) 0 is the OTS layer next to the silica surface. Subscript T stands for the sum over all layers. b Containing 0.01 mg/mLâ-casein. c The errors given reflect the maximum uncertainty in the fitting and any possible coupling of the fitting parameters.


are similar in the presence of both divalent electrolytes(Figures 4 and 5), while it is substantially less in the presenceof NaCl (Figure 2). A further decrease is observed if theconcentration of BCN is lowered (Figure 3). Thus, it appearsas if the largest effect of changing electrolyte and proteinconcentration is in the outer parts of the adsorbed layer.However, the picture is different in terms of protein volumefraction (see Discussion). Rinsing with pure buffer withoutadded electrolyte does not have any effect on the layeradsorbed from the protein solution containing CaCl2. For allother conditions a slight decrease of the adsorbed amount isobserved in the outer layer only. In all cases very smallchanges are observed in the thickness of the two outer layers.There is however one exception and that is for the presenceof NaCl, where the outermost layer seems to disappear.However, it should be emphasized that there is very littleprotein in the outermost layer and the accuracy in judgingthe amount in such a layer is low.

The Effect of Adding Endoproteinase AspN.For all theadded electrolytes, endoproteinase Asp N only affects theoutermostâ-casein layer (Table 4). In fact the data couldnow be fitted to a three-layer model. The largest reduction

in amount by a third of the total mass and by half the layerthickness is observed forâ-casein adsorbed from a solutioncontaining CaCl2 (Figure 4). The reduction in the totalamount is only about 10% for the other conditions, althoughthe reduction in total thickness was larger. It should beemphasized that the sample cell was rinsed with the samebuffer, that is, buffer without added electrolyte, before theendoproteinase Asp-N dissolved in 0.02 M imidazole bufferwas added. This was done to make sure that enzyme activitywas not affected by the added electrolyte ensuring that theenzymatic reaction was carried out in equal conditions.Rinsing with enzyme free buffer without added electrolytehad no effect on the enzyme-treated layer.

The Effect of a Second Addition ofâ-Casein.To assessfurther the effect of the enzyme treatment, and hence thestructure of the adsorbed layer, a second addition ofâ-caseinto the enzyme-treated layer was made. The protein was addedfrom buffer solution without added electrolyte. In every casethe second addition led to large increases in amountsadsorbed and in the thickness of the adsorbed layers. In fact,the layer became significantly thicker and contained signifi-cantly larger amounts ofâ-casein than the layer initiallyformed on the d-OTS, hydrophobic surface (Table 5).

The largest increase was observed in the presence of thedivalent electrolyte, MgCl2 (Figure 5). Here the second

Figure 2. Properties of the adsorbed layer obtained from fittingneutron reflectivity data to a four-layer model, where layer no. 0 isclosest to the surface (mixed d-OTS-â-casein layer) (solid black) andlayer 1 (hatched lines with narrow spacing), layer 2 (hatched lineswith broad spacing), and layer 3 (light gray) are indicated in the barcharts. The extension of the bars indicates the (a) thickness of thedifferent layers, τ, and (b) the amount of â-casein adsorbed, Γ.â-Casein was adsorbed from a solution containing 0.1 mg/mL proteinin 0.02 M imidazole-HCl and 0.05 M NaCl in D2O at pH 7 (BC),followed by rinse with 0.02 M imidazole-HCl in D2O at pH 7 (BCr),addition of endoproteinase Asp-N (0.04 µg/mL 0.02 M imidazole-HClin D2O at pH 7) (E), and rinse with 0.02 M imidazole-HCl in D2O atpH 7 (Er). A second addition of â-casein (0.1 mg/mL in 0.02 Mimidazole-HCl and in D2O at pH 7) was then made (2BC), followedby rinse with 0.02 M imidazole-HCl in D2O at pH 7 (2BCr).

Figure 3. Properties of the adsorbed layer obtained from fittingneutron reflectivity data to a four-layer model, where layer no. 0 isclosest to the surface (mixed d-OTS-â-casein layer) (solid black) andlayer 1 (hatched lines with narrow spacing), layer 2 (hatched lineswith broad spacing), and layer 3 (light gray) are indicated in the barcharts. The extension of the bars indicates the (a) thickness of thedifferent layers, τ, and (b) the amount of â-casein adsorbed, Γ. Thesame experimental procedure was used as the one used to obtainthe data in Figure 2, except that the â-casein concentration was 0.01mg/mL protein.


addition almost doubled the adsorbed amount, and a smallportion of this, about 8%, could be removed by rinsing withbuffer. Adsorption from a solution containing NaCl of thesame ionic strength also gives quite large amounts adsorbedupon the second addition ofâ-casein (Figure 2), but nowabout 28% of the mass was removed when rinsing with purebuffer. When the protein concentration is lowered 10-fold,the adsorption at the second addition is also lower (Figure3) and about 15% of the amount could be desorbed. Thelowest increase in adsorbed amount on second addition ofâ-casein is observed for the enzyme-treatedâ-casein layer,adsorbed in the presence CaCl2. No desorption upon subse-quent rinsing with protein-free buffer could be observed.Here we note that the largest reduction in adsorbed amountupon endoproteinase Asp-N addition was observed on thelayer adsorbed from CaCl2 solution. In all cases, apart fromadsorption to initially adsorbed layer formed in the presenceof NaCl, the second addition only causes changes in the threeoutermost layers. In the case of NaCl the volume fractionof theâ-casein in the innermost layer,φ0, also increases fromabout 0.6 to 0.9 or 0.8 at the lowerâ-casein concentration.

Discussion

In an earlier neutron reflectivity study Fragneto et al. useda two-layer model to evaluate their data on the adsorption

of â-casein on a d-OTS surface.12 Although our data arequalitatively almost identical to theirs, we have used a four-layer model in this study. The reason is that we wanted tomake a fair comparison with our neutron reflective study ofâ-casein adsorption on a hydrophilic surface.32 In that studyit turned out that the quality of fit increased significantlywhen increasing the number of layers from two to three. Dueto the presence of an imperfect outer layer of d-OTS, anextra layer was added in the model to account for penetrationof â-casein into this layer. The volume fraction ofâ-caseinsegments versus distance from the crystalline inner d-OTSlayer is plotted for different experimental conditions in Figure6.

The data are taken from Table 3 and the figure plots thevolume fraction at the center of each layer at the corre-sponding distance. The volume fraction ofâ-casein in theouter d-OTS layer has been calculated as the volume fractionremaining after subtraction of that of the OTS. In any case,the volume fraction ofâ-casein in this layer is substantiallysmaller than that in the innermost pureâ-casein layer, andthis causes the maximum in Figure 6. This is expected, asthe presence of d-OTS islands is likely to make packing ofâ-casein at the surface less efficient. The most obviousdifference between the results forâ-casein adsorption on thehydrophobic surface compared with the corresponding data

Figure 4. Properties of the adsorbed layer obtained from fittingneutron reflectivity data to a four-layer model, where layer no. 0 isclosest to the surface (mixed d-OTS-â-casein layer) (solid black) andlayer 1 (hatched lines with narrow spacing), layer 2 (hatched lineswith broad spacing), and layer 3 (light gray) are indicated in the barcharts. The extension of the bars indicate the (a) thickness of thedifferent layers, τ, and (b) the amount of â-casein adsorbed, Γ. Thesame experimental procedure was used as the one used to obtainthe data in Figure 2, except that 0.1 mg/mL â-casein in 0.02 Mimidazole-HCl and 0.0167 M CaCl2 in H2O at pH 7 were used in thefirst step (BC).

Figure 5. Properties of the adsorbed layer obtained from fittingneutron reflectivity data to a four-layer model, where layer no. 0 isclosest to the surface (mixed d-OTS-â-casein layer) (solid black) andlayer 1 (hatched lines with narrow spacing), layer 2 (hatched lineswith broad spacing), and layer 3 (light gray) are indicated in the barcharts. The extension of the bars indicate the (a) thickness of thedifferent layers, τ, and (b) the amount of â-casein adsorbed, Γ. Thesame experimental procedure was used as the one used to obtainthe data in Figure 2, except that 0.1 mg/mL â-casein in 0.02 Mimidazole-HCl and 0.0167 M MgCl2 in D2O at pH 7 were used in thefirst step (BC).


for the hydrophilic surface is the very different distributionof the volume fraction versus distance. Close to the surface,the density ofâ-casein segments seems to be less or as denseon the hydrophilic as on the hydrophobic surface. Althoughthere are a small number of data points, making comparisonuncertain, the volume fraction on the hydrophilic surfaceseems to decrease more or less linearly with distance whereasa more drastic decrease at about 40 Å from the inner d-OTSlayer is observed for the hydrophobic surface. The additionof endoproteinase Asp-N leads to a relatively smallerreduction in the amount and layer thickness ofâ-casein onthe hydrophobic (present study) than on the hydrophilicsurface.32 Similar findings were found in our previousellipsometry study.15 This indicates the difference structureof the adsorbed layer, which also is indicated by the muchslower adsorption kinetics on the hydrophilic surface.15,32Thetotal thickness of the adsorbed layer also seems to be largeron the hydrophilic surface than on a hydrophobic surface(Figure 6), which was also observed in our earlier studywhere the forces between twoâ-casein covered surfaces weredetermined by using the interferometric surface force ap-paratus (SFA).16 However, in contrast to the SFA and neutronreflectivity measurements, in the ellipsometry study we didnot observe any difference inâ-casein thickness betweenthe hydrophobic and hydrophilic surfaces.15 Also the thick-ness values determined by ellipsometry were somewhat lowerthan those observed in the present study and in previousneutron scattering and reflectivity studies.6,11,12Even largerthicknesses were observed in the SFA studies,≈110-125Å,16,18 light scattering studies of adsorbedâ-casein layerson emulsion droplets and particles,≈100-120 Å,5-7,9 andthickness of emulsion films as determined by microinter-ferometry,≈110 Å.18 These discrepancies have to do withhow well each technique responds to low segment densities

and whether it gives just an average thickness, as for instance,ellipsometry. Although segment density profiles can, inprinciple, be calculated from neutron reflectivity, the modelsusually employed consist of just a small number of layers.The resolution is not sufficient to distinguish stepwise andsmooth distributions.

Leermakers et al.13,14 used self-consistent field modelingto estimate the segment density profiles of adsorbed layersof â-casein on hydrophobic surfaces. The total segmentdensity profile, æ(z), where z is the distance from thehydrophobic surface, was found to fall off in a featurelessfashion from very high values (≈0.95) close to the interface(smallz) to values approaching the protein bulk concentrationfor z > 50 Å. A very dilute tail region (æ(z) < 0.01),corresponding to the N-terminal hydrophilic sequence of≈40amino acid residues, was found to extend out fromz ) 30-70 Å to z ≈ 200 Å, depending on pH, ionic strength, andprotein bulk concentration. A typical result for the modelused to representâ-casein on a hydrophobic surface at pH7, taken from Atkinson et al.,13 is inserted in Figure 6 as asolid line, where their data have been shifted 20 Å to allowfor the d-OTS layer. This is to account for the 20 Å thickincomplete layer of d-OTS. Their calculated curve agreesquite well with our experimental data suggesting that thereal â-casein layer conforms well to the model protein andthat the self-consistent field modeling satisfactorily accountsfor the protein segment density profile.

The volume fraction versus distance profiles have the sameshape whetherâ-casein is adsorbed in the presence of NaCl,CaCl2, and MgCl2 of the same ionic strength or whether theprotein concentration is lowered 10 times. In particular atlarger distances from the surface, the volume fraction valuesare low and similar. This is also reflected in the fact thatendoproteinase Asp-N reduces the outer part of the adsorbedlayer in all cases. The low volume fraction of proteinsegments in the outer layer makes it possible for the enzymeto penetrate and cleave at the N-terminal hydrophilic partof â-casein, residues 43 and/or 47. However, we note thatthe profile recorded at the lowerâ-casein concentration, 0.01mg/mL, is less extended. If we ignore the innermost mixedâ-casein and d-OTS layer, it is obvious that the largestdifference between results from the experiments carried outunder different conditions is in the region close to thehydrophobic surface. The volume fraction in this layerdecreases in the order CaCl2 > MgCl2 > NaCl at constantionic strength. This confirms the findings in our previousellipsometry study, where the adsorbed amounts were foundto decrease in this order, while the layer thickness remainedconstant. It should be borne in mind that the binding of bothcalcium and magnesium ions to the five serine phosphateresidues onâ-casein is similar and of high affinity, asdetermined by multinuclear magnetic resonance spectros-copy.26 Thus the large molar electrolyte-â-casein ratio ofabout 800:1, used in the present study, suggests that all thesebinding sites on the protein are saturated.

In contrast to our study, Brooksbank et al.,9 Atkinson etal. 11 and Velev et al.18 found that theâ-casein layerthickness decreases in the presence of calcium. However inall of these studies different experimental conditions were

Figure 6. Volume fraction of adsorbed layers of â-casein obtainedfrom neutron reflectivity measurements plotted versus the distancefrom the surface. The data are given in Table 3, and the distancevalues are taken as half the thickness of the layer plus the totalthickness of inner layers. Neutron reflectivity data from â-caseinadsorption on a hydrophilic surface is also inserted (0).32 The â-caseinconcentration was 0.1 mg/mL 0.02 M imidazole-HCl at pH 7, unlessstated otherwise. The data were recorded in the presence of addedelectrolyte: 0.05 M NaCl (2); 0.05 M NaCl and 0.01 mg/mL â-casein(4); 0.0167 M CaCl2 (b); 0.00167 M MgCl2 ([). For comparison, thetotal segment density profile versus distance for â-casein look alikeat pH 7 on a hydrophobic surface calculated by the self-consistent-field theory by Atkinson et al.13 is introduced as a solid line. It shouldbe noted that for fair comparison their data have been shifted 20 Å.This is to account for the 20 Å thick incomplete layer of d-OTS.


used compared to those employed in the present study, whichmight have resulted in different packing on the hydrophobicsurface. In the study of Brooksbank et al., the calcium ionswere added after adsorbingâ-casein to the “hydrophobic”latex particles. Atkinson et al. studied the adsorption at theair/water interface for which they reported a more compactlayer, that is, thinner and denser, compared with the resultsin our study. They reported a significant decrease of theamount ofâ-casein adsorbed when adding 2.0 mM calciumto a solution containing 0.05 mg/mLâ-casein. However, itshould be borne in mind that they studied the adsorption ata liquid interface. This means that the lateral mobility isconsiderably higher than at the solid-liquid interface.Although, the formation elastic protein networks at the air/water has been reported, these are much weaker for aâ-casein than a globular protein likeâ-lactoglobulin.33 Thus,by proper orientation of the serine phosphate moieties thenumber of intermolecular calcium bridges can be increased.Such a reorientation can, if it means thatâ-casein protrudesless into the subphase, lead to a lateral expansion of the layer,which in turn means an increase in area per molecule. Incontrast to the findings of Brooksbank et al.9 and Atkinsonet al.,11 Velev et al.18 found that the adsorbed amount ofâ-casein at the oil-water interface did not seem to beaffected by the presence of calcium. They measured theirthickness with different surface force techniques, for whichthe value of the thickness may be sensitive to the compress-ibility of the layer. They found a decrease of thickness withincreasing calcium ion concentration until 12 mM, afterwhich no further decrease of the thickness was observed.Interestingly, above this concentration they observed a strongadhesive force from which they concluded that cross-bindingof the film surfaces occurs. It should also be noted that theyused a slightly lower pH of 6.5 in their study. One shouldalso be aware that calcium can cause precipitation (formationof calcium bridges) even in imidazole buffer under conditionswhere magnesium does not.26,34,35 This could also explainwhy calcium gives a higher volume fraction in the first layerthan magnesium. In addition we note that, although the timeresolution of the neutron reflection measurements is poor,the adsorption in the presence of calcium seemed to beslightly time dependent, which might indicate some kind ofrearrangement, possibly cross-linking. It is noteworthy thatthe largest reduction in adsorbed amount upon endoproteinaseAsp-N addition was observed on the layer adsorbed fromCaCl2 solution. Cross-linking of the released N-terminalpeptide, which, in turn might precipitate in the bulk solution,could facilitate this proteolytic reaction. The second additionof â-casein to the enzyme-treatedâ-casein layer seems torestore theâ-casein layer because no desorption uponsubsequent rinsing with protein-free buffer could be ob-served. Such behavior suggests a strong interaction with theremaining layer or with small uncovered regions on thehydrophobic surface. The very large increase observed onthe second addition ofâ-casein to an enzyme treated layeradsorbed from a solution containing MgCl2 implies thatelectrostatic forces somehow control the formation of thesecond layer. It might be that some magnesium ions remainat the surface, which might reduce or even reverse the

negative charge on the surfaces and hence favor formationof the second layer. The relative large reduction by endopro-teinase Asp-N of theâ-casein layer adsorbed in the presenceof calcium might lead to relative larger loss of bound calciumions from the adsorbed layer. Hence, we did not observethe same effect of second addition ofâ-casein to anendoproteinase Asp-N treated protein layer formed in thepresence of calcium compared to the one formed in thepresence of magnesium. The large adsorption during theaddition ofâ-casein after cleaving portions of the N-terminalpart shows that this part is essential forâ-casein to act as aprotective colloid. This confirms the findings from our earlierstudy, where we demonstrated the same behavior by additionof â-lactoglobulin to an endoproteinase Asp-N treatedâ-casein surface.10

Acknowledgment. The financial support from the Swed-ish Research Council for Engineering Sciences (TFR) isgratefully acknowledged.

References and Notes

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BM0056308


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