278 Biomacromolecules 2001, 2, 278-287

β-Casein Adsorption at the Hydrophobized Silicon

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

Introduction There are strong similarities between β-casein and am-

The milk proteins and casein in particular have, due to
their excellent properties as stabilizers of emulsions, foams,
and dispersions, come to be used in a range of applications
both in foods and in other applications, such as glue, paint,
and putty.1-3 One of the major constituents of milk casein
is β-casein.4 Therefore, quite a number of studies have been
devoted to the behavior of β-casein at the air-aqueous and
oil-aqueous interfaces and the interfaces between hydro-
phobic solid surfaces and an aqueous phase.5-20 These studies
have been conducted under various conditions, using a range
of different experimental techniques and various types of
hydrophobic 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 reason
could be that the protein has such a strong amphiphilic
character, with the amino acid sequence divided into one
hydrophilic and one hydrophobic domain. Thus, β-casein
forms quite well structured monolayers on hydrophobic
surfaces, in which the hydrophobic protein segments are
attached to the surface, forming a densely packed inner layer.
The highly charged N-terminal portion of this casein extends
into the aqueous solution to form a brushlike structure. The
consequence is that the surface becomes more hydrophilic,
and this is manifested by a change in the wetting properties.20
* To whom correspondence should be addressed. E-mail:
Tommy.Nylander@fkem1.lu.se.
† Lund University.
‡ Institute for Surface Chemistry.
§ University of Surrey.
| Physical and Theoretical Chemistry Laboratory.
10.1021/bm0056308 CCC: $20.00
Published on Web 02/15/2001
phiphilic (di-)block copolymers in terms of their ability to
associate in solution and in their interfacial behavior.
β-Casein, for instance, forms micellar-like aggregates (cmc
≈ 0.5 mg/mL) in aqueous solutions.21,22 In comparison with
block copolymers the structure of interfacial layers of
proteins 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 that β-casein contains 209 amino acid
residues in a particular order giving it a molecular mass of
24 020 Da,4,23 although different genetic variants, where a
few of the amino acid residues are different, exist. Dickinson
et al. have modeled the structure of the adsorbed layer of a
model polymer, resembling β-casein, on a hydrophobic
surface,13,14,17 by using the self-consistent field theories of
Scheutjens and Fleer.24 Their results qualitatively confirm
the structure based on experimental studies. However, the
model calculations showed no evidence of two distinct layers
with markedly different volume fractions of protein segments.
Instead, the volume fraction of protein segments decreases
exponentially with the distance from the surface. They also
found significant effects of ionic strength and pH on the
segment density profiles. Similar effects have been demon-
strated in a number of experimental studies.3,9,11,15,18 This is
due to the large number of charged and chargeable residues
in the N-terminal 50 residues of the protein. In fact the net
negative charge of this part is at least -16 at pH 7.4 These
charge residues include five phosphorylated serines, which
gives the protein a specific affinity to divalent ions such as
© 2001 American Chemical Society
Casein Adsorption
calcium and magnesium.25,26 Our earlier study indicated that
the surface excess increased in the order NaCl < MgCl2 <
CaCl2 at constant ionic strength, although no significant
change in the layer thickness was observed.15 However, other
studies have indicated a decrease in layer thickness on
calcium addition.11,18 To investigate further the effect of
added electrolyte on the structure of β-casein layers on
hydrophobic surfaces, the present neutron reflectivity study
was undertaken. This has previously been demonstrated to
be a powerful technique for studying the structure of β-casein
layers at hydrophobic surfaces12 as well as the air-water
interface.8,11,13 We have also made use of a specific pro-
teolytic enzyme, endoproteinase Asp-N,27 as a tool to reveal
the interfacial structure of β-casein.10,15,20 This protein has
four potential cleavage sites for the enzyme, where two are
located in the hydrophilic part (residues 43 and 47) and two
in the hydrophobic part (residues 129-184).
Materials and Methods
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,10 where
also details of the analyses of the sample are given. The
sample was free from contamination of other proteins as
determined by FPLC and in addition the protein was applied
on a Chelex-100 (200-400 mesh, BioRad) column for
complete removal of cations before being lyophilized.
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 or 0.01 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 the
appropriate amount of sodium chloride, calcium chloride,
and magnesium chloride as described in the Results. Fresh
solutions were always prepared immediately before the
adsorption measurement was started.
The procedure for polishing the large face (111) of the
silicon block and hydrophobizing with deuterated octa-
decyltrichlorosilane (d-OTS, C18H37SiCl3) has been described
earlier.28 We will describe only the basic steps. The silicon
block was initially soaked for 10 min in nitric acid followed
by etching for 10 min in a pH-buffered mixture of NH4F
(40%)/HF (10%) at volume ratio of 7:1. The substrate was
then 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 rinsing
by water and finally CH2Cl2. The self-assembled layer of
d-OTS on the silica surface was formed by immersing the
block in a freshly prepared solution of d-OTS in CH2Cl2 at
a concentration of 9.4 × 10-4 M for 1 h. The block was
then rinsed in CH2Cl2, ethanol, and water. Before use and
between each adsorption experiment, the surface was cleaned
in 5% Decon 90 solution and the whole block was then
thoroughly rinsed in UHQ water.
The neutron reflection measurements were made on the
“white beam” time-of-flight reflectometer CRISP at the
Biomacromolecules, Vol. 2, No. 1, 2001 279
Rutherford Appleton Laboratory, ISIS, Didcot, U.K. A
detailed description of the experimental procedure and data
evaluation employed in this study is given elsewhere.29-32
The sample cell consisted of a Teflon trough clamped against
a silicon block of dimensions 12.5 × 5 × 2.5 cm3.28 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. The neutron reflectivity, that is the ratio of the
reflected and incoming beam intensities, is determined as a
function of momentum transfer (wave vector), κ, where κ
) 4π sin θ/λ (θ is the incidence angle and λ is the
wavelength). Neutron wavelengths from 1 to 6 Å were used
in these experiments. Each reflectivity profile was measured
at three different glancing angles of 0.35°, 0.8°, and 1.8°,
and the results were combined. The beam intensity was
calibrated using the totally reflected beam below the critical
angle with D2O in the cell. A flat background determined
by extrapolation to high values of momentum transfer, κ,
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
the background for H2O was 3.5 × 10-6, given in terms of
reflectivity.
The structural parameters of the d-OTS layer on the surface
of the silicon block were fully characterized before any
adsorption of the protein. To remove entrapped air on the
hydrophobic surface, the cell was initially filled with ethanol,
which was swept from the cell with excess of water. The
characterization was then done in different isotopic composi-
tions of water: pure D2O, water CMSiO2 (contrast matching
for SiO2 with H2O/D2O weight ratio of 0.401/0.599), water
CMSi (contrast matching for Si with H2O/D2O weight ratio
of 0.595/0.405), and finally pure H2O. The cell was then
filled with protein solution (about 40 mL), generally prepared
from D2O, and the reflectivity profile was recorded versus
time until no changes with time could be observed. It took
about 15 min after the protein was added until the reflectivity
measurement was started, and each measuring cycle took
about 40 min (depending on the neutron flux). At least two
measuring cycles were performed, and the reflectivity profiles
were compared. If they were similar, the protein solution
was removed and the cell was filled with pure buffer prepared
from D2O (or H2O for the experiment with CaCl2). This
solution was then removed from the cell, and the cell was
filled with a fresh aliquot of buffer. The reflectivity profile
was then recorded as for the protein solution. The buffer
was then changed to one prepared from H2O, and the
reflectivity profile was recorded. This experimental protocol
was followed for the addition of enzyme and the second
addition of β-casein.
The reflectivity profile, R(κ), is determined by the variation
of the scattering length density, F, along the normal to the
surface, z, by
16π2
R(κ) ) |F̂(κ)|2 (1)
κ2
280 Biomacromolecules, Vol. 2, No. 1, 2001 Nylander et al.

Table 1. Constants Used for the Fitting of the Reflectivity Profiles Table 2. Characterization of the Hydrophobic Interface (d-OTS
(Taken from Reference 12) (95% D)/SiO2)a
density volume τ(3
material (g/cm3) (Å3) b (10-4 Å) F (10-6 Å-2) layer (Å)b FD2O FH2O φOTS ( 0.1b φw ( 0.1b φair ( 0.1b
H2O 0.9975 30 -0.168 -0.56 oxide 18 4.00 2.62 0.2
D2O 1.105 30 1.905 6.35 OTS (inner) 17 6.35 6.35 1.0 0.0 0.0
water CMSi 1.038 30 0.621 2.07 OTS (outer) 20 5.40 1.41 0.3 0.6 0.2
water CMSiO2 1.059 30 1.023 3.41 a τ is the layer thickness (Å), F
D2O and FH2O are the scattering length
Si 2.32 20 0.415 2.07 densities in D2O and H2O, respectively, and φOTS, φw, and φair are the
SiO2 2.16 47 1.585 3.41 volume fraction of OTS, water, and air, respectively b The errors given
-C18D37 0.7768 542 36.65 6.76 reflect the maximum uncertainty in the fitting and any possible coupling
β-casein 1.365 29594 532.6 1.80 of the fitting parameters.

where F(κ) is the one-dimensional Fourier transform of F-
(z), that is
F̂(κ) ) ∫-∞+∞ e-iκz F(z) dz
In turn the scattering length density depends on the chemical
composition of the sample as (cf. ref 30)
F) ∑nibi
where ni is the number density of element i and bi is its
scattering amplitude (scattering length). After assumption of
a structural model for the adsorbed layer, the reflectivity
profile is calculated. The calculated reflectivity is then
compared with the measured data, and the structural param-
eters are varied until the fit is optimized. 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 surface excess, Γ, for each layer can
be deduced directly from the derived scattering length density
and thickness of the layer using
Fτ
Γ)
∑
Na( mibi + nwbw)
where ∑mibi denotes the total scattering length of the protein
molecule, Na is Avogadro’s number, 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 number mi of component i has a scattering length bi.
The volume fraction of protein, φp, can for each layer be
obtained from
F ) φpFp + (1 - φp)Fw
where Fp and Fw are the scattering length densities of protein
and water, respectively. The parameters used for the calcula-
tions are given in Table 1.
Results
The characteristics of the silicon sample block with the
OTS layer are given in Table 2.
As reported previously,12,28 the only model that could fit
the reflectivity profiles recorded for all four of the different
water 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 discussed
by Fragneto et al.,12,28 the d-OTS layer could be divided into
two layers, where the layer next to the silicon oxide surface
is crystalline. Consequently, this 17 Å thick layer consists
almost entirely of d-OTS with only 0.01 volume fraction
entrapped air. This outer layer, however, is defective and
contains a significant amount of water and air. The thickness
of this layer was found to be about 20 Å, and the volume
(2) fraction of d-OTS is about 0.27. This defective layer is
thicker than the value, 11 Å, reported earlier by Fragneto et
al.28 However, they reported a higher surface coverage of
5.9 µmol/m2, compared with a d-OTS coverage of 1.7 µmol/
m2 calculated from the values given in Tables 1 and 2. The
outer d-OTS layer in the present study can therefore be
considered to be more defective than that obtained in the
earlier studies. It is worth commenting at this stage that the
division 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 on
the surface, and the overall thickness, would be little changed
from the simpler two-layer division. Here, and in the
discussion of the β-casein results, we have opted for using
(3)
the minimum number of distinct layers required to fit the
data. While this may not be entirely realistic, it is the standard
practice unless there are other reasons for describing the
system in terms of smooth distributions (for example to fit
a theoretical model). The errors quoted in Table 2 reflect
the maximum uncertainty in the fitting and any possible
coupling of the fitting parameters. In the fitting we have also
allowed for the presence of air. We do normally see some
air present at the hydrophobic surface unless it has been
specially treated with ethanol and water. It is displaced when
adsorption of other material occurs. We note that the surfaces
(4) were 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,
β-casein was likely to penetrate into the outer part of the
OTS. Thus when it came to fitting the reflectivity data for
adsorbed protein, we used the parameters already given for
the oxide and lower OTS layer but allowed the scattering
length density of the outer layer to vary according to how
much protein was in it. It was immediately clear when fitting
the data for the protein that the volume fraction declines away
from the surface. This allows the choice of using a functional
form for the decay or of dividing the protein into uniform
layers each containing less protein than the last. The
resolution of the experiment cannot distinguish these two
Casein Adsorption Biomacromolecules, Vol. 2, No. 1, 2001 281

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

adsorption rinse
0.05 M 0.05 M 0.017 M 0.017 M 0.05 M 0.05 M 0.017 M 0.017 M
NaCl NaClb CaCl2c MgCl2 NaCl NaClb CaCl2 MgCl2
τ0 ( 3d 20 20 20 20 20 20 20 20
τ1 ( 3d 8 8 6 8 8 8 6 8
τ2 ( 5d 25 15 25 25 25 15 25 25
τ3 ( 10d 25 25 30 30 25 30 25
F0 4.7 4.7 2.44-3.02 5.1 4.7 4.7 3.9 5.1
F1 4.2 4.4 1.47 3.8 4.2 4.4 3.4 3.8
F2 5.8 5.8 0.03 5.5 6.0 5.9 5.5 5.7
F3 6.1 6.1 -0.39 6.1 6.2 6.1 6.2
φ0 ( 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 0.24-0.37
φ1 ( 0.1d 0.6 0.6 0.9 0.8 0.6 0.6 0.9 0.8
φ2 ( 0.05d 0.16 0.16 0.25 0.25 0.1 0.13 0.25 0.19
φ3 ( 0.03d 0.07 0.07 0.07 0.07 0.04 0.07 0.04
Γ0 ( 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 0.7-1.0
Γ1 ( 0.1d 0.7 0.6 0.7 0.8 0.7 0.6 0.7 0.8
Γ2 ( 0.15d 0.6 0.4 0.9 0.9 0.3 0.3 0.9 0.6
Γ3 ( 0.1d 0.2 0.2 0.3 0.3 0.1 0.3 0.2
τT 58 48 61 63 33 48 61 58
ΓT/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 β-casein layers were obtained from a four-layer model fit to data obtained from neutron reflectivity measurements (in 0.02

M imidazole-HCl buffered D2O, pH ) 7, with added electrolyte) after exposure to 0.1 mg/mL β-casein solution 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, 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/mL β-casein. c In H2O. dThe errors given reflect the
maximum uncertainty in the fitting and any possible coupling of the fitting parameters.

cases and would not be able to distinguish at all clearly the

functional form of the decay. We therefore opted to use a
description in terms of the minimum number of distinct layers
required to fit the data. The experiment is sensitive to the
overall thickness of the layer until the volume composition
drops below about 10%; i.e., the uncertainty in each layer
thickness increases as there is less protein in it. It is also
sensitive to the total amount adsorbed and to the more
detailed distribution within the OTS layer. On the basis of
this we have estimated the errors as shown in the tables,
and these estimates include contributions that could arise
from coupling of the fitting parameters. Our data were
analyzed by implementing a four-layer model, where the
lower layer is the outer, defective d-OTS-layer. Typical
scattering curves for the pure d-OTS layer at various stages
of the β-casein adsorption study are given in Figure 1.
The results from the fit to the experimental scattering
curves are given in Tables 3-5. For the sake of clarity, the
evolution of thickness and the surface excess in the four
different layers during the different steps in the adsorption
experiments are illustrated by bar charts in Figure 2-5. The
thickness of the outer d-OTS layer could in all cases be fitted
to 20 Å. However, during the course of adsorption the density
of this layer changed, which indicates penetration of BCN.
The Adsorption of BCN. The adsorption data for BCN
in the various electrolytes could all be fitted to the four-
layer model (Table 3).
In every case the adsorption of BCN on the OTS surface
reaches a plateau after the first measuring cycle, which is
completed about 55 min after adding the protein. Here, we
note that the time resolution of neutron reflectivity measure-
ments is rather limited and the plateau might have been
reached after a significantly shorter time. The largest total
amount adsorbed is found in the presence of CaCl2 (Figure
Figure 1. Neutron reflectivity versus momentum transfer curves in
0.02 M imidazole-HCl buffered D2O solution, pH 7: the curve for the
clean d-OTS block (×); the layer adsorbed from a 0.1 mg/mL β-casein
solution containing 0.0167 M CaCl2 after changing solution to 0.02
M imidazole-HCl buffered D2O solution (+); the layer after treatment
with endoproteinase Asp-N (0.04 µg/mL) and subsequent rinsing in
buffered D2O solution (4); the layer formed after second addition of
a 0.1 mg/mL β-casein solution and subsequent rinsing in buffered
D2O solution (O) are shown together with the corresponding fits, which
are inserted as solid lines.
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 three
electrolytes is quite similar (Figures 4a, 2a, and 5a, respec-
tively). To see the effect of the protein concentration, one
additional experiment was performed in the presence of 0.05
M NaCl, with a 10-fold lower protein concentration of 0.01
mg/mL. This led to a decrease in the adsorbed amount
(Figure 3b) as well as in the total thickness of the protein
layer (Figure 3a). The adsorbed amount in the first two layers
is basically the same at this lower protein concentration
(Figure 3b) as in the presence of NaCl and MgCl2 (Figures
2b and 5b), while it is slightly higher in the presence of CaCl2
282 Biomacromolecules, Vol. 2, No. 1, 2001 Nylander et al.

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

endoproteinase rinse
0.05 M 0.05 M 0.017 M 0.017 M 0.05 M 0.05 M 0.017 M 0.017 M
NaCl NaClb CaCl2 MgCl2 NaCl NaClb CaCl2 MgCl2
τ0 ( 3c 20 20 20 20 20 20 20 20
τ1 ( 3c 8 8 6 8 8 8 6 8
τ2 ( 5c 20 15 25 25 20 15 25 25
τ3 ( 10c
F0 4.7 4.7 3.9 4.3 4.7 4.7 3.9 4.3
F1 4.2 4.4 4.2 4.6 4.2 4.4 4.2 4.6
F2 6.2 6.1 6.0 6.1 6.2 6.1 6.0 6.1
F3
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 0.47-0.6
φ1 ( 0.1c 0.6 0.6 0.6 0.5 0.6 0.6 0.6 0.5
φ2 ( 0.02c 0.04 0.07 0.1 0.07 0.04 0.07 0.1 0.07
φ3
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 1.3-1.6
Γ1 ( 0.1c 0.7 0.6 0.5 0.6 0.7 0.6 0.5 0.6
Γ2 ( 0.07c 0.14 0.14 0.34 0.24 0.14 0.14 0.34 0.24
Γ3
τT 28 23 31 33 28 23 31 33
ΓT/(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 β-casein layers after addition of an endoproteinase Asp-N solution, followed by rinsing with buffered D O. The results
2
were obtained from a four-layer model fit to data obtained from neutron reflectivity measurements in 0.02 M imidazole-HCl buffered D2O, pH ) 7. The
β-casein layers were adsorbed from buffers containing different electrolytes and a protein concentration of 0.1 mg/mL β-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, 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/mL β-casein. cThe errors given
reflect the maximum uncertainty in the fitting and any possible coupling of the fitting parameters.

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

second β-casein addition rinse
0.05 M 0.05 M 0.017 M 0.017 M 0.05 M 0.05 M 0.017 M 0.017 M
NaCl NaClb CaCl2 MgCl2 NaCl NaClb CaCl2 MgCl2
τ 0 ( 3c 20 20 20 20 20 20 20 20
τ1 ( 3c 8 8 6 8 8 8 6 8
τ2 ( 5c 30 23 25 34 30 23 25 34
τ3 ( 10c 35 30 35 37 25 25 35 30
F0 4.2 4.1 3.8 4.3 4.2 4.1 3.8 4.3
F1 3.2 3.8 3.2 3.1 3.2 3.8 3.2 3.1
F2 5.0 5.3 5.2 4.8 5.7 5.7 5.2 4.8
F3 5.9 6.1 6.1 5.6 6.2 6.2 6.1 5.8
φ0 ( 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 0.47-0.6
φ1 ( 0.1c 0.9 0.8 0.9 1.0 0.9 0.8 0.9 1.0
φ2 ( 0.05c 0.39 0.31 0.34 0.45 0.19 0.19 0.34 0.45
φ3 ( 0.03c 0.13 0.07 0.07 0.22 0.04 0.04 0.07 0.16
Γ0 ( 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 1.3-1.6
Γ1 ( 0.1c 1.0 0.8 0.8 1.0 1.0 0.8 0.8 1.0
Γ2 ( 0.15c 1.6 1.0 1.2 2.1 0.8 0.6 1.2 2.1
Γ3 ( 0.1c 0.6 0.3 0.3 1.1 0.1 0.1 0.3 0.7
τT 73 61 66 79 63 56 66 72
ΓT/(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 β-casein layers after (second) addition of 0.1 mg/mL β-casein solution, followed by rinsing with

buffered D2O. The results were obtained from a four-layer model fit to data obtained from neutron reflectivity measurements in 0.02 M imidazole-HCl
buffered D2O, pH ) 7. The β-casein layers were adsorbed from buffers containing different electrolytes and a protein concentration of 0.1 mg/mL β-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, 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/mL
β-casein. c The errors given reflect the maximum uncertainty in the fitting and any possible coupling of the fitting parameters.

(Figure 4b). Here, we also note that the build-up of β-casein
layer in the presence of CaCl2 was time dependent unlike in
the other conditions studied. It took about 2 h until the
constant reflectivity profile was obtained. The slower equili-
bration time is related to the fact that a more dense layer is
formed, which set demands on interfacial ordering and thus
requires more extensive rearrangements of casein molecules
at the surface. The thicknesses of the three inner layers are
basically the same for the different electrolytes, while the
lower protein concentration leads to a substantial decrease
in thickness of the outermost layer. However, the amounts
adsorbed are different and below we will discuss this further
in terms of protein volume fraction versus distance. The
adsorbed amount and the thickness of the two outer layers
Casein Adsorption
Figure 2. Properties of the adsorbed layer obtained from fitting
neutron reflectivity data to a four-layer model, where layer no. 0 is
closest to the surface (mixed d-OTS-β-casein layer) (solid black) and
layer 1 (hatched lines with narrow spacing), layer 2 (hatched lines
with broad spacing), and layer 3 (light gray) are indicated in the bar
charts. The extension of the bars indicates the (a) thickness of the
different layers, τ, and (b) the amount of β-casein adsorbed, Γ.
β-Casein was adsorbed from a solution containing 0.1 mg/mL protein
in 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-HCl
in D2O at pH 7) (E), and rinse with 0.02 M imidazole-HCl in D2O at
pH 7 (Er). A second addition of β-casein (0.1 mg/mL in 0.02 M
imidazole-HCl and in D2O at pH 7) was then made (2BC), followed
by rinse with 0.02 M imidazole-HCl in D2O at pH 7 (2BCr).
are similar in the presence of both divalent electrolytes
(Figures 4 and 5), while it is substantially less in the presence
of NaCl (Figure 2). A further decrease is observed if the
concentration of BCN is lowered (Figure 3). Thus, it appears
as if the largest effect of changing electrolyte and protein
concentration is in the outer parts of the adsorbed layer.
However, the picture is different in terms of protein volume
fraction (see Discussion). Rinsing with pure buffer without
added electrolyte does not have any effect on the layer
adsorbed from the protein solution containing CaCl2. For all
other conditions a slight decrease of the adsorbed amount is
observed in the outer layer only. In all cases very small
changes are observed in the thickness of the two outer layers.
There is however one exception and that is for the presence
of NaCl, where the outermost layer seems to disappear.
However, it should be emphasized that there is very little
protein in the outermost layer and the accuracy in judging
the amount in such a layer is low.
The Effect of Adding Endoproteinase AspN. For all the
added electrolytes, endoproteinase Asp N only affects the
outermost β-casein layer (Table 4). In fact the data could
now be fitted to a three-layer model. The largest reduction
Biomacromolecules, Vol. 2, No. 1, 2001 283
Figure 3. Properties of the adsorbed layer obtained from fitting
neutron reflectivity data to a four-layer model, where layer no. 0 is
closest to the surface (mixed d-OTS-β-casein layer) (solid black) and
layer 1 (hatched lines with narrow spacing), layer 2 (hatched lines
with broad spacing), and layer 3 (light gray) are indicated in the bar
charts. The extension of the bars indicates the (a) thickness of the
different layers, τ, and (b) the amount of β-casein adsorbed, Γ. The
same experimental procedure was used as the one used to obtain
the data in Figure 2, except that the β-casein concentration was 0.01
mg/mL protein.
in amount by a third of the total mass and by half the layer
thickness is observed for β-casein adsorbed from a solution
containing CaCl2 (Figure 4). The reduction in the total
amount is only about 10% for the other conditions, although
the reduction in total thickness was larger. It should be
emphasized that the sample cell was rinsed with the same
buffer, that is, buffer without added electrolyte, before the
endoproteinase Asp-N dissolved in 0.02 M imidazole buffer
was added. This was done to make sure that enzyme activity
was not affected by the added electrolyte ensuring that the
enzymatic reaction was carried out in equal conditions.
Rinsing with enzyme free buffer without added electrolyte
had no effect on the enzyme-treated layer.
The Effect of a Second Addition of β-Casein. To assess
further the effect of the enzyme treatment, and hence the
structure of the adsorbed layer, a second addition of β-casein
to the enzyme-treated layer was made. The protein was added
from buffer solution without added electrolyte. In every case
the second addition led to large increases in amounts
adsorbed and in the thickness of the adsorbed layers. In fact,
the layer became significantly thicker and contained signifi-
cantly larger amounts of β-casein than the layer initially
formed on the d-OTS, hydrophobic surface (Table 5).
The largest increase was observed in the presence of the
divalent electrolyte, MgCl2 (Figure 5). Here the second
284 Biomacromolecules, Vol. 2, No. 1, 2001
Figure 4. Properties of the adsorbed layer obtained from fitting
neutron reflectivity data to a four-layer model, where layer no. 0 is
closest to the surface (mixed d-OTS-β-casein layer) (solid black) and
layer 1 (hatched lines with narrow spacing), layer 2 (hatched lines
with broad spacing), and layer 3 (light gray) are indicated in the bar
charts. The extension of the bars indicate the (a) thickness of the
different layers, τ, and (b) the amount of β-casein adsorbed, Γ. The
same experimental procedure was used as the one used to obtain
the data in Figure 2, except that 0.1 mg/mL β-casein in 0.02 M
imidazole-HCl and 0.0167 M CaCl2 in H2O at pH 7 were used in the
first step (BC).
addition almost doubled the adsorbed amount, and a small
portion of this, about 8%, could be removed by rinsing with
buffer. Adsorption from a solution containing NaCl of the
same ionic strength also gives quite large amounts adsorbed
upon the second addition of β-casein (Figure 2), but now
about 28% of the mass was removed when rinsing with pure
buffer. When the protein concentration is lowered 10-fold,
the adsorption at the second addition is also lower (Figure
3) and about 15% of the amount could be desorbed. The
lowest increase in adsorbed amount on second addition of
β-casein is observed for the enzyme-treated β-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 amount
upon endoproteinase Asp-N addition was observed on the
layer adsorbed from CaCl2 solution. In all cases, apart from
adsorption to initially adsorbed layer formed in the presence
of NaCl, the second addition only causes changes in the three
outermost layers. In the case of NaCl the volume fraction
of the β-casein in the innermost layer, φ0, also increases from
about 0.6 to 0.9 or 0.8 at the lower β-casein concentration.
Discussion
In an earlier neutron reflectivity study Fragneto et al. used
a two-layer model to evaluate their data on the adsorption
Nylander et al.
Figure 5. Properties of the adsorbed layer obtained from fitting
neutron reflectivity data to a four-layer model, where layer no. 0 is
closest to the surface (mixed d-OTS-β-casein layer) (solid black) and
layer 1 (hatched lines with narrow spacing), layer 2 (hatched lines
with broad spacing), and layer 3 (light gray) are indicated in the bar
charts. The extension of the bars indicate the (a) thickness of the
different layers, τ, and (b) the amount of β-casein adsorbed, Γ. The
same experimental procedure was used as the one used to obtain
the data in Figure 2, except that 0.1 mg/mL β-casein in 0.02 M
imidazole-HCl and 0.0167 M MgCl2 in D2O at pH 7 were used in the
first step (BC).
of β-casein on a d-OTS surface.12 Although our data are
qualitatively almost identical to theirs, we have used a four-
layer model in this study. The reason is that we wanted to
make a fair comparison with our neutron reflective study of
β-casein adsorption on a hydrophilic surface.32 In that study
it turned out that the quality of fit increased significantly
when increasing the number of layers from two to three. Due
to the presence of an imperfect outer layer of d-OTS, an
extra layer was added in the model to account for penetration
of β-casein into this layer. The volume fraction of β-casein
segments versus distance from the crystalline inner d-OTS
layer is plotted for different experimental conditions in Figure
6.
The data are taken from Table 3 and the figure plots the
volume fraction at the center of each layer at the corre-
sponding distance. The volume fraction of β-casein in the
outer d-OTS layer has been calculated as the volume fraction
remaining after subtraction of that of the OTS. In any case,
the volume fraction of β-casein in this layer is substantially
smaller than that in the innermost pure β-casein layer, and
this causes the maximum in Figure 6. This is expected, as
the presence of d-OTS islands is likely to make packing of
β-casein at the surface less efficient. The most obvious
difference between the results for β-casein adsorption on the
hydrophobic surface compared with the corresponding data
Casein Adsorption
Figure 6. Volume fraction of adsorbed layers of β-casein obtained
from neutron reflectivity measurements plotted versus the distance
from the surface. The data are given in Table 3, and the distance
values are taken as half the thickness of the layer plus the total
thickness of inner layers. Neutron reflectivity data from β-casein
adsorption on a hydrophilic surface is also inserted (0).32 The β-casein
concentration was 0.1 mg/mL 0.02 M imidazole-HCl at pH 7, unless
stated otherwise. The data were recorded in the presence of added
electrolyte: 0.05 M NaCl (2); 0.05 M NaCl and 0.01 mg/mL β-casein
(4); 0.0167 M CaCl2 (b); 0.00167 M MgCl2 ([). For comparison, the
total segment density profile versus distance for β-casein look alike
at 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 should
be 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.
for the hydrophilic surface is the very different distribution
of the volume fraction versus distance. Close to the surface,
the density of β-casein segments seems to be less or as dense
on the hydrophilic as on the hydrophobic surface. Although
there are a small number of data points, making comparison
uncertain, the volume fraction on the hydrophilic surface
seems to decrease more or less linearly with distance whereas
a more drastic decrease at about 40 Å from the inner d-OTS
layer is observed for the hydrophobic surface. The addition
of endoproteinase Asp-N leads to a relatively smaller
reduction in the amount and layer thickness of β-casein on
the hydrophobic (present study) than on the hydrophilic
surface.32 Similar findings were found in our previous
ellipsometry study.15 This indicates the difference structure
of the adsorbed layer, which also is indicated by the much
slower adsorption kinetics on the hydrophilic surface.15,32 The
total thickness of the adsorbed layer also seems to be larger
on the hydrophilic surface than on a hydrophobic surface
(Figure 6), which was also observed in our earlier study
where the forces between two β-casein covered surfaces were
determined by using the interferometric surface force ap-
paratus (SFA).16 However, in contrast to the SFA and neutron
reflectivity measurements, in the ellipsometry study we did
not observe any difference in β-casein thickness between
the hydrophobic and hydrophilic surfaces.15 Also the thick-
ness values determined by ellipsometry were somewhat lower
than those observed in the present study and in previous
neutron scattering and reflectivity studies.6,11,12 Even larger
thicknesses were observed in the SFA studies, ≈110-125
Å,16,18 light scattering studies of adsorbed β-casein layers
on emulsion droplets and particles, ≈100-120 Å,5-7,9 and
thickness of emulsion films as determined by microinter-
ferometry, ≈110 Å.18 These discrepancies have to do with
how well each technique responds to low segment densities
Biomacromolecules, Vol. 2, No. 1, 2001 285
and whether it gives just an average thickness, as for instance,
ellipsometry. Although segment density profiles can, in
principle, be calculated from neutron reflectivity, the models
usually employed consist of just a small number of layers.
The resolution is not sufficient to distinguish stepwise and
smooth distributions.
Leermakers et al.13,14 used self-consistent field modeling
to estimate the segment density profiles of adsorbed layers
of β-casein on hydrophobic surfaces. The total segment
density profile, φ(z), where z is the distance from the
hydrophobic surface, was found to fall off in a featureless
fashion from very high values (≈0.95) close to the interface
(small z) to values approaching the protein bulk concentration
for z > 50 Å. A very dilute tail region (φ(z) < 0.01),
corresponding to the N-terminal hydrophilic sequence of ≈40
amino acid residues, was found to extend out from z ) 30-
70 Å to z ≈ 200 Å, depending on pH, ionic strength, and
protein bulk concentration. A typical result for the model
used to represent β-casein on a hydrophobic surface at pH
7, taken from Atkinson et al.,13 is inserted in Figure 6 as a
solid line, where their data have been shifted 20 Å to allow
for the d-OTS layer. This is to account for the 20 Å thick
incomplete layer of d-OTS. Their calculated curve agrees
quite well with our experimental data suggesting that the
real β-casein layer conforms well to the model protein and
that the self-consistent field modeling satisfactorily accounts
for the protein segment density profile.
The volume fraction versus distance profiles have the same
shape whether β-casein is adsorbed in the presence of NaCl,
CaCl2, and MgCl2 of the same ionic strength or whether the
protein concentration is lowered 10 times. In particular at
larger distances from the surface, the volume fraction values
are low and similar. This is also reflected in the fact that
endoproteinase Asp-N reduces the outer part of the adsorbed
layer in all cases. The low volume fraction of protein
segments in the outer layer makes it possible for the enzyme
to penetrate and cleave at the N-terminal hydrophilic part
of β-casein, residues 43 and/or 47. However, we note that
the profile recorded at the lower β-casein concentration, 0.01
mg/mL, is less extended. If we ignore the innermost mixed
β-casein and d-OTS layer, it is obvious that the largest
difference between results from the experiments carried out
under different conditions is in the region close to the
hydrophobic surface. The volume fraction in this layer
decreases in the order CaCl2 > MgCl2 > NaCl at constant
ionic strength. This confirms the findings in our previous
ellipsometry study, where the adsorbed amounts were found
to decrease in this order, while the layer thickness remained
constant. It should be borne in mind that the binding of both
calcium and magnesium ions to the five serine phosphate
residues on β-casein is similar and of high affinity, as
determined by multinuclear magnetic resonance spectros-
copy.26 Thus the large molar electrolyte-β-casein ratio of
about 800:1, used in the present study, suggests that all these
binding sites on the protein are saturated.
In contrast to our study, Brooksbank et al.,9 Atkinson et
al. 11 and Velev et al. 18 found that the β-casein layer
thickness decreases in the presence of calcium. However in
all of these studies different experimental conditions were
286 Biomacromolecules, Vol. 2, No. 1, 2001
used compared to those employed in the present study, which
might have resulted in different packing on the hydrophobic
surface. In the study of Brooksbank et al., the calcium ions
were added after adsorbing β-casein to the “hydrophobic”
latex particles. Atkinson et al. studied the adsorption at the
air/water interface for which they reported a more compact
layer, that is, thinner and denser, compared with the results
in our study. They reported a significant decrease of the
amount of β-casein adsorbed when adding 2.0 mM calcium
to a solution containing 0.05 mg/mL β-casein. However, it
should be borne in mind that they studied the adsorption at
a liquid interface. This means that the lateral mobility is
considerably 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 a
β-casein than a globular protein like β-lactoglobulin.33 Thus,
by proper orientation of the serine phosphate moieties the
number of intermolecular calcium bridges can be increased.
Such a reorientation can, if it means that β-casein protrudes
less into the subphase, lead to a lateral expansion of the layer,
which in turn means an increase in area per molecule. In
contrast to the findings of Brooksbank et al.9 and Atkinson
et al.,11 Velev et al.18 found that the adsorbed amount of
β-casein at the oil-water interface did not seem to be
affected by the presence of calcium. They measured their
thickness with different surface force techniques, for which
the value of the thickness may be sensitive to the compress-
ibility of the layer. They found a decrease of thickness with
increasing calcium ion concentration until 12 mM, after
which no further decrease of the thickness was observed.
Interestingly, above this concentration they observed a strong
adhesive force from which they concluded that cross-binding
of the film surfaces occurs. It should also be noted that they
used a slightly lower pH of 6.5 in their study. One should
also be aware that calcium can cause precipitation (formation
of calcium bridges) even in imidazole buffer under conditions
where magnesium does not.26,34,35 This could also explain
why calcium gives a higher volume fraction in the first layer
than magnesium. In addition we note that, although the time
resolution of the neutron reflection measurements is poor,
the adsorption in the presence of calcium seemed to be
slightly time dependent, which might indicate some kind of
rearrangement, possibly cross-linking. It is noteworthy that
the largest reduction in adsorbed amount upon endoproteinase
Asp-N addition was observed on the layer adsorbed from
CaCl2 solution. Cross-linking of the released N-terminal
peptide, which, in turn might precipitate in the bulk solution,
could facilitate this proteolytic reaction. The second addition
of β-casein to the enzyme-treated β-casein layer seems to
restore the β-casein layer because no desorption upon
subsequent rinsing with protein-free buffer could be ob-
served. Such behavior suggests a strong interaction with the
remaining layer or with small uncovered regions on the
hydrophobic surface. The very large increase observed on
the second addition of β-casein to an enzyme treated layer
adsorbed from a solution containing MgCl2 implies that
electrostatic forces somehow control the formation of the
second layer. It might be that some magnesium ions remain
at the surface, which might reduce or even reverse the
Nylander et al.
negative charge on the surfaces and hence favor formation
of the second layer. The relative large reduction by endopro-
teinase Asp-N of the β-casein layer adsorbed in the presence
of calcium might lead to relative larger loss of bound calcium
ions from the adsorbed layer. Hence, we did not observe
the same effect of second addition of β-casein to an
endoproteinase Asp-N treated protein layer formed in the
presence of calcium compared to the one formed in the
presence of magnesium. The large adsorption during the
addition of β-casein after cleaving portions of the N-terminal
part shows that this part is essential for β-casein to act as a
protective colloid. This confirms the findings from our earlier
study, where we demonstrated the same behavior by addition
of β-lactoglobulin to an endoproteinase Asp-N treated
β-casein surface.10
Acknowledgment. The financial support from the Swed-
ish Research Council for Engineering Sciences (TFR) is
gratefully acknowledged.
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