β-Lactoglobulin Adsorption Layers at the Water/Air Surface: 3. Neutron Reflectometry Study on the Effect of pH

Georgi G. Gochev,*,1,2,a Ernesto Scoppola,1 Richard A. Campbell,3,4 Boris A. Noskov,5 Reinhard Miller1 and Emanuel Schneck*,1,b

1 Max-Planck-Institute of Colloid and Interface Science, 14476 Golm, Germany

2 Institute of Physical Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

3 Institut Laue-Langevin, 71 avenue des Martyrs, CS20156, 38042 Grenoble, France

4 Division of Pharmacy and Optometry, University of Manchester, Manchester M13 9PT, UK

5 Institute of Chemistry, St. Petersburg State University, Saint-Petersburg, Russia

* Corresponding authors, e-mail: gochev@ipc.bas.bg , schneck@fkp.tu-darmstadt.de

a Current affiliation address: Institute of Physical Chemistry, WWU Münster, 48149 Münster, Germany

b Current affiliation address: Physics Department, TU Darmstadt, 64289 Darmstadt, Germany

Abstract

Several characteristics of β-lactoglobulin (BLG) layers adsorbed at the air/water interface exhibit a strong pH dependence, but our knowledge on the underlying structure-property relations is still fragmental. Here, we therefore extend our recent studies by neutron reflectometry (NR) and provide a comprehensive overview through direct measurements of the surface excess Γ and the layers’ molecular structure. This enables comparison with available literature data in order to draw general conclusions. The NR experiments were performed at various pH values and within a wide range of protein concentrations, CBLG. Adsorption kinetics measurements in air-contrast-matched-water enabled direct quantification of the dynamic surface excess Γ(t) and are found to be consistent with ellipsometry data. Near the isoelectric point, pI, the rate of adsorption and Γ are maximal, but only at sufficiently high CBLG. NR data collected over a wider Qz-range and in two aqueous isotopic contrasts revealed the structure of adsorbed BLG layers close to equilibrium. Independently of the pH, BLG was found to form dense monolayers with average thicknesses of 1.5 nm, suggesting flattening of the BLG globules upon adsorption as compared to their bulk dimensions (≈ 3.5 nm). Near pI and at sufficiently high CBLG, a thick (≈ 5.5 nm) but looser secondary sublayer is additionally formed adjacent to the dense primary monolayer. The thickness of this sublayer can be interpreted in terms of disordered BLG dimers. The results obtained and notably the specific interfacial structuring of BLG near pI complement previous observations relating the impact of solution pH and CBLG on other interfacial characteristics such as surface pressure and surface dilational viscoelasticity modulus.

1. Introduction2

Proteins are of great interest in food colloids due to their nutritional values and ability to stabilize foams and emulsions.1,2 The whey protein β-lactoglobulin (BLG) is one of the most frequently studied biomacromolecules and it appears to be a good candidate for a model globular protein in interface and colloid studies.3,4 Its molecular structure5 and solution bulk properties6,7 as well as its adsorption at liquid/fluid interfaces3,4,8,9 have been extensively investigated in dependence on various factors (pH, ionic strength, surfactants, polyvalent metal ions, etc.).

BLG molecules adopt globular conformations in aqueous environments and carry a certain net charge |Z|. Both the molecular conformation and |Z| are governed by the solution pH.10-13 Furthermore, monomers can associate into dimers, which can further associate into octamers. These transitions are reversible and are characterized by pH-dependent dissociation constants sensitive to the concentration(s) of electrolyte or/and protein.6,13-22 Sawyer5 recently summarized BLG crystalline structures of monomers and dimers including their spatial dimensions in solution, and information about their intra- and inter- molecular interactions and conformational transitions.

BLG strongly adsorbs to the water/air interface forming viscoelastic layers3,8,9,23-29 and stabilizes foam films,30-35 thus providing stability of foams3,23-27,32,33. The thermodynamics, electrostatics and mechanics (e.g. rheological properties) of BLG adsorption layers as well as the kinetics of their formation are significantly modified by variations in environmental physicochemical factors like pH and electrolyte content.23-29,32,36-38

Variations of the solution pH lead to non-monotonic changes in the dynamical, mechanical and electrical properties of BLG adsorption layers.23-29,32,37 Fastest adsorption kinetics and peak values of the surface pressure Π and the dilational elasticity E’ are found at pH-values close to the isoelectric point of BLG in aqueous solution (pI ≈ 5.1)24.23,24,28,29 However, the surface pressure isotherms Π(CBLG) at pH 5 and 7 show that the surface activity of BLG increases for pH 5 only above certain protein concentration CBLG ≳ 10-7 M.28

At the same time the layer thickness d and the surface excess Γ, as measured by ellipsometry, reach maximum values24,32 corresponding to minimum values of the amplitudes |AK(OH)| of the O-H stretching vibrations of interfacial water molecules as measured by vibrational sum frequency generation (SFG) spectroscopy24. Apparently, the net charge of the protein controls to a great extent its surface activity as manifested in most of the physico-chemical observables mentioned above. Engelhardt et al. 24,25 concluded from SFG and ellipsometry experiments that at pH-values away from the isoelectric point, charged monolayers are formed causing highly polar ordering of the first-neighbour water molecules, whereas at pH→pI formation of disordered and presumably agglomerated BLG multilayers takes place. However, the existing knowledge about the influence of pH on the structure of BLG interfacial layers is still fragmented.

Mackie et al.39 and Perriman et al.40 studied the surface properties of BLG solutions by tensiometry and neutron and x-ray reflectometry (NR and XRR, respectively). They discussed their results in terms of linkage between the monomer-dimer equilibrium in the bulk and the surface properties and structure. They concluded that the BLG monomers exhibit preferential adsorption and dimers presumably dissociate into monomers upon contact with the water/air interface.

Resolving the structure of adsorption layers on the molecular scale is an essential step to get deeper insight into the behaviour of BLG at the water/air interface. This is achievable by surface scattering methods, such as NR.41 BLG adsorption layers at planar water/air interfaces have been studied by these means in several works.40,43-47 Although results at various pH-values in the range 2 – 7 have been reported, it appears difficult to derive a systematic pH-dependent trend in the structure of the adsorption layers, mostly because of a large variety of experimental conditions used (e.g. different protein and electrolyte concentrations). Moreover, either one-slab or two-slab structures have been reported but there is no clear information about the conditions governing the formation of these distinct types of interfacial structures. Finally, to the best of our knowledge, data measured at pH values close to pI are lacking.

Here, we therefore aim to reveal systematically key structure-property relations that can explain the observed trends and the extremum in the pH dependence of several surface characteristics that appears around the

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isoelectric point. In particular, we are interested in quantifying variations in the surface excess and in the corresponding adsorption layer thickness and structure. For this purpose, we performed measurements by NR as well as by ellipsometry for solutions at pH-values near and further away from pI (at constant buffer concentration of 10 mM) for a wide range of BLG concentrations (10-7–2×10-4 M). Based on the obtained results, we attempt to link the existing knowledge on BLG bulk solution properties to properties of the corresponding adsorption layers in order to reveal the molecular origin of the unique pH effect.

2. Experimental2.1. Preparation of solutions

Native BLG purified from whey protein isolate48 was kindly supplied by U. Kulozik (TU Munich, Germany) and used in all experiments. The sample contains total protein ≈98.9 % (from which BLG content >99%, BLG-A/BLG-B ≈ 1.22), salts ≈0.7 %, traces of lactose (<0.05 %). Stock solutions with a protein concentration CBLG = 10-4 M (≈1.83 mg/ml; ≈0.183 wt. %) were prepared at pH 3 or pH 7 in Na2HPO4/citric acid buffer with concentration of 10 mM. For each pH, three types of aqueous phases were used, namely Milli-Q H2O, D2O (Sigma Aldrich) or 8.1% v/v D2O in H2O (called air contrast matched water, ACMW). The ‘pH’-values of all samples in different isotopic aqueous media were adjusted by measurements with commercial glass-electrode based instruments (standardized to read pH for H2O solutions) and without corrections for pD.49 In all cases, to eliminate low-molecular-weight surface active contaminations, the protein solutions were purified with activated charcoal50 (BLG/charcoal mass ratio 1/3, stirred for 20 min and filtered through 0.45 µm pore size filter). These stock solutions were left to rest overnight at 4o C and then used either directly for measurements or for the preparation of samples with lower protein concentrations (10-5 and 10-7 M), see Table S1 in the SI. In the case of the highest BLG concentration of 2×10 -4 M (pH 3) used in this work, samples were prepared in D2O and ACMW and left to rest overnight at 4o C prior to measurements. Due to the tendency for precipitation of solutions at pH close to the BLG isoelectric point (pI ≈ 5.1), the samples at pH 5 were freshly prepared prior measurement from a stock solution pH 7 by dilution with buffer at appropriate pH (in the respective aqueous phase) in order to obtain a final pH value of 5. All experiments were performed at 23-25o C.

2.2. Neutron reflectometry

NR can be used to quantify the surface excess of (macro)molecules adsorbed at the water/air interface and to resolve their interfacial conformation in terms of density profiles normal to the interface. The reflectivity (R) is the ratio between the intensities of the reflected and the incident neutron beams for a given scattering vector component perpendicular to the interface, Qz = (4π/λ)sinθ, where λ is the neutron wavelength and θ is the angle of incidence. The measurements were performed on the time-of-flight reflectometer FIGARO at the Institut Laue-Langevin (Grenoble, France).41,42 Reflectivity data in a sufficiently large Qz-range for ACMW contrast is in principle suited to extract the interfacial protein distributions, e.g. for BLG.40,43-45,47

More robust structural information can be obtained by co-refinement of a model to experimental data recorded in different isotopic contrasts. A recent study on the adsorption kinetics of lysozyme involved deuterated protein to enhance the scattering contrast of the adsorbed material,51 but deuterated BLG was not custom-synthesized for this work, so measurements were recorded simply using hydrogenous BLG with different contrasts of the subphase: D2O and ACMW (having the same null scattering length density as air). It has been shown that data from these two subphase contrasts is sufficient to resolve the interfacial structure of hydrogenous human serum albumin (HSA) at the water/air interface.52 The use of isotopic contrasts other than ACMW (e.g. D2O or suitable H2O/D2O mixtures) was shown to be a powerful approach to resolve the interfacial structure of adsorbed protein layers.46,53,54 In this study we combined measurements in ACMW and D2O. Detailed structural information is then extracted from a self-consistent analysis of both contrasts, which provides structural results with high reliability.

A sample changer with 6 adsorption troughs was used. For every cycle of experiments, 3 troughs contained BLG solutions in ACMW and the other 3 contained BLG solutions in D2O. The samples in ACMW were

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monitored in a kinetic mode using data recorded at a small incident angle (θ = 0.62 deg) to resolve the initial changes in Γ during adsorption (see further below). When a reasonable plateau (i.e. approaching equilibrium) was achieved, typically after several hours, data were recorded also at a higher incident angle (θ = 1.98 deg for ACMW and θ = 3.78 deg for D2O). These data were acquired in order to model the interfacial structure through the acquisition of data over a wide Qz-range. A wavelength range of 2–16 Å was used for the data recorded at θ = 0.62 deg and 1.98 deg, and a range of 2–30 Å was used for the data recorded at θ = 3.78 deg.

The obtained reflectivity curves R(Qz) were analysed with a fitting procedure based on parameterized volume fraction profiles of the chemical components,55 in which the components are water and protein, while the buffer constituents were neglected. The model assumed that the interface is laterally homogeneous on the length scale of the in-plane coherence length of the neutron beam. As a first conceptual step, the volume fraction profile of a bare (protein-free) water/air interface was described with an error function:

ΦW0 ( z )=1

2¿, (1)

where z denotes the distance to the interface and σ∫¿¿ is an adjustable parameter specifying the interfacial roughness, which was constrained to values above 3 Å, according to an earlier estimate of the lower limit of capillary wave amplitudes at the water/air interface.56,57 As shown in section 3.1 in the supporting information, decrease of the surface tension due to protein adsorption has modest influence on the capillary roughness (< 1 Å) and the ensuing interpretation of the results.

In the second step, the volume fraction profile of the adsorbed protein, ΦBLG ( z ) was modelled as a rough

layer with adjustable thickness d and adjustable plateau value of the volume fraction ΦBLG0 . Mathematically,

this layer is represented by the difference of two error functions:

ΦBLG ( z )=ΦBLG

0

2 [erf ( z−z0+d /2√2 σ a )−erf ( z−z0−d /2

√2 σw )], (2)

where σ a and σ w are adjustable parameters specifying the roughnesses of the layer towards air and water, respectively. Note that the centre of the protein distribution was allowed to be offset from the “interface” (z = 0, see Eq. 1), by an adjustable increment z0. Whenever required (see section 3.2), a second layer-like protein distribution adjacent to the first one was allowed, with an additional set of adjustable parameters d2, ΦBLG, 2

0 , z2. In the final step, the volume not occupied by the protein layer(s), 1−ΦBLG ( z ), was filled up with water according to the definition of the interface in Eq. 1:

ΦW ( z )=ΦW0 ( z )⋅ (1−ΦBLG ( z ) ). (3)

With both volume fraction profiles resolved, the corresponding SLD profiles, ρ(z), were then calculated as:

ρ ( z )=ρBLG ⋅ΦBLG (z )+ρW ⋅ΦW ( z ), (4)

where ρW and ρBLG are the SLDs of the water and the protein, respectively; note that the latter has different values in D2O and in ACMW according to the dynamic exchange of labile hydrogens (see Table 1). The theoretical reflectivity curves in each water contrast were then calculated by discretizing the SLD profiles into 1 Å layer of constant SLD and subsequent application of Parratt’s iterative procedure.58 Finally, the best-matching model parameters were obtained in a least-square minimization of the theoretical reflectivity curves with respect to the experimental ones, performed simultaneously for both contrasts. To sample properly the entire physically-plausible parameter space, parameters were varied randomly, and the changes were accepted following the Metropolis-MC criteria.59 Statistical errors in Table 3 were estimated by the bootstrap method60 and correspond to the 68% (1-sigma) confidence interval. These purely statistical errors

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typically represent an underestimation of the actual error because they do not reflect systematic uncertainties associated to the experiment and the model employed for the data analysis. 61 For the layer thickness, we consider 3 Å a reasonable error estimate.

Some molecular parameters of BLG such as the empirical chemical formula (C817N206O244S9H1275), molecular volume VBLG and molecular weight MBLG (that give the molecular mass density DBLG), and the number of exchangeable hydrogen atoms (271), required for the processing of the NR measured data, were calculated with the “ISIS Biomolecular Scattering Length Density Calculator”* on the basis of the 162 amino acids sequence:

LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI

taken from the UniProtKB database (sp|P02754|17-178)†. In these calculations we assumed 100 % exchange of the labile hydrogens.45 The molecular volume VBLG was calculated as the sum of the volumes of its constituent amino acids from its primary sequence and represents the actual volume of BLG at the water/air interface, i.e., no compaction of the molecule. This assumption was made in spite of known changes in volume of molecules with hydrogen bonds upon adsorption to an interface; yet the assumption is tested as described below. Any pH-driven changes in the VBLG-values can be estimated as less than 1 % in the pH range 3–7,10 and are thus neglected in our calculations. Then, using VBLG = 22 700 Å3, values of the SLD of BLG were calculated with the web-based “Neutron activation and scattering calculator”‡. The output values from these web-based calculators for parameters involved in the processing of the experimental data are listed in Table 1.

The obtained protein volume fraction profiles are directly linked to the surface excess Γ and the area A per BLG molecule according to the relations:

ΓM BLG

= 1A

= 1V BLG

∫−∞

∞

ΦBLG ( z ) dz. (5)

For the low-Qz NR data recorded in the kinetic mode, simpler reflectivity fits based on a single, roughness-free homogeneous protein layer with adjustable thickness were performed as described previously.52 While they do not resolve the structural details of the BLG layers, the obtained -values are nonetheless determined with high precision.

Table 1. Values of the molecular parameters for BLG in different aqueous media.Aqueous phase Molecular volume*

VBLG [Å3]Molecular weight*

MBLG [kg/mol]Molecular

densityDBLG [g/ml]

SLD‡

ρBLG 10-6 [Å-2]

H2O22 700

18.3 1.34 1.74ACMW 18.3 1.34 1.88

D2O 18.5 1.36 3.02

2.3. Ellipsometry

Measurements of the ellipsometric angles Δ and Ψ 62 were conducted with an Optrel Multiskop ellipsometer (Germany)63 working with a wavelength of λ = 633 nm. Experiments at the water/air interface were

*http://psldc.isis.rl.ac.uk/Psldc/ †http://www.uniprot.org/uniprot/P02754 ‡https://www.ncnr.nist.gov/resources/activation/

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performed with solutions either in a Petri glass dish or in a circular Teflon vessel equipped with a lid in order to reduce evaporation of the sample and to allow for long time measurements.

Samples in the Petri glass dish were measured for 1 hour at an angle of incidence θ elli = 49 deg. After gently pouring the solution, the surface was aspirated (to remove any kinetically-trapped protein already present at the interface and set the origin t0), and an automated measuring mode was then started. In this way, we were able to gain the first measured point at around 10 s after the aspiration of the surface at t0 and further data were acquired with a time resolution of 10 s.

Samples in the teflon vessel were measured for ~ 22 h at an angle of incidence of θ elli = 56.5 deg. The geometry of the compartment where the sample rests allowed for approximately the same (Teflon)surface-to-(solution)volume ratio to be maintained as that of the troughs used for the NR measurements. The lid of the vessel has two small circular windows (diameter ~1 mm) which are open to ensure the incident and the reflected beams travel undisturbed in air. After pouring the solution and aspirating the surface, some time was needed to mount the lid and ensure that the incident and the reflected beams transmit through the small windows. In this way, we were able to gain the first measuring point within 2 min after the aspiration ( t0) of the surface; further data were acquired with a time resolution of 60 s.

The phase shift (Δ) of light is much more sensitive than its attenuation (Ψ) to the properties of thin transparent layers at the water/air interface. Here therefore we focus on the parameter Δ BLG(t) = Δ(t) − Δ0, which is defined as the change in phase shift resulting from BLG assembled at the water/air interface, and Δ0

is the reference value of the phase shift for the solvent used; in the present case we measured Δ 0 = 179.2 deg at θelli = 49 deg and Δ0 = 359.6 deg at θelli = 56.5 deg for 10 mM buffers at all studied pH-values. The small deviation from 360 deg or 180 deg, respectively, accounts for the roughness of the liquid interface,64 and hence subtraction of Δ0 aims approximately to remove the effects of surface roughness from the processed data.

In the thin layer limit, ΔBLG is linearly proportional to the ellipsometric thickness as the pre-factor is a function that depends only on the laser wavelength, bulk optical properties (refractive indices of solution n w

and air na) and the angle of incidence θelli.62 Knowing these parameters for a single uniform isotropic protein layer with refractive index nBLG separated by sharp stratified interfaces, one can calculate the relation between ΔBLG and the layer thickness d; detailed explanation is given in the SI of ref.65. Further, ΔBLG can be related to the surface excess Γ via the de Feijter’s formalism66 by knowing the refractive index increment ∂nw/∂CBLG, where CBLG is the protein bulk concentration; see also the Supporting Information of ref.65. A model with an “oil-like” layer (constant density with changing thickness) results in a linear relation between Γ and ΔBLG while a model with a “particle-like” layer (constant thickness with changing density) results in a second order relation. Following a recent approach used in the modelling of surface layers of HAS, 65 we used the “particle-like” model being as more likely to represent the physical characteristics of protein adsorption. A key input parameter in this model is the layer thickness d and here we made use of the layer thickness values dFWHM (for details see in section 3.2) obtained from the NR experiments and listed in Table 3. In the case of the sample at 10-5 M BLG at pH 5, where a two-slab structure was resolved, as discussed in details in section 3.2, we used an effective thickness deff, defined as:

deff =Γ

DBLG Φeff, (6)

which corresponds to a single interfacial layer with effective protein volume fraction Φeff and total surface excess Γ. Here the effective volume fraction of such layer is:

Φeff=Φ1 Γ1

Γ+

Φ2 Γ2

Γ(7)

where subscripts 1 and 2 denote the volume fraction and the surface excess of the primary and secondary isotropic slabs in a multilayer interfacial structure (see Table 3).

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The dynamic surface excess Γ(t) may be related to Δpro in terms of the refractive index of the protein, the effective layer thickness and the protein molecular density. By making use of the estimated deff-values and the values of the following parameters: Refractive indices: air, na = 1; water solution, nw= 1.33; BLG, nBLG = 1.58; the latter is calculated by the

relation nBLG = nw + DBLG(∂nw/∂CBLG), where CBLG is the BLG bulk concentration; Refractive index increment for BLG solutions: ∂nw/∂CBLG = 0.18 cm3/g (neglecting any effect of pH) –

this value has been experimentally derived for BLG solutions with electrolyte content of 10 mM phosphate buffer pH 6.7 66 and practically the same as that estimated for pH 5.2,67 and also used in ref.68;

Protein mass density: DBLG= 1.34 g/cm3 (see Table 1);a set of “particle-like layer” second order relations of the type:

Γ=a ΔBLG+b ΔBLG2 (8)

were derived; the values of the coefficients a and b are listed in Table 2.

Table 2. Values of the coefficients a and b in eq. (8) evaluated with the given θelli and deff for different BLG concentrations and pH.

CBLG [M] pH deff [Å] θelli [deg] a [mg/(m2 deg)] b [mg/(m2 deg2)]10-5 3 10.9 49 0.84 – 0.09310-5 5 38.5 49 0.84 – 0.02610-5 7 11.9 49 0.84 – 0.08510-5 3 10.9 56.5 – 0.66 – 0.05610-5 5 38.5 56.5 – 0.66 – 0.01610-5 7 11.9 56.5 – 0.66 – 0.05210-7 3 9.4 56.5 – 0.66 – 0.06510-7 5 10.1 56.5 – 0.66 – 0.06110-7 7 10.3 56.5 – 0.66 – 0.060

Before presenting and discussing the results, we recall here that we used different aqueous isotopic contrasts in the kinetic experiments with NR (ACMW) and ellipsometry (H2O), and in the measurements for the structural characterization of the adsorbed layers (ACMW and D2O). Therefore, it should be noted that partial or entire substitution of H2O by D2O could affect the adsorption of proteins, however, mostly the rates of adsorption at comparatively short time scales. Indeed, it has been shown that the adsorption kinetics of proteins on a hydrophobic solid surface, as measured by surface plasmon resonance (SPR), can be slower in D2O than in H2O, e.g. for bovine serum albumin and RNase the adsorption rate constants in the initial stages of adsorption are respectively 1.7-fold and 2.6-fold smaller in D2O.69 However, the measured SPR signal for each of these proteins become indistinguishable in both isotopic aqueous media after about 10 min of adsorption and the amount of adsorbed protein does not show a major solvent isotope effect. Ganzevles et al.46 reported that the adsorption kinetics at the water/air interface for BLG solutions at CBLG ≈ 5.510-6 and pH 4.5 is about 3-fold slower in D2O than in H2O, but the major part of the adsorption process was found to take place within the first hour. Since our kinetic experiments with NR were performed in ACMW, which contains only 8.1% v/v D2O in H2O), we assume eventually only weak effect of the aqueous media on the adsorption kinetics as measured by NR and ellipsometry. The measurements in the full Qz-range were performed after at least 9 hours of adsorption (see more details in section 3.2).

3. Results3.1. Adsorption kinetics

Kinetic Γ(t) data for BLG concentrations of 10-7 M and 10-5 M at pH 3, 5 and 7 were recorded with NR and ellipsometry. The agreement between the results from the two techniques is very good (with the exception

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of pH 5 at high BLG concentration, see further below). In addition, two higher concentrations of 10 -4 M and 2×10-4 at pH 7 and pH 3, respectively, were measured with NR. All these results are presented in Fig. 1.

Fig. 1. Dynamic surface excess Γ(t) at the water/air interface for BLG solutions at different BLG concentrations and pH 5 (A), pH 7 (B) and pH 3 (C) in 10 mM buffer. NR, symbols: (□) 10 -7 M, (○) 10-5 M, (×) 10-4 M, (+) 2×10-4 M; the last data points for 10-7 M and 10-5 M are those obtained from the co-refinement analysis of the reflectivity curves in Fig. 2 (for details see in the next section). Ellipsometry, lines: ( ●●●●●) 10-7

M, θelli = 56.5 deg; 10-5 M: (----) θelli = 49 deg and ( ) θ⸺ elli = 56.5 deg. Insets show the same data as in the main graphs, but on a logarithmic time scale.

Γ increases monotonically with time,36,43,44,67,70 which is in agreement with interpretations of previously obtained dynamic surface pressure Π(t) data for the same solution compositions28 (shown in Fig. S1 in the SI). It should be noted that protein adsorption is a continuous process lacking any commonly accepted criterion for the point at which a protein layer is sufficiently close to equilibrium. Usually, one refers to a steady state in the kinetic dependences of the surface tension γ or the surface excess Γ. Importantly, such curves never show a plateau region when presented on a logarithmic time axis (see the insets in Fig. 1 and also Fig. S1 in SI). For the lowest protein concentration (10 -7 M, Fig. 1-A) the kinetic Γ(t) curves for pH 5 and 7 are comparable, while the data at pH 3 shows slower adsorption kinetics and lower surface excess both consistent with the highest molecular net charge |Z|pH3

10-12. This picture is in agreement with interpretations of previously reported dynamic surface pressure data (see also Fig. S1 in SI).3,24,28 However, some peculiarities in the surface pressure isotherm Π(CBLG) were reported at low concentrations (CBLG < 10−8 M) where an ‘anomalously’ lower surface activity of BLG was observed at pH 5 as compared to the case at pH 7. 28

Furthermore, it seems that at CBLG = 10−7 M the surface excess and the corresponding surface pressure are not much affected by changing pH from 5 to 7, but they are strongly pH-dependent at higher concentrations (Fig. 1 and Figs. S1 and S2 in SI) – the Γ(t) curves shift to larger values in the order Γ(ti)pH3 < Γ(ti)pH7 < Γ(ti)pH5. This general trend shows that the net charge |Z| of a protein molecule in solution controls its surface activity and the larger |Z| the lower the surface activity, as measured in terms of surface pressure and surface excess (see also Fig. S3 in SI).28,36 Thus, since |Z|pH7 < |Z|pH3 (ZpH7 ≈ –10 and ZpH3 ≈ +30),10-12 the surface excesses at

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given BLG concentration are larger at pH 7 than at pH 3 (Fig. 1). This trend is expected to be valid also for a wider protein concentration range as suggested by the surface pressure isotherm Π(CBLG) measured at pH 3 and 7 (Fig. S3 in SI).28

At pH values close to the isoelectric point, BLG reaches its highest surface activity reflected in the highest values of Π and Γ (see also Fig. S3 in SI). Based on results from sum frequency generation (SFG) spectroscopy24 and ellipsometry24,32 it was earlier proposed that disordered and agglomerated BLG multilayers are formed, which apparently result in maximum Γ-values. Indeed, the BLG surface excesses for pH 7 and pH 3, even at the highest measured concentrations (10 -4 M and 2×10-4 M, respectively), do not reach the high Γ-values measured for pH 5 at one order of magnitude lower BLG concentration of 10 -5 M (Fig. 1; for better comparison these data are plotted together in Fig. S2 in SI).

As mentioned above, the agreement between ellipsometry and NR results as shown in Fig. 1 is generally very good. However, for the higher BLG concentration at pH 5, Γ as obtained by ellipsometry is significantly larger than the corresponding NR values for long time scales (t > ~3 h). A possible explanation for this discrepancy could be the presence of BLG aggregates in the vicinity of the interface at low densities, which are not captured by the layer model used for the analysis of the NR data obtained at the late stage of the adsorption process (see next section). As described in the supporting information, at the lowest initial protein concentration investigated (CBLG = 10-7 M), protein adsorption to the surfaces can lead to bulk depletion of 20–30 % depending on pH. Strictly, this result indicates the breakdown of the assumption of a time-independent bulk concentration, which may slightly affect the shape of the adsorption curves. On the other hand, such depletion effect is not drastic either, and becomes insignificant at higher CBLG.

3.2. Structure of the adsorption layers

The full accessible Qz-range of reflectivity with contrast variation (ACMW and D2O) was measured after 9 h (pH 5), 11 h (pH 7), and 13 h (pH 3) for CBLG = 10-5 M, and after 16 h (pH 5), 17 h (pH 7) and 18 h (pH 3) for CBLG = 10-7 M. The durations of the scans for obtaining the full Qz-range are indicated with error bars in Fig. 1. Within these time periods, the change in surface tension is no more than 1 mN/m until the end of the data acquisition after 22 hours and the surface excesses for all samples were assumed to have reached virtually constant values, and the interfacial layers were assumed to be indistinguishable in both ACMW and D2O (see the discussion in the beginning of section 3), allowing for application of a co-refinement analysis of the reflectivity data obtained in both aqueous isotopic contrasts. Such analysis allows optimization between surface excess (reflectivity from ACMW solutions) and layer’s structure (reflectivity from D2O solutions).

The measured reflectivity curves (symbols) are shown in Fig. 2 in a log(RQz4) vs. Qz representation. Only the

curves at pH 5 and CBLG = 10-5 M (panel A2) exhibit distinct reflectivity minima at Qz ≈ 0.07 Å-1 and Qz ≈ 0.09 Å-1 in D2O and ACMW, respectively, indicating the formation of a thick protein layer (see text below and Table 3). These features are not seen for the other samples in Fig. 2, because they are shifted towards higher Qz-values for the thin protein layer formed under the given conditions.

The solid lines in Fig. 2 are the calculated reflectivity curves according to the best-matching parameters in the common model for the two contrasts (see section 2.2) that yield the volume fraction profiles ΦBLG(z ) and ΦW (z) for protein and water, respectively. These profiles are shown in Fig. 3. For all studied pH and CBLG conditions, a thin and dense protein layer is formed at the water/air interface, in the following termed primary monolayer. These layers are characterized by a full width at half maximum (FWHM) thickness of dFWHM ≈ 11 ± 5 Å (see Table 3). The FWHM of the distribution, in contrast, is conserved in all cases, and can therefore be considered a much more meaningful and robust specification of a protein distribution

10

Fig. 2. log(RQz4) vs. Qz fits (solid lines) to the experimental NR data (symbols) for solutions at 10-7 M

(A1,B1,C1) and 10-5 M (A2,B2,C2) BLG at pH 5 (A), pH 3 (B) and pH 7 (C) in ACMW (light blue triangles) and D2O (purple squares). Protein concentrations and pH-values are shown in legend.

Fig. 3. Volume fraction profiles of protein ΦBLG(z ) (red solid lines) and water ΦW (z) (blue dash-dotted lines) at the water/air interface (z = 0), obtained from the fits in Fig. 2.

11

Table 3. Present and literature data from NR measurements in ACMW single contrast (unless otherwise mentioned) for BLG adsorption layers at the water/air interface. Legend: CBLG – BLG bulk concentration; SL – single layer; L1 – primary monolayer exposed to the air; L2 – secondary sublayer exposed to the solution; dFWHM – values for the layer’s thickness in terms of the FWHM deduced in the present study ; ΦBLG

max – maximum protein volume fraction (see section 2.2); Γ – surface excess.

pH Source Salt[mM]

CBLG

[M]Mo-del

Thickness a [Å]

dFWHM

[Å]ΦBLG

max A[Å2 /molecule]

Γ[mg/m2]

2.0 ref.45 - 5.510-5 SL 29 1.63

2.6 ref.47 - 0.27-2.710-

4SL 25 ±6 1.54 ±0.14 b

3.0 This study c 10

1.010-7

1.010-5

2.010-4

SL

SL

-

9

11

-

0.65

0.91

-

3530

2220

0.86

1.37

1.89

4.5 ref.46 c 2 5.510-6 SL 40 3.20

5.0 This study c 10

1.010-7

1.010-5

SL

L1

L2

10

12

55

0.87

0.99

0.22

2490 1.22

2.80 d

5.7 ref.44

20 5.510-5

SL 44.4 2.45

6.0 ref.43 SL 18 3.3

ref.44

L1

L2

14.0 ±1.2

22.7 ±1.6

2.43 d

7.0

L1

L2

10.1

21.1

1.69 d

ref.45 - SL 28 1.58

ref.47 - 0.27-2.710-

4

SL 22 ±5 1.64 ±0.14 b

ref.40 50 5.510-4 L1

L2

18.2

18

2.1 d

This study c 10

1.010-7

1.010-5

1.010-4

SL

SL

-

10

12

-

0.85

0.92

-

2550

2030

1.19

1.49

2.07

a This “layer thickness” coincides with the thickness parameter of a single protein “slab”. The corresponding values for the present study are summarized in the supporting information. This concept, however, is valid and practicable only when the slab thickness is much larger than the interfacial roughness. As soon as they become comparable, considerable ambiguities arise. The FWHM of the distribution, in contrast, is conserved in all cases, and can therefore be considered a meaningful and robust specification of a protein distribution. b Estimated according to the reported data in the respective publication c Both in D2O and ACMWd Total value for all layers

12

Only in the single case at pH 5 and the higher BLG concentration (Fig. 3-A2), an additional, thicker but less dense layer is formed adjacent to the primary monolayer, as evidenced from the bimodal protein distribution, in the following termed secondary sublayer. Extended surface density profiles have been found as well for layers of other proteins: lysozyme53 bovine serum albumin (BSA),54,71 HSA65 and myoglobin72. For BLG such profiles (with ACMW subphase) have been previously resolved for adsorption layers at pH 7, but only at higher protein concentrations of ~5.510-5 M 44 and ~5.510-4 M 40 and in the presence of 20 and 50 mM buffer, respectively (note that at comparable BLG concentrations and pH 7, but at very low electrolyte content, homogeneous layers were found45,47). However, the interpretation of such extended profiles, which correspond to inhomogeneous (in density) surface layers, can be different. For BLG (at pH 7) Atkinson et al.44 concluded that denaturation-induced rearrangements of the protein’s tertiary structure at the interface – rather than additional multilayer formation – takes place. A similar assumption has been made by Eaglesham et al.71 for the case of spread layers of BSA, but the authors consider as well the option that the second layer can contain ‘solubilized’ protein molecules, i.e. discrete molecular layer; however, it was emphasised that the technique (with ACMW subphase) provides no direct method of differentiating between these possibilities. On the other hand, Holt et al.72 showed by NR measurements with D2O subphase that for myoglobin a discrete molecular secondary sublayer is formed at pH conditions close to the isoelectric point, which is consistent with our results for BLG at pH 5. Similar situation was reported for lysozyme 53 and BSA54. Therefore, the secondary sublayer of BLG identified in our results is not surprising, but its structure seems to be unique as discussed further below.

For the case of 10-5 M BLG at pH 5, while the model fits in Fig. 2 describe satisfactorily the measured reflectivity data, slightly better agreement (χ2 reduction by 37 %) can be obtained when the model allows for a slight increase in the protein SLD, i.e., a slight reduction in the volume per protein (see SI). In fact, such an increase in the molecular packing would be consistent with a recent report on phospholipid monolayers at the water/air interface, where poor model fits were shown to result if an assumption of no molecular compaction was made a priori in cases where it could be demonstrated to occur.73 Slightly better agreement with the experimental data (χ2 reduction by 87 %) is also achieved when assuming that patches of adsorbed proteins coexist with bare water/air interfacial regions (see the SI). Indeed, such patch-like structure has been found for adsorbed layers of lysozyme at the water/air interface,51 but there is no indication that BLG could behave in a similar manner.

4. Discussion4.1. Bulk properties vs. interfacial structure of BLG solutions

The structural data in Table 3 obtained via the model described above are given in terms of layer thickness dFWHM and maximal volume fractions. Further interpretation of these results and conclusions about the molecular structuring at the interface require information about the shape and dimensions of the adsorbing entities. Therefore, here we give firstly a brief overview about the molecular structure of BLG in bulk solutions and then propose possible interfacial structures.

The spatial dimensions of BLG have been the subject of investigations by crystallographic methods as well as light scattering and small-angle X-ray and neutron scattering (SAXS and SANS, respectively). The crystal structure has been resolved by X-ray diffraction.5,74-78 The linear dimensions of BLG in solution were reported in terms of the radius of gyration Rg or the hydrodynamic radius Rh: Rh = 19.15 Å (monomer),79 Rh = 20.4 Å (monomer) and 31.9 Å (dimer),14 Rh = 18 Å (monomer),80 Rg = 21-22 Å (dimer)81,82 and Rg = 34-35 Å (compact cubic octamer)74,81. Vogtt et al.21 performed SAXS and SANS experiments with BLG solutions at pH 7 and found that the dimer structure can be well approximated by two attached spheres each with a radius of 17.2 Å (corresponding to a monomer) that well agrees with the results of Timasheff and Townend74,81 who reported a monomer sphere radius of 17.9 Å. In the following we assume the consensus shape and size of a

13

BLG monomer in bulk solution to be a sphere with a diameter of 35 Å. A dimer is approximated as a prolate ellipsoid (consisting of two impinging such spheres) with a long axis of 69 Å and a short axis of 35 Å.74,81 An octamer is approximated by a cube with sides of 69 Å that comprises four dimers (see Fig. 4). At this point we recall that the solution pH does not significantly affect the dimensions of the individual BLG entities (monomer, dimer and octamer) but does control the transitions between them. Below pH 4 and above pH 5.2, BLG exists in a dynamic monomer-dimer equilibrium,5,14-21 while a dimer-octamer equilibrium is established within this pH range19,74. Changes in pH further away from pI result in an increase of the dissociation constant of dimers, i.e. the monomer is favoured.22 For a given pH, the respective equilibrium constants are sensitive to the concentrations of protein14,17,22 and electrolyte,6,14,18 and to the temperature14,18,19,21. With this information at hand, one can readily estimate the prevalence of the respective bulk entities in the studied samples and make a link to the interfacial structure. For pH 7, we can assume dimer fractions of ca. 10 % at 10-7 M, 75 % at 10-5 M and 90 % at 10-4 M BLG (note that these values, given by Aymard et al.,14 are relevant to BLG solutions containing 100 mM CH3OONH4). At pH 3 the equilibrium is strongly shifted towards the monomer and the dimer fraction was estimated to be <5 % at 10-5 M and negligible at 10-7 M BLG.17,18 It is not straightforward to predict the octamer fraction in our samples at pH 5, but we could accept it as in the order of few percent.19,83 Therefore, we assume that in our experiments the predominant species that arrive at the interface are monomers for the case of pH 3, dimers for pH 5 and a mixture of monomers and dimers for pH 7.

Based on these assumptions and according to the protein volume fraction profiles ΦBLG(z ) in Fig. 3, obtained from the NR data analysis, we can propose possible BLG interfacial structures. At a first glance, the monolayers at any pH and BLG concentrations appear very thin (dFWHM ≈ 11 ± 2 Å) as compared to the dimensions just of a spherical monomer in the bulk. This fact strongly indicates flattening of the BLG globules upon adsorption at the water/air interface and intuitively leads to the conclusion that if a dimer adsorbs at the interface it should adopt orientation with its polar (minor) axis normal to the surface (side-on); and eventually dissociates into monomers.39,40 Further, the shape of the flattened globules can be approximated by an oblate ellipsoid. For an adsorbed layer of such identical ellipsoids, a calculated FWHM-value of the layer is significantly thinner, by a factor of √2, than the ellipsoid’s polar diameter, because of the heterogeneous matter distribution, i.e., dFWHM ≈ 9–12 Å corresponds to a polar diameter of ≈ 13–17 Å (schematically illustrated in Fig. 4). This requires that a deformed spheroidal globule adsorbs in a side-on configuration, i.e. with its polar diameter being perpendicular to the interface. In the same framework, it was shown that BSA, lysozyme and myoglobin globules also adopt flattened configuration upon adsorption.51,55,72

Note that flattening of BLG globules has also been observed upon adsorption at a solid hydrophobic surface in all cases of solutions with pH 3, 5 and 7.84 However, that study reported that flattening was much less pronounced for the hydrophobic surface investigated.

Fig. 4 schematically illustrates BLG layers corresponding to the structural results in Fig. 3. It is evident that any BLG oligomeric entities (dimer or octamer) present in solution (at any of the used pH values) experience strong interaction with the interface and adsorb forming a primary monolayer (Fig. 4-A). The obtained monolayer thicknesses demonstrate flattening of the native globular structure at the interface where it occupies areas per molecule A in the range from ≈ 17 nm2 (CBLG = 10-5 M pH 5) to ≈ 35 nm2 (CBLG = 10-7 M pH 3). It still does not seem clear whether a dimer dissociates into monomers upon adsorption (some evidence is discussed by Perriman et al.40) or just adopts a side-on configuration. Even less clear is the surface behaviour of octamers. The fact that a monolayer was found for the case of CBLG = 10-7 M at pH 5 excludes the presence of octamers at the interface. Indeed, some indications that octamers disintegrate into dimers/monomers upon adsorption onto methylated silica have been reported,85 but this is not necessarily valid for liquid-gas interfaces. For the higher measured protein concentration of 10-5 M at pH 5, the profiles in Fig 3-A2 reveal the formation of a looser secondary sublayer adhered onto the dense primary monolayer. The FWHM-thickness of the secondary sublayer (d2 ≈ 54 Å) is larger than the dimensions of the monomer but smaller than the side of a cubic octamer or the longer axis of a dimer. It is worth noting that according to

14

the total thickness (≈ 67 Å) of adsorbed protein molecules one could consider adsorption of cubic octamers (side of ≈ 69 Å), but this is not supported by our results which clearly show a double layer structure with rather different protein volume fractions. Therefore, the most plausible scenario is that the secondary loose sublayer is composed of disordered dimers (Fig. 4-B1). However, a more complicated structure where un-dissociated dimers are incorporated into the monolayer in configurations other than side-on could also be possible (Fig. 4-B2).

Fig. 4. Schematic representation of adsorbed BLG entities from aqueous solutions with different pH and protein concentrations: (A1,2) monolayers, (B1,2) possible multilayer structures.

4.2. Comparisons with literature results

Although BLG has been studied both in solutions and at interfaces for decades, the influence of protein concentration, ionic strength I and pH on the adsorption at the water/air interface has eluded a comprehensive description. In many cases, one or two of these factors have been fixed at a constant value while others varied. As a result, it can be helpful to consider a multidimensional parameter space achieved from different works to date in order to identify general trends. The pH-dependences of key surface layer characteristics such as the surface pressure Π and the surface dilational elasticity E’ as well as the surface excess Γ and the corresponding layer thickness d are of special importance. In the present work we used a fixed buffer concentration (Cbuff = 10 mM) and measured the dynamic surface excess Γ(t) as a function of pH and CBLG. On the basis of the obtained results and those from literature, we attempt to discuss the Γ(CBLG, pH, I) type of parameter space for surface layers close to equilibrium. Further, we use previously obtained results for the surface pressure isotherm Π(CBLG, pH) and for the dynamic surface dilational elasticity E’(t) to complement the adsorption data.

Investigations of the adsorption in solutions with BLG concentrations as low as 10 -7 M, as studied in the present work, have been reported to our best knowledge only for close to neutral pH.36,86-89 In those works, Γ(t) data for solutions with different protein concentrations and comparatively high ionic strengths were reported. In several studies, ellipsometry experiments have been performed for different BLG concentrations in solutions at pH 7 and at the same buffer concentration as in the present work, 27,32,67,70 therefore, a comparison with those results is helpful. Indeed, the results of Lech et al. 32 on the variation of Γ within a wide pH-range for CBLG ≈ 2.710-5 are in good agreement with our results. On the other hand, general features of the Γ(CBLG)pH7 dependence are not always obvious.

15

Adsorption kinetics of BLG solutions have been measured earlier using NR for CBLG ≈ 5.510-5 M at pH 6 (in 20 mM buffer).43,44 The authors observed an increase of the surface excess while the layer thickness d was virtually unchanged. A consistent comparison of our results with those of Atkinson et al.44 shows that the surface excess generally increases when the pH approaches the isoelectric point. For BLG layers at pH 6, Horne et al.43 found a monolayer with a thickness of ≈ 20 Å and a comparatively large surface excess (≈ 3.3 mg/m2), while Atkinson et al.44 were able to fit their reflectivity data either with single-slab or two-slab models. However, in both works, the authors concluded that the results indicate time-dependent rearrangements of the protein tertiary structure at the interface via a denaturation process rather than to additional multilayer formation, as we found at pH 5 in the present work.

Fig. 5. Surface excess Γ vs. pH for BLG layers at the water/air interface – comparison of literature results from NR measurements. Circles are data at different CBLG (in M) from the present study; other symbols are literature data from Table 3: red triangles (buffer-free).45 dark-blue stars (buffer-free),47 green diamonds (20

mM buffer),44 light-blue square (50 mM buffer),40 dark-red hexagon (I = 2 mM)46. Labels: CBLG in µM.

The adsorption and the structure of BLG layers close to equilibrium at the water/air interface have been studied by NR.40,43-47 The experiments in these works have been performed at ionic strengths I in the range from few mM, i.e., just by HCl/NaOH adjustment of pH, up to 50 mM buffer, which lessens the value of any comparisons, because the known I-dependence of the surface activity of BLG, which affects both Π23,28,36,38

and Γ27. However, a pH-dependent trend can be outlined pointing out the highest surface activity of BLG in the pH region 4.5–5 (close to pI) for sufficiently high BLG concentrations (Fig. 5).

BLG has been studied with NR the most at pH 7.40,44,45,47 Interestingly, Tucker et al.47 observed that the surface excess and thickness of BLG monolayers at low I are independent of the studied protein concentrations in the range from 2.710-5 M to 2.710-4 M. The average surface excess Γ ≈ 1.6 ± 0.14 mg/m2 is practically the same as that obtained by Holt et al.45 for 5.510-5 M BLG (also at low I). These results are qualitatively consistent with the constancy of the surface pressure within the CBLG range between 10-7 and 10-4 M in the surface pressure isotherm Π(CBLG) for salt-free BLG solutions (pH ≈ 6.5).28

These findings together suggest that monolayers on BLG solutions with near-neutral pH and at very low ionic strength become saturated at a surface pressure of ca. 20 mN/m and a surface excess Γ ≈ 1.6 mg/m2. The pronounced repulsive intermolecular interactions in the plane of the monolayer are consistent with a relatively loose protein lateral packing. Screening of the protein net charge by higher ionic strength leads to closer packing and respectively to higher Π28 and larger Γ27. At 10 mM buffer, pH 7 and CBLG =10-4 M, the adsorption is already Γ ≈ 2.1 mg/m2. Virtually the same surface excess was reported by Perriman et al. 40 for solutions at even higher concentrations of BLG (5.510-4 M) and buffer (50 mM). These results demonstrate

16

that the BLG primary monolayer observed at pH 7 is close to saturation at Γ ≈ 2.1 mg/m2 (see Table 3 and Fig. 5).

BLG surface layers have been studied at pH 2 45 and pH 2.6 47 for buffer-free solutions (low I) with protein concentrations ranging from 2.710-5 to 2.710-4 M. The surface excesses reported are similar to those at pH 7 investigated in the same studies and again do not exceed Γ ≈ 1.6 mg/m2. An increase to Γ ≈ 1.9 mg/m2

was found in the present work at pH 3 upon addition of 10 mM buffer (210-4 M BLG), see Fig. 1-C. This surface excess is still slightly lower than that (Γ ≈ 2.1 mg/m2) at pH 7 (10-4 M BLG), which is consistent with the larger protein net charge │Z│pH7 < │Z│pH3 and is also reflected in the surface pressure isotherm (see Fig. S4 in SI).28

Neither measurements of the dynamic surface pressure (Fig. S1 in SI) nor those of the dynamic surface excess (Figs. 1 and S2) were able to detect the onset of the multilayer formation since they both show monotonic kinetic dependences. Still, some evidence can be obtained from the kinetic dependence of the surface dilational rheological parameters – E’(t) and E”(t), being the real and the imaginary parts of the dilational viscoelasticity modulus – and in particular the obtained ‘dynamic’ E’(Π) and E”(Π) dependences (Fig. S1 in the SI).29 The E’(Π) data both for pH 3 (all measured BLG concentrations) and for pH 7 (CBLG < 10-4 M) agree well with a master curve, whose initial linear slope is weakly pH-dependent. 29 For pH 5 the situation is similar but only for relatively low BLG concentrations and at CBLG ≥ 10-6 M a local drop from the master curve of the dilational elasticity appears that can be related to the onset of multilayer formation. However, at longer times the dilational elasticity increases again (Fig. S1 in SI). The appearance of such a local drop in the dilational elasticity is accompanied by a distinct increase of the observed maximum in the E”(Π) dependence. We discuss these finding in more details in the SI and emphasize that they can be indicative of a transient step – due to fast adsorption of dimers – at the onset of the formation of the found BLG bilayer structure.

On the basis of our experimental data one cannot draw unambiguous conclusions about the most likely pathways in the evolution of BLG layers. Neither can one discern with certainty between the multilayer structures illustrated in Figs. 4-B1 and B2. In this context, the well-defined E’(Π)-dependence for monolayers at pH 3 (Fig. S1 in SI) can be related to the predominating monomer fraction in solution. On the other hand, for pH 7 (monomer-dimer mixture), we observed deviations from the E’(Π) master curve as well, but only for a relatively high BLG concentration (CBLG = 10-4 M). Indeed, under similar solution conditions a two-slab structure of the surface layer was found, but as discussed above it has not been assigned to a discreet secondary layer.40,44

5. Conclusions

We measured with ellipsometry and NR the adsorption kinetics Γ(t) of BLG layers at the water/air interface as a function of protein concentration CBLG and pH. The obtained results corroborate inferences from previously reported pH-dependent trends in the dynamic surface pressure Π(t) and surface pressure isotherm Π(CBLG, pH), but go an important step further to quantify the surface excess Γ and distinguish systems that exhibit primary monolayer structures from those featuring an additional secondary sublayer.

The highest surface activity of BLG is achieved at pH 5,23,24,32 but we show by surface pressure and surface excess measurements that this is valid only above a certain protein concentration ca. CBLG > 10-7 M. The structure of the protein adsorption layers studied, as obtained by NR with isotopic contrast variation, revealed the formation of a monolayer with dFWHM ≈ 11 ± 5 Å, which after accounting for the heterogeneous matter distribution in a slab of adsorbed ellipsoidal globules, corresponds to a physical thickness of the layer of ≈ 16 ± 7 Å. This suggests flattening of the native globular structure of a BLG monomer (diameter ≈ 35 Å) upon adsorption at the water/air interface. Only for high protein concentrations at pH 5, formation of a looser secondary sublayer (d2 ≈ 55 Å) adhered to the dense primary monolayer was found. Its thickness suggests that it consists of disordered dimers.

17

In a parameter space map (Γ, CBLG, pH, I) for interfacial layers we can conclude some limiting conditions in the adsorption behaviour of BLG at the water/air interface:

1) Under conditions of significant protein net charge (pH well away from pI) and low electrostatic screening the surface excess does not exceed ≈ 1.6 mg/m2 and relatively loose BLG monolayers are formed with thicknesses reported between 16 Å 47 (also in this study) and 30 Å 45,47.

2) Under the conditions of significant protein net charge and higher electrostatic screening the surface excess grows but still does not exceed Γ ≈ 2.1 mg/m2 40 (also in this study). In this case, screening of the protein net charge enhances the surface activity of BLG, which results in denser protein monolayers.

3) When approaching the isoelectric point close to pH 5 from either direction, the surface excess increases until it reaches values of up to Γ ≈ 3.2 mg/m2 46. The striking difference between these results is not just in the distinct Γ-values but rather in the revealed structures of the interfacial layers. For pH 4.5 46 and pH 5.7 44 the BLG interfacial layers were reported to have a uniform thickness of about 4 nm, which is comparable to the BLG monomer dimensions in the bulk solution, while for pH 5 we found a non-uniform volume fraction distribution in the interfacial zone consistent with a dense monolayer and a discrete looser secondary sublayer53,54.

Supporting Information

Tables S1–S3; Fig. S1, data for the kinetic dependencies of the surface pressure Π( t) and the dilational rheology parameters E’(t) and E”(t); Fig. S2, Γ(t) data from Fig. 1 for the highest BLG concentrations at pH 3, 5 and 7 measured in this study; Fig. S3, reflectivity curves and volume fraction distribution profiles obtained from the fitting test; Fig. S4, comparison of previous surface pressure isotherms data with the present surface excess data; short explanations.

Acknowledgements

The authors thank Institut Laue-Langevin (ILL) for beam time allocation https://doi.ill.fr/10.5291/ILL-DATA.9-13-640. Financial support by the Max Planck Society and by the German Research Foundation (DFG) via Emmy-Noether grant (SCHN 1396/1) is gratefully acknowledged. G.G. and R.M. acknowledge also the DFG/AiF cluster project on “Protein Foams” Mi418/20-1 and thank U. Kulozik (TU Munich) for supplying the high-quality protein sample. B.A.N. is grateful to the Russian Foundation of Basic Research, and the Ministry of Science and Technology of Taiwan (project no. 19-53-52006 MNT_a) for the financial support.

References

1. Heertje, I. Structure and function of food products: A review. Food Structure 1 (2014) 3-23.2. Foegeding, E.A. Food Protein Functionality-A New Model. J. Food Sci. 80 (2015) C2670-C2677.3. Wierenga, P.A., Gruppen, H. New views on foams from protein solutions Curr. Opin. Colloid Interface

Sci. 15 (2010) 365-373.4. Tcholakova, S., Denkov, N.D., Ivanov, I.B., Campbell, B. Coalescence stability of emulsions containing

globular milk proteins. Adv. Colloid Interface Sci.123-126(2006) 259-293.5. Sawyer, L. β-Lactoglobulin. in: Advanced Dairy Chemistry: Vol. 1A: Proteins: Basic Aspects. 3rd

Ed.Fox P.F. and McSweeney P.L.H. (eds.), Kluwer Academic/Plenum Publishers, 2003,Ch. 7, pp. 319; 4th Ed. McSweeney P.L.H. and Fox P.F. (eds.), Springer, New York, 2013, Ch. 7, pp. 211.

6. Piazza, R. Protein interactions and association: An open challenge for colloid science. Curr. Opin. Colloid Interface Sci. 8 (2004) 515-522.

7. Zhang, F., Weggler, S., Ziller, M. J., Ianeselli, L., Heck, B. S., Hildebrandt, A., Kohlbacher, O., Skoda, M.W.A., Jacobs, R.M.J., Schreiber, F. Universality of protein reentrant condensation in solution induced by multivalent metal ions. Proteins 78 (2010) 3450-3457.

8. Lucassen-Reynders, E.H., Benjamins, J., Fainerman, V.B. Dilational rheology of protein films adsorbed at fluid interfaces. Curr. Opin. Colloid Interface Sci. 15 (2010) 264-270.

18

9. Mitropoulos, V., Mütze, A., Fischer, P. Mechanical properties of protein adsorption layers at the air/water and oil/water interface: A comparison in light of the thermodynamical stability of proteins. Adv. Colloid Interface Sci. 206 (2014) 195-206.

10. Nozaki, Y., Bunville, L.G., Tanford, C. Hydrogen ion titration curves of β-lactoglobulin. J. Am. Chem. Soc. 81 (1959) 5523-5529.

11. Basch, J.J., Timasheff, S.N. Hydrogen ion equilibria of the genetic variants of bovine β-lactoglobulin. Arch. Biochem. Biophys. 118 (1967) 37-47.

12. Ghosh, S.K., Chaudhuri, S. Roy, J., Sinha, N.K., Sen, A. Physicochemical investigations on buffalo β-lactoglobulin. Studies on sedimentation, diffusion, and hydrogen ion titration. Arch. Biochem. Biophys. 144 (1971) 6-15.

13. Taulier, N., Chalikian, T.V. Characterization of pH-induced Transitions of β-Lactoglobulin: Ultrasonic, Densimetric, and Spectroscopic Studies. J. Mol. Biol. 314 (2001) 873-889.

14. Aymard, P., Durand, D., Nicolai, T. The effect of temperature and ionic strength on the dimerisation of β-lactoglobulin. Int. J. Biol. Macromolecules 19 (1996) 213-221

15. Renard, D., Lefebvre, J., Griffin, M.C.A., Griffin W.G. Effects of pH and salt environment on the association of β-lactoglobulin revealed by intrinsic fluorescence studies. Int. J. Biol. Macromol. 22 (1998) 41-49.

16. Verheul, M., Pedersen, J.S., Roefs, S.P.F.M., de Kruif, K.G. Association behavior of native β-lactoglobulin. Biopolymers 1999, 49, 11-20.

17. Sakai, K., Sakurai, K., Sakai, M., Hoshino, M., Goto, Y. Conformation and stability of thiol-modified bovine β-lactoglobulin. Prot. Sci. 9 (2000) 1719-1729.

18. Sakurai, K., Oobatake, M., Goto, Y. Salt-dependent monomer-dimer equilibrium of bovine β-lactoglobulin at pH 3. Prot. Sci. 10 (2001) 2325-2335.

19. Gottschalk, M., Nilsson, H., Roos, H., Halle, B. Protein self-association in solution: The bovine β-lactoglobulin dimer and octamer. Prot. Sci. 12 (2003) 2404-2411.

20. Mehalebi, S., Nikolai, T., Durand, D. Light scattering study of heat-denatured globular protein aggregates. Int. J. Biol. Macromol. 43 (2008) 129-135.

21. Vogtt, K., Javid, N., Alvarez, E., Sefcik, J., Bellissent-Funel, M.-C. Tracing nucleation pathways in protein aggregation by using small angle scattering methods. Soft Matter 7 (2011) 3906-3914.

22. Mercadante, D., Melton, L.D., Norris, G.E., (...), Dobson, R.C.J., Jameson, G.B. Bovine β-lactoglobulin is dimeric under imitative physiological conditions: Dissociation equilibrium and rate constants over the pH range of 2.5-7.5. Biophys. J. 103(2012) 303-312.

23. Davis, J.P., Foegeding, E.A., Hansen, F.K. Electrostatic effects on the yield stress of whey protein isolate foams, Colloids Surfaces B 34 (2004) 13-23.

24. Engelhardt, K., Lexis, M., Gochev, G., Ch. Konnerth, R. Miller, N. Willenbacher, W. Peukert and B. Braunschweig, pH effects on the molecular structure of -lactoglobulin modified air-water interfaces and its impact on foamrheology. Langmuir 29 (2013) 11646-11655.

25. Engelhardt, K., Peukert, W. Braunschweig, B. Vibrational sum-frequency generation at protein modified air–water interfaces: Effects of molecular structure and surface charging. Curr. Opin. Colloid Interface Sci. 19 (2014) 207-215.

26. Lexis, M., Willenbacher, N. Relating foam and interfacial rheological properties of β-lactoglobulin solutions. Soft Matter 10 (2014) 9626-9636.

27. Delahaije, R.J.B.M., Gruppen, H., Giuseppin, M.L.F., Wierenga, P.A. Quantitative description of the parameters affecting the adsorption behaviour of globular proteins. Colloids Surfaces B 123 (2014) 199-206.

28. Ulaganathan, V., Retzlaff, I., Won, J.Y., G. Gochev, C. Gehin-Delval, M.E. Leser, B.A. Noskov and R. Miller. β-Lactoglobulin adsorption layers at the water/air surface: 1. Adsorption kinetics and surface pressure isotherm: Effect of pH and ionic strength. Colloids Surfaces A 519 (2017) 153-160.

19

29. Ulaganathan, V., Retzlaff, I., Won, J.Y., G. Gochev, D.Z. Gunes, C. Gehin-Delval, M. Leser, B.A. Noskov and R. Miller. β-Lactoglobulin adsorption layers at the water/air surface: 2. Dilational rheology: Effect of pH and ionic strength. Colloids Surfaces A 521 (2017) 167-176.

30. Dimitrova, T.D., Leal-Calderon, F., Gurkov, T.D., Campbell, B. Disjoining pressure vs thickness isotherms of thin emulsion films stabilized by proteins. Langmuir 17 (2001) 8069.

31. Gochev, G., Retzlaff, I., Exerowa, D., Miller, R. Electrostatic stabilization of foam films from β-lactoglobulin solutions. Colloids Surfaces A 460 (2014) 272–279.

32. Lech, F.J., Delahaije, R.J.B.M., Meinders, M.B.J., Gruppen, H., Wierenga, P.A. Identification of critical concentrations determining foam ability and stability of β-lactoglobulin. Food Hydrocolloids 57 (2016) 46-54.

33. Braunschweig, B., Schulze-Zachau, F., Nagel, E., K. Engelhardt, S. Stoyanov, G. Gochev, K.. Khristov, E. Mileva, D. Exerowa, R. Miller and W. Peukert. Specific effects of Ca2+ ions and molecular structure of β-lactoglobulin interfacial layers that drive macroscopic foam stability. Soft Matter 12 (2016) 5995-6004.

34. Petkova, V., Sultanem, C., Nedyalkov, M., Benattar, J.-J., Leser, M.E., Schmitt, C. Structure of a freestanding film of β-lactoglobulin. Langmuir 19 (2003) 6942-6949

35. Kolodziejczyk, E., Petkova, V., Benattar, J.-J., Leser, M.E., Michel, M. Effect of fluorescent labeling of β-lactoglobulin on film and interfacial properties in relation to confocal fluorescence microscopy. Colloids Surfaces A 279 (2006) 159-166.

36. Song K.B., Damodaran, S. Influence of electrostatic forces on the adsorption of succinylated -lactoglobulin at the air-water interface. Langmuir 7 (1991)2737–2742.

37. Jara, F.L., Sánchez, C.C., Patino, J.M.R., Pilosof, A.M.R. Competitive adsorption behavior of -lactoglobulin α-lactalbumin, bovin serum albumin in presence of hydroxypropylmethylcellulose. Influence of pH. Food Hydrocolloids 35 (2014) 189–197.

38. Beierlein, F.R., Clark, T., Braunschweig, B., Engelhardt, K., Glas, L., Peukert, W. Carboxylate ion pairing with alkali-metal ions for -lactoglobulin and its role on aggregation and interfacial adsorption. J. Phys. Chem. B 119 (2015) 5505–5517.

39. Mackie, A. R.; Husband, F. A., Holt, C., Wilde, P. J. Adsorption of β-Lactoglobulin variants A and B to the air-water interface. Int. J. Food Sci. Technol. 34 (1999) 509-516.

40. Perriman, A.W., Henderson, M.J., Holt, S.A., White, J.W. Effect of the air-water interface on the stability of β-lactoglobulin. J. Phys. Chem. B 111 (2007) 13527-13537.

41. Braun, L., Uhlig, M., von Klitzing, R., Campbell, R.A. Polymers and surfactants at fluid interfaces studied with specular neutron reflectometry. Adv. Colloid Interface Sci. 247 (2017) 130-148.

42. Campbell, R.A., Wacklin, H. P., Sutton, I., Cubitt, R., Fragneto, G. FIGARO: the New Horizontal Neutron Reflectometer at the ILL. Eur. Phys. J. Plus 126 (2011) 107.

43. Horne, D. S., Atkinson, P. J., Dickinson, E., Pinfield, V. J., Richardson, R. M. Neutron reflectivity of competitive adsorption of β-lactoglobulin and non-ionic surfactant at the air-water interface. Int. Dairy J. 8 (1998) 73-77.

44. Atkinson, P.J., Dickinson, E., Horne, D.S., Richardson, R.M. Neutron reflectivity of adsorbed β-casein and β-lactoglobulin at the air/water interface. J. Chem. Soc., Faraday Trans. 91 (1995) 2847-2854.

45. Holt, S.A., Henderson, M.J., White, J.W. Thermal denaturation of interfacial protein layers. Aust. J. Chem. 55 (2002) 449-459.

46. Ganzevles, R.A., Fokkink, R., van Vliet, T., Cohen Stuart, M.A., de Jongh, H.H.J. Structure of mixed β-lactoglobulin/pectin adsorbed layers at air/water interfaces; a spectroscopy study. J. Colloid Interface Sci. 317 (2008) 137-147.

47. Tucker, I.M., Petkov, J.T., Penfold, J., Thomas R.K., Cox A.R., Hedges N. Adsorption of hydrophobin-protein mixtures at the air-water interface: The impact of pH and electrolyte. Langmuir 31 (2015) 10008-10016.

48. Toro-Sierra, J., Tolkach, A., Kulozik, U. Fractionation of α-Lactalbumin and β-Lactoglobulin from Whey Protein Isolate Using Selective Thermal Aggregation, an Optimized Membrane Separation

20

Procedure and Resolubilization Techniques at Pilot Plant Scale. Food Bioprocess Technol. 6 (2013) 1032-1043.

49. Glasoe, P.K., Long, F.A. Use of Glass Electrodes to Measure Acidities in Deuterium Oxide. J. Phys. Chem. 64 (1960) 188-190.

50. Clark, D.C., Husband, F., Wilde, P., Cornec, M., Miller, R., Krägel, J., Wüstneck, R. Evidence of Extraneous Surfactant Adsorption altering Adsorbed Layer Properties of β-Lactoglobulin. J. Chem. Soc., Faraday Trans. 91 (1995) 1991-1996.

51. Campbell, R. A., Tummino, A., Varga, I., Milyaeva, O.Y., Krycki, M.M., Lin, S.-Y., Laux, V., Haertlein, M., Forsyth, V.T., Noskov, B.A. Adsorption of Denaturated Lysozyme at the Air-Water Interface: Structure and Morphology. Langmuir 34 (2018) 5020-5029.

52. Ang, J.C., Henderson, M.J., Campbell, R.A., Lin, J.-M., Yaron, P.N., Nelson, A., Faunce, T., White, J.W. Human serum albumin binding to silica nanoparticles-effect of protein fatty acid ligand. Phys. Chem. Chem. Phys. 16 (2014) 10157-110168.

53. Lu, J.R., Su, T.J., Thomas, R.K., Penfold, J., Webster, J. Structural conformation of lysozyme layers at the air/water interface studied by neutron reflection. J. Chem. Soc., Faraday Trans. 94 (1998) 3279-3287.

54. Lu, J.R., Su, T.J., Thomas, R.K. Structural Conformation of Bovine Serum Albumin Layers at the Air–Water Interface Studied by Neutron Reflection. J. Colloid Interface Sci. 213 (1999) 426-437.

55. Schneck, E., Schollier, A., Halperin, A., Moulin, M., Haertlein, M., Sferrazza, M., Fragneto, G. Neutron reflectometry elucidates density profiles of deuterated proteins adsorbed onto surfaces displaying poly(ethylene glycol) brushes: Evidence for primary adsorption. Langmuir 29 (2013) 14178-14187.

56. Braslau, A., Deutsch, M., Pershan, P. S., Weiss, A. H., Als-Nielsen, J., Bohr J. Surface roughness of water measured by x-ray reflectivity. Phys. Rev. Lett. 54 (1985) 114-117.

57. Sinha, S.K., Sirota, E.B., Garoff, S., Stanley, H.B. X-ray and neutron scattering from rough surfaces. Phys. Rev. B 38 (1988) 2297-2311.

58. Parratt, L.G. Surface studies of solids by total reflection of x-rays. Phys. Rev. 95 (1954) 359-369.59. Metropolis, N., Rosenbluth, A.W., Rosenbluth, M.N., Teller, A.H., Teller, E. Equation of state

calculations by fast computing machines. J. Chem. Phys. 21 (1953) 1087-1092.60. Gerelli, Y. Aurore: new software for neutron reflectivity data analysis. J. Appl. Cryst. 49 (2016) 330-

339.61. Rodriguez-Loureiro, I., Scoppola, E., Bertinetti, L., (...), Fragneto, G., Schneck, E. Neutron

reflectometry yields distance-dependent structures of nanometric polymer brushes interacting across water. Soft Matter 13 (2017) 5767-5777.

62. Motschmann, H., Teppner, R. Ellipsometry in Interface Science. In: Studies in Interface Science , vol. 11, Elsevier: New York, 2001, 1-42.

63. Harke, H., Teppner, R., Schulz, O., Orendi, H., Motschmann, H. Description of a single modular optical setup for ellipsometry, surface plasmons, waveguide modes, and their corresponding imaging techniques including Brewster angle microscopy. Rev. Sci. Instrum. 68 (1997) 3130-3134.

64. Meunier, J. Light Scattering by Liquid Surfaces and Complementary Techniques. Marcel Dekker: New York, 1992.

65. Campbell, R.A. Ang, J.C., Sebastiani, F., Tummino, A., White J.W. Spread Films of Human Serum Albumin at the Air−Water Interface: Optimization, Morphology, and Durability. Langmuir 31 (2015) 13535-13542.

66. De Feijter, J.A., Benjamins, J., Veer, F.A. Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the air-water interface. Biopolymers 17 (1978) 1759-1772.

67. van Dijk, J.A.P.P., Smit, J.A.M. Size-exclusion chromatography–multiangle laser light scattering analysis of β-lactoglobulin and bovine serum albumin in aqueous solution with added salt. J. Chromatogr. A 867 (2000) 105-112.

21

68. Wierenga, P.A., Egmond, M.R., Voragen, A.G.J, de Jongh H.H.J. The adsorption and unfolding kinetics determines the folding state of proteins at the air–water interface and thereby the equation of state. J. Colloid Interface Sci. 299 (2006) 850-857.

69. Grunwald, C., Kuhlmann, J., Wöll, C. In Deuterated Water the Unspecific Adsorption of Proteins Is Significantly Slowed Down:  Results of an SPR Study Using Model Organic Surfaces. Langmuir 21 (2005) 9017-9019.

70. Noskov, B.A., Grigoriev, D.O., Latnikova, A.V., Lin, S.-Y., Loglio, G., Miller, R. Impact of Globule Unfolding on Dilational Viscoelasticity of β-Lactoglobulin Adsorption Layers. J. Phys. Chem. B113 (2009) 13398–13404.

71. Eaglesham, A., Herrington, T.M., Penfold, J. A neutron reflectivity study of a spread monolayer of bovine serum albumin. Colloids Surfaces A 65 (1992) 9-16.

72. Holt, S.A., McGillivray, D.J., Poon, S., White, J.W. Protein deformation and surfactancy at an interface. J. Phys. Chem. B 104 (2000) 7431-7438.

73. Campbell, R.A., Saaka, Y., Shao, Y., Gerelli, Y., Cubitt, R., Nazaruk, E., Matyszewska, D., Lawrence, M.J. Structure of Surfactant and Phospholipid Monolayers at the Air/Water Interface modeled from Neutron Reflectivity Data. J. Colloid Interface Sci. 531 (2018) 98-108.

74. Timasheff, S.N., Townend, R. Structure of the β-Lactoglobulin Tetramer. Nature 203 (1964) 517-519.75. Brownlow, S., Morais Cabral, J.H., Cooper, R., (...), North, A.C.T., Sawyer, L.Bovine β-lactoglobulin at

1.8 Å resolution - still an enigmatic lipocalin. Structure 5 (1997) 481-495.76. Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, H.M., Baker, E.N. and Jameson, G.B. Structural basis

of the Tanford transition of bovine β-lactoglobulin. Biochem. 37 (1998) 14014-14023.77. Adams, J. J.; Anderson, B. F.; Norris, G. E.; Creamer, L. K.; Jameson, G. B. Structure of bovine β-

lactoglobulin (variant A) at very low ionic strength. J. Struct. Biol.154 (2005) 246-254.78. Kontopidis, G., Gilliver, A.N., Sawyer, L., Ovine β-lactoglobulin at atomic resolution. Acta

Crystallogr. F: Struc. Biol. Commun. 70 (2014) 1498-1503.79. Baldini, G., Beretta, S., Chirico, G., Franz, H., Maccioni, E., Mariani, P., Spinozzi, F. Salt-Induced

Association of β-Lactoglobulin by Light and X-ray Scattering. Macromolecules 32 (1999) 6128-6138.80. Ruiz-Henestrosa, V.M.P., Martinez, M. J., Carrera Sánchez, C., Rodríguez Patino, J.M., Pilosof, A.M.R.

Mixed soy globulins and β-lactoglobulin systems behavior in aqueous solutions and at the air/water interface. Food Hydrocolloids 35 (2014) 106-114.

81. Witz, J., Timasheff, S.N., Luzzati, V. Molecular interactions in β-lactoglobulin, VIII: Small-angle X-ray scattering investigation of the geometry of β-lactoglobulin A tetramerization. J. Am. Chem. Soc. 86 (1964) 168-173.

82. Panick, G., Malessa, R., Winter R. Differences between the Pressure- and Temperature-Induced Denaturation and Aggregation of β-Lactoglobulin A, B, and AB Monitored by FT-IR Spectroscopy and Small-Angle X-ray Scattering. Biochem. 38 (1999) 6512-6519.

83. Townend, R., Timasheff, S.N. Molecular Interactions in β-Lactoglobulin. III. Light Scattering Investigation of the Stoichiometry of the Association between pH 3.7 and 5. J. Amer. Chem. Soc.82 (1960) 3168-3174.

84. Fragneto, G., Su, T.J., Lu, J.R., Thomas, R.K., Rennie A.R. Adsorption of proteins from aqueous solutions on hydrophobic surfaces studied by neutron refection. Phys. Chem. Chem. Phys. 2 (2000) 5214-5221.

85. Elofsson, U.M.; Paulsson, M.A.; Arnebrant, T. Adsorption of β-Lactoglobulin A and B in Relation toSelf-Association: Effect of Concentration and pH. Langmuir 13 (1997) 1695-1700.

86. Sengupta, T., Razumovsky, L., Damodaran, S. Energetics of Protein-Interface Interactions and Its Effecton Protein Adsorption. Langmuir 15 (1999) 6991-7001.

87. Cornec, M., Narsimhan, G. Effect of Contaminant on Adsorption of Whey Proteins at theAir-Water Interface. J. Agric. Food Chem. 46 (1998) 2490-2498.

88. Cornec, M., Cho, D., Narsimhan, G. Adsorption Dynamics of α-Lactalbumin and β-Lactoglobulinat Air–Water Interfaces. J. Colloid Interface Sci. 214 (1999) 129-142.

22

89. Cornec, M., Narsimhan, G. Adsorption and Exchange of β-Lactoglobulin onto Spread Monoglyceride Monolayers at the Air-Water Interface. Langmuir 16 (2000) 1216-1225.

23