Article pubs.acs.org/Langmuir

Adsorption of Bovine Serum Albumin (BSA) at the Oil/Water Interface: A Neutron Reflection Study M. Campana,†,‡ S. L. Hosking,‡ J. T. Petkov,§ I. M. Tucker,‡ J. R. P. Webster,∥ A. Zarbakhsh,⊥ and J. R. Lu*,† †

Biological Physics Group, School of Physics and Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom ‡ Unilever Research and Development Laboratory, Port Sunlight, Quarry Road East, Bebington, Wirral CH63 2JW, United Kingdom § KLK Oleomas SDN BHD, Level 8, Menara KLK, Jalan PJU7/6, Mutiara Damansara, 47810 Petaling Jaya, Selangor Malaysia ∥ ISIS Neutron Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot OX11 0QX, United Kingdom ⊥ School of Biological and Chemical Sciences, Queen Mary University of London, Joseph Priestley Building, Mile End Road, London E1 4NS, United Kingdom S Supporting Information *

ABSTRACT: The structure of the adsorbed protein layer at the oil/water interface is essential to the understanding of the role of proteins in emulsion stabilization, and it is important to glean the mechanistic events of protein adsorption at such buried interfaces. This article reports on a novel experimental methodology for probing protein adsorption at the buried oil/ water interface. Neutron reflectivity was used with a carefully selected set of isotopic contrasts to study the adsorption of bovine serum albumin (BSA) at the hexadecane/water interface, and the results were compared to those for the air/water interface. The adsorption isotherm was determined at the isoelectric point, and the results showed that a higher degree of adsorption could be achieved at the more hydrophobic interface. The adsorbed BSA molecules formed a monolayer on the aqueous side of the interface. The molecules in this layer were partially denatured by the presence of oil, and once released from the spatial constraint by the globular framework they were free to establish more favorable interactions with the hydrophobic medium. Thus, a loose layer extending toward the oil phase was clearly observed, resulting in an overall broader interface. By analogy to the air/water interface, as the concentration of BSA increased to 1.0 mg mL−1 a secondary layer extending toward the aqueous phase was observed, possibly resulting from the steric repulsion upon the saturation of the primary monolayer. Results clearly indicate a more compact arrangement of molecules at the oil/water interface: this must be caused by the loss of the globular structure as a consequence of the denaturing action of the hexadecane.

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interface.12 The oil layer was kept in contact with a hexane pool adjacent to the aqueous phase; the thickness of the oil layer was then maintained by balancing the condensation rate and the film drainage rate. However, maintaining a constant oil film thickness was a challenging task, and the methodology was applicable only to volatile oils. In recent years, an alternative methodology has been developed to control the oil via film coating,13 allowing more systematic studies of surfactant adsorption at the oil/water interface.14−16 To avoid drastic attenuation of the neutron beam upon traversing the oil phase, a thin oil (hexadecane) layer is coated and then sandwiched between a silicon substrate and the aqueous phase. However, reflectivity from the silicon/oil interface interferes with that from the oil/water interface and must thus be minimized by matching the scattering length density (SLD or Nb) of the oil

INTRODUCTION Many proteins readily adsorb to interfaces and are extensively used as emulsifiers.1−3 Proteins are often the first choice in personal care, pharmaceuticals, and food preparation because of their low cost, biocompatibility, and good nutritional value.4−9 Against many synthetic surfactants, polymers, and their mixtures, proteins can also offer low toxicity and reduced environmental impact in both personal care and pharmaceuticals.10 The study of protein adsorption at fluid/fluid interfaces is essential to the fuller understanding of the relationship between protein molecular structures and emulsifying properties. Despite the extensive literature dealing with protein adsorption at fluid/fluid interfaces, attention has only recently moved to studying the conformation of proteins upon adsorption.11 This is mainly because of the lack of suitable techniques and methodologies to probe such buried interfaces. In a pioneering neutron reflectivity experiment Dickinson et al. discussed the adsorption of β-casein at the hexane/water © 2015 American Chemical Society

Received: February 27, 2015 Revised: April 15, 2015 Published: April 15, 2015 5614

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particularly the random-coil structure (17 to 27%) upon adsorption at the water/hexadecane interface.23 Because of small changes in secondary structures taking place at the air/water interface and the large extent of structural rearrangements upon exposure to the oil phase,23 it has been suggested that the oil plays a key role in the ability of proteins to self-assemble at oil/water interfaces. However, the synchrotron-based study has examined only the adsorption of BSA at a relatively low surface adsorbed amount (∼1.9 mg m−2). It remains unclear how the extent of structural rearrangement could be affected by increasing protein surface coverage. The aim of this study was to investigate in detail BSA adsorption at the oil (hexadecane)/water interface as a function of BSA concentration and compare to the results from the air/ water interface. In particular, the neutron work will unravel how the adsorbed interfacial layer structure varies with the amount of BSA adsorbed.

to that of silicon. This process limits the sample choices to molecules whose Nb significantly deviates from that of silicon. The Nb of the average protein changes from ∼1.8 × 10−6 to ∼3.0 × 10−6 Å−2 depending on whether the solution is prepared in H2O or D2O or from any intermediate values for their mixtures due to labile H/D exchanges.17 This implies that by using silicon (Nb = 2.07 × 10−6 Å−2) only a small amount of sensitivity to the adsorbed layer can be achieved, making it impractical to determine the surface adsorbed amount. For this task, the Nb of the aqueous solution is typically contrast matched to that of the oil, and one must rely on the maximum difference in Nb between the surface-active species and the bulk phase.15,18 An elegant solution could be achieved by moving away from silicon as a solid substrate to provide the maximum ΔNb between the protein and the bulk phases. This could be achieved by using an optically flat sapphire block that presents a much higher Nb than silicon (Nbsapphire = 5.65 × 10−6 Å−2). Both the oil and the water are then contrast matched to sapphire (CMSa). Besides opening new possibilities for the analysis of novel samples, this methodology has a second major advantage: by using predominantly deuterated oil (∼85% dhexadecane) the attenuation of the neutron beam upon traversing the oil phase is substantially reduced.14 This allows either a significant reduction in the run time for measuring normal samples or better statistics in this case from the use of smaller cells for expensive samples. This study was thus designed to test these ideas by illustrating how protein adsorbs at the oil/water interface under different solution conditions. For these reasons, it is convenient to choose proteins whose behavior has been well described in the literature. Bovine serum albumin (BSA) is a 66.5 kDa protein derived from cow serum.19 It contains 35 cystein residues that form 17 disulfide bridges, leaving one free cystein.20 These disulfide bridges are formed between residues not far from each other in the protein backbone, allowing the identification of three main domains.21 These domains are held together by intramolecular forces that promote a globular structure.22 The secondary structure of BSA has been described, and it contains predominantly α-helix structures (73%) with only 2% β-sheets.23 The rest is composed of u turns and random-coil structures. The shape adopted by the protein in solution has been reported to be roughly cylindrical, with dimensions of 40 × 40 × 140 Å3, but the exact size and shape in aqueous solution may vary with pH. Because of its structural flexibility, BSA manifests good surface activity (surface adsorption and surface tension reduction), resulting in good foaming24 and emulsification.25 It is widely believed that the protein secondary structure is hardly affected upon adsorption at the air/water interface.26 In fact, the secondary structure is lost only when a relatively high concentration of denaturants is added to the solution.27 BSA molecules adsorb with the major axis parallel to the water surface, forming a relatively compact monolayer.28 Not even with increasing protein concentration do the molecules in the primary monolayer lose their structure, but a secondary, diffuse layer forms underneath it, extending toward the aqueous phase. When BSA is adsorbed at the oil (hexadecane)/water interface, on the other hand, the secondary structure changes significantly. A change (12%) in overall secondary structure was reported from FTIR studies.29 Recent Synchrotron radiation circular dichroism measurements have also indicated a reduction in the α-helix (73 to 52%), together with an increase in the β-sheet (2% to 9%), u turns (8 to 12%), and

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MATERIALS

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METHODS

Hydrogenous hexadecane was purchased from Sigma-Aldrich UK and purified by passing it through an alumina column before its use. Deuterated d34-hexadecane was obtained from Cambridge Isotopes Laboratories (>98 atom % D). Fatty acid-free bovine serum albumin (BSA) was purchased from Aldrich. The absence of fatty acids, although claimed by the supplier, was tested in the laboratories at the University of Manchester (Supporting Information). D2O was provided by Fluorochem, and ultrapure H2O was produced using a laboratory-based Elgastat water purification unit. All samples were used as received unless specified. All solutions and mixtures were prepared by mass. Measurements were carried out at the protein isoelectric point (pI = 4.8). The possible presence of ions in the starting material was buffered by the addition of 10 mM NaCl. The pH adjustment was performed by adding the minimum amount of 3% HCl in H2O and 3% DCl in D2O (mixtures with the appropriate Nb were prepared before the addition to match the Nb of the aqueous solutions). A single-crystal sapphire block (Al2O3) with dimensions of 70 × 50 × 20 mm3 was obtained from Dimitri Styrkas and used for the experiment. One of the large surfaces (C type) was polished by using an Engis “Hyprez” polishing apparatus in Dr. Bob Thomas’ laboratory at the University of Oxford. The polishing started with the lapping accompanied by the injection of a suspension of large alumina particles (1 μm) to gain surface flatness. After 5−10 min, the surface was rinsed with tap water, followed by a visual inspection of the lapping outcome. The process was repeated until the surface was free of unevenness or large defects. Following the thorough cleaning of the mat, injection system, and ring holder with detergent and distilled water, the sapphire surface was further polished with a fine diamond suspension (0.1 μm) for 10−15 min. Before a visual inspection of any sign of scratches and defects, the surface was rinsed with ethanol and plenty of tap water and a final round of distilled water. The fine polishing process was also repeated until there was no sign of scratches to the naked eye.

Neutron Reflectometry. Neutron reflectometry is an optical technique that enables the determination of the neutron refractive index profile normal to an interface, Nb(z), averaged over the whole sample region.30 The neutron refractive index, n, is often expressed as n(λ) ≈ 1 −

λ2 Nb 2π

(1)

N is the atomic number density, b is the coherent scattering length, and λ is the neutron wavelength. The multiplier Nb, commonly referred to as the scattering-length density, varies linearly with the volume fraction composition: 5615

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Figure 1. Schematic representation of the cell (a) and the experimental geometry (b) used for this study. Image b is not to scale because the oil layer is several orders of magnitude thicker than the adsorbed protein layer.

Nb ≈

reflectivity experiment was performed using the INTER reflectometer32 at the ISIS spallation source, Rutherford Appleton Laboratory, Didcot, U.K. The instrument uses a polychromatic neutron beam with a wavelength λ of between 1.5 and 20 Å. The sample was always underilluminated, maintaining a fixed resolution of δQ/Q ≈ 3%. Two measurements at θ = 0.7 and 2.3° were taken for the characterization of the air/sapphire interface. The two profiles were then customarily overlapped and combined to cover a broader Q range. Unless otherwise stated, for all of the oil/water measurements the neutron beam was deflected by means of a supermirror to give an incident angle θ = 1.4° with respect to the sample position. This permits us to maintain the sample horizontal geometry and hence the stability of the oil layer. Because of the attenuation coefficient being wavelength-dependent, all reflectivity profiles and data analysis were performed as a function of wavelength. Data are, however, presented conventionally as a function of Q, defined as

∑ Φj(Nb)j j

(2)

(Nb)j is the scattering-length density, and Φj is the volume fraction of component j, respectively. Whereas electrons and photon radiation interact with electrons, neutrons interact with nuclei. This is of upmost importance because the scattering length b not only varies significantly between different elements but also varies between different isotopes. The difference in b between hydrogen (bH = −3.74 fm) and deuterium (bD = 6.67 fm) is the basis of the so-called contrast variation. With this methodology, hydrogen and deuterium can be exchanged so that the Nb of molecules can be fine-tuned to match the Nb of other components of the system (or to maximize the difference between them). The method used for handling the oil film was based on the spin− freeze−thaw technique pioneered by Zarbakhsh et al.14 Instead of using a silicon block, a sapphire block was chosen to provide better contrast and to ensure maximum sensitivity to the buried adsorbed layer. The sapphire block was thoroughly cleaned with methanol; this limited the presence of free hydroxyl groups on the sapphire surface, hence boosting its hydrophobic nature. It has been reported that a thin oil film trapped between a sapphire block and a surfactant solution is only metastable:31 the surfactant solution would break through the oil and preferentially wet the sapphire surface within minutes. However, very low values of the interfacial tension, typical of those achieved by surfactants, are required to observe this phenomenon. The values for interfacial tension achieved by protein solutions are generally higher and the dynamics are much slower. Thus, such an effect was not recorded in this experiment. Figure 1SI (in Supporting Information) shows two droplets of hexadecane (left) and water (right) deposited onto the sapphire block surface. The spreading nature of the hexadecane droplet clearly demonstrated the higher affinity of the sapphire surface for the more hydrophobic medium. A thin layer of CMSa hexadecane (contrast matched to sapphire) was deposited by spin-coating onto the sapphire block. The film was then frozen in place, and the sample cell was assembled with the oil still frozen. The aqueous solution containing BSA was carefully injected into the sample cell, sandwiching the oil film between the solid substrate and the aqueous phase. Only then was the oil film allowed to melt. The temperature was maintained at about 5 °C by means of a circulating water bath during the assembly procedure. The temperature was then raised to 25 ± 1 °C for the neutron reflectivity measurement. A schematic representation of the cell used is shown in Figure 1 along with a highlighted sketch of the interfacial region. For a sufficiently thick oil film the reflectivity R is given by the thickfilm approximation12 (Supporting Information). The neutron

Q=

4π sin θ λ

(3)

A flat background was subtracted from the reflectivity profiles. The data analysis was performed using the model fitting procedure. The interface is divided into layers, each characterized by a certain thickness (d), scattering-length density (Nb), and roughness (σ). The reflectivity is then calculated using the optical matrix method.33 The use of the simplest model is generally preferable to maintain the number of fitting parameters to a minimum. The expression of deuterated proteins can be very expensive and time-consuming,34 hence only the hydrogenous BSA was used for this study. The Nb of the oil phase was also maintained constant (CMSa oil, obtained by mixing 14.8% h-hexadecane (Nb = −0.43 × 10−6 Å−2) with 85.2% d-hexadecane (Nb = 6.70 × 10−6 Å−2) by volume) for all measurements to minimize the reflectivity from the sapphire/oil interface. Therefore, the contrast variation was performed by changing the Nb of the aqueous subphase. Three different contrasts were used: (1) Contrast CMSa: water CM sapphire, prepared by mixing 10.1% H2O (Nb = −0.56 × 10−6 Å−2) with 89.9% D2O (Nb = 6.35 × 10−6 Å−2) by volume. This contrast enables the determination of the interfacial adsorbed amount. (2) Contrast CM2.2: water CM 2.2 × 10−6 Å−2 (60.0% H2O, 40.0% D2O). The aqueous phase is contrast matched to the protein, making the two species indistinguishable by neutrons. This contrast allows sensitivity solely to the layer immersed in the oil phase. (3) Contrast H2O: H2O. These runs present the greatest ΔNb between the two bulk phases and allow maximum sensitivity to the structure of the adsorbed layer. 5616

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Figure 2. Reflectivity profiles for the different contrasts are grouped in separate figures. Solid lines represent the fit to the data. (a) Reflectivity profile for the sapphire/air interface. (b) Reflectivity profiles for the CMSa water contrast, with fitting parameters given in Table 1. (c) Reflectivity profiles for the CM 2.2 contrast. The two profiles overlay each other, indicating that the layer immersed in the oil phase does not change as the [BSA] increases from 0.1 to 1.0 mg mL−1. The fitting parameters are shown in Table 2. (d) Reflectivity profiles for the H2O contrast, with parameters also given in Table 2. The bare CMSa oil/H2O interface is also shown as a reference (⧫).

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Results from contrast CMSa were used to determine the surface adsorbed amount, Γ, calculated using eq 4:35

Γ = dρ

RESULTS AND DISCUSSION The air/sapphire interface was initially measured to characterize the cleaned sapphire block. The reflectivity profile is shown in Figure 2a, and the solid line represents the best fit to the data. The optimum fit for the critical edge was obtained using a scattering-length density for sapphire of Nb = 5.65 × 10−6 Å−2. The profile was fitted only using 1 Å of roughness with no interfacial layer. This result indicated that the block surface was extremely flat and smooth. Contrast CMSa. Measurements in 30 min intervals and 1 h apart indicated that a 30 min equilibration time was sufficient. This observation agrees with the trend observed from the interfacial tension measurements (shown in Figure 3). At the air/water interface the reflectivity profiles in null reflecting water (NRW) for the lower BSA concentrations could be adequately represented using a single-layer model. Around or above 1 mg mL−1, a two-layer model was required for BSA concentrations to account for the inhomogeneous layer structures formed.28 However, at the oil/water interface the single-layer model could not produce an adequate fit to any of the measured profiles; therefore, a two-layer model was deployed. Figure 2SI (Supporting Information) shows the improvement in the fitting quality achieved by moving from a one-layer (dashed line) to a two-layer model (solid line) for the adsorbed layer from 0.3 mg L−1 at the isoelectric point of pH 4.7. The use of the two-layer model, with both layer thickness at d = 45 Å, significantly improved the quality of the fit, making an appropriate representation of the feature observed at Q > 0.08 Å−1, which was neglected when adopting the uniform layer fit.

Nb layer − Nb b NbBSA − Nb b

(4)

ρ is the protein density (a value of 1.40 g mL−1 was used in H2O),36 d and Nblayer are the experimentally determined layer thickness and scattering length density, Nbb is the scattering-length density of the two bulk phases, and NbBSA is the scattering-length density of BSA. Both ρ and NbBSA were adequately adjusted for H/D substitution.17 Values for NbBSA were 2.87 × 10−6, 2.20 × 10−6, and 1.80 × 10−6 Å−2 in CMSa, CM2.2, and H2O, respectively. All Nb values are grouped in Table 1SI. Drop Volume Measurements for the Determination of Interfacial Tension. Measurements were performed on a Krüss DVT50 drop volume tensiometer at Unilever R&D, Port Sunlight, Wirral, U.K. The instrument enables the measurement of interfacial tension between two immiscible liquids. The hydrophobic liquid flows under controlled flux through a capillary of known diameter d and forms a drop in contact with the aqueous phase to be tested. The buoyancy force pushes the drop upward, but the drop does not detach because the lifting force is compensated for by the interfacial tension on the wetted length of the capillary. The interfacial tension γ can be calculated at the moment when the drop detaches from the capillary

γ=

V Δρg πd

(5)

where V is the drop volume, Δρ is the density difference between the two bulk phases, and g the acceleration of gravity. If the flux through the capillary is carefully adjusted, then the instrument can yield information regarding adsorption dynamics on a time scale ranging from milliseconds to about 1 h.37 5617

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Figure 3. Changes in interfacial tension against time measured at the oil/water interface from the three different BSA solutions using the drop-volume technique.

Figure 4. Surface adsorbed amount for BSA solutions at the pI at the air/water (•, ref 28) and oil/water interfaces (□) as determined by neutron reflectivity. Values are listed in Table 3.

A similar two-layer fit, using no roughness and allowing only the layer Nb to vary, was used to represent all reflectivity profiles for contrast CMSa. This contrast allowed very limited sensitivity to the structure of the adsorbed layer and was solely used to determine the surface adsorbed amount. For the determination of the interfacial structures, it was necessary to involve other measurements under different contrasts, as discussed in the following section. Reflectivity profiles measured from different BSA solutions under the same pH and ionic strength are shown in Figure 2b, with the fitted parameters shown in Table 1. The intensity of the reflected signal increases with concentration, indicating the increase in the adsorbed mass. The calculated adsorbed amount Γ from data analysis is shown in Figure 4 together with the results obtained at the air/water interface. A significant increase in Γ was observed when the BSA concentration increased from 0.1 to 0.3 mg mL−1, followed by only minor changes as detected upon further increases to 1.0 mg mL−1. These changes broadly match the trend from interfacial tension measurements (Figure 3), where only small variations were observed upon increasing the concentration of BSA from 0.3 to 1.0 mg mL−1. Strictly speaking, however, Γ is associated with the change in interfacial tension arising from the activity increase of the protein which is inaccessible due to the unknown activity coefficient. The measured values for Γ are similar to those reported at the air/water interface. It should be stressed that for the experiments at the air/water interface the ionic strength was maintained at 0.02 M, whereas it was kept at 0.01 M for these measurements. This difference does not seem to jeopardize the comparability between the two measurements, both because of the relatively low salt concentration and the fact that salt has

major effects only in the presence of charged species (the proteins are at the pI, thus no net charges are present). The pH was found to play an important role in the adsorption pattern of BSA at the air/water interface,28 where a maximum in Γ was clearly identified at the pI, the effect being more pronounced at high protein concentration. This was observed because at the pI there is a minimum electrostatic repulsion between proteins, which do not possess a net charge. As the pH moves away from the pI, lateral repulsion prevents the accommodation of many molecules per area unit, hence the decrease in the adsorbed amount. In addition, as the protein charge increases with the solution pH being shifted away from the pI, the protein molecules become more hydrophilic with less tendency to adsorb. With the general pattern of BSA adsorption on the surface of water well characterized, this work was set to examine how its adsorption at the oil/water interface was affected by pH variation. The reflectivity profiles measured for BSA adsorption at 0.3 mg mL−1 at pH 4.7 and 6.0 are shown in Figure 3SI (in Supporting Information), with the fitted parameters listed in Table 2. Figure 4SI shows how Γ decreases with pH shifting away from the pI at the oil/water interface, in excellent agreement with the observation at the air/ water interface. Contrast CM2.2. In contrast CM2.2 the aqueous phase has Nb = 2.2 × 10−6 Å−2, which is the same as for the protein in solution. This enables us to determine the extent of protein penetration into the oil phase. Two reflectivity profiles measured for [BSA] = 0.1 and 1.0 mg mL−1 together with the best fit are shown in Figure 2c, with the fitted parameters given in Table 2. The two profiles overlay each other, indicating that the extent of BSA penetration into the oil phase does not

Table 1. Fitted Parameters for Contrast CMSa from the Reflectivity Profiles Shown in Figure 2b and Figure 3SI pH 4.7 layer

thickness (Å, ± 2)

0.1 mg mL−1

pH 6

0.3 mg mL−1

1 mg mL−1

0.3 mg mL−1

roughness (Å)

5.35 4.89

0 0 0

Nb (±0.04) × 10−6 Å−2 CMSa hexadecane layer 1 layer 2 CMSa water

5.65 45 45

5.49 4.90

5.35 4.66

5.32 4.62 5.65

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Langmuir Table 2. Fitting Parameters for Contrast CM2.2 (above) and Contrast H2O (below)a Contrast CM2.2 0.1 mg mL−1 and 1 mg mL−1 layer

thickness (Å , ±2)

Nb (±0.05) × 10−6 Å−2

roughness (Å)

hexadecane layer in oil H2O

75

5.65 5.41 2.20

0 8

Contrast H2O 1 mg mL−1 layer

thickness (Å , ±2)

hexadecane layer in oil layer in water diffuse layer H2O

75 37 30

a

0.1 mg mL−1

Nb (±0.05) × 10−6 Å−2 5.65 4.76 1.50 −0.26 −0.56

4.76 0.77

roughness (Å)

Figure 5. Scattering-length density (Nb) profiles for the contrast CM2.2 and contrast H2O reflectivity profiles; data are shown in Figure 2(c), (d) for consistency. Where no interlayer roughness was used, a 1 Å roughness was assigned solely to provide a better visualization of the profiles.

0 8 0 0

Reflectivity profiles and fits are shown in Figure 2c,d, respectively.

Structure of the Adsorbed Layer. We have previously shown that BSA remains almost fully immersed in water upon adsorption at the air/water interface,28 with their secondary structures remaining intact. At the oil/water interface, on the other hand, the loss or rearrangement of the secondary structures must be associated with the formation of a relatively thick layer (75 Å) in the oil phase. Thus, it is in this region that the oil (hexadecane) plays an important role in altering the protein secondary and tertiary structures. The more hydrophobic domains of BSA, now released from the spatial constraints given by the globular nature, are free to establish more favorable interactions with the hydrophobic medium. The nature of the hydrophobic medium must therefore play an important role in how the protein secondary and tertiary structures change. This supports the observation that the degree of structural reorganization scales with the hydrophobicity of the medium.23 The lack of changes in the reflectivity profiles as the concentration of BSA was increased from 0.1 to 1.0 mg mL−1 when water is contrast matched to the protein clearly shows that the layer in the oil phase reaches saturation at low BSA concentration ([BSA] ≤ 0.1 mg mL−1). Unless further experiments are performed, it is not prudent to discuss the BSA concentration at which saturation of the oil layer may be achieved. However, what can be deduced from combining the information obtained from contrast CM2.2 and contrast H2O is that at low BSA concentrations the protein forms a monolayer at the oil/water interface. Under the action of the hexadecane, the adsorbed molecules start denaturing and part of the protein is accommodated in the oil phase; here a mixed top region of the layer is formed that reaches saturation at [BSA] ≤ 0.1 mg mL−1. As the BSA concentration increases, no more protein molecules can penetrate the oil phase; hence the adsorbed molecules remain confined to the aqueous side of the interface where they form an increasingly compact layer with thickness d = 37 ± 2 Å, comparable to the values obtained at the air/water interface.28 These two layers together describe the conformation of BSA at the interface as a primary monolayer which is broadened by the action of hexadecane. At a concentration of between 0.1 and 1.0 mg mL−1 the primary monolayer reaches full coverage and a secondary layer starts to form on the

change in the concentration range studied. A 75 Å layer was required to model the reflectivity profiles, with 8 Å roughness between the oil layer and the aqueous phase. These values were used for the fitting procedure for contrast H2O, which possesses the largest ΔNb between the oil and the aqueous phases and is most sensitive to the interfacial structure. Contrast H2O. For contrast H2O all solutions were prepared in H2O. By analogy to contrast CM2.2, two reflectivity profiles were measured for [BSA] = 0.1 and 1.0 mg mL−1. The reflectivity profiles are shown in Figure 2d, and fitting parameters are listed in Table 2. The bare oil/water interface was also measured for this system as a reference, and the reflectivity profile is reported together with the fit in Figure 2d. (Note that because the latter profile was measured at θ = 2.3° instead of 1.4° there is a slight difference in the Q range.) No layer between the oil and the water was used to fit the reflectivity profile for the bare interface: a roughness of 5 ± 1 Å was solely used to model the data, in excellent agreement with both X-ray38 and previous neutron reflectivity39 results. Using the single-layer fit from contrast CM 2.2 as the starting point, the reflectivity profiles for contrast H2O were fitted by assuming no oil extended further than the first layer. At low BSA concentration a two-layer model adequately represented the reflectivity data. When the concentration of BSA reached 1 mg mL−1 the interfacial structure could not be described using a two-layer model: the dashed line in Figure 5SI shows the best two-layer fit obtained for 1 mg mL−1 BSA for the H2O contrast. Despite allowing all parameters for the second layer to vary independently (d, Nb, and roughness), the quality of the fit is very poor. In light of these results, the interface was modeled by adding a third layer with thickness 30 ± 2 Å extending toward the bulk aqueous phase. This change in the model is analogous to what was observed at the air/water interface, where the transition from monolayer to “monolayer + diffuse structure” took place at BSA concentrations of between 0.05 and 1 mg mL−1.28 To help the visual representation of the interfacial layers, the Nb profiles for contrast CM2.2 and contrast H2O are shown in Figure 5. 5619

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Langmuir aqueous side of the interface. This layer, with a low Nb, represents a diffuse structure with low BSA volume fraction. This transition from primary monolayer to “primary monolayer + secondary layer” with increasing BSA concentration is consistent with what was previously reported at the air/water interface at the isoelectric point.28 To verify the lack of secondary layer at lower protein concentration, it was decided to apply the three-layer model to the 0.1 mg mL−1 BSA. Results presented together with the fitting procedure in Figure 6SI (Supporting Information) show that the optimum fitting (minimum χ squared per point) was clearly obtained by adopting the two-layer fit. Although the presence of a very small amount of BSA in the layer (equivalent to a BSA volume fraction of ΦBSA ≤ 0.035) could still be considered within error, the quality of the fit progressively worsens as ΦBSA in the layer increases, indicating that if any protein is present in this interfacial region it must be in a very small amount and it is beyond the technical resolution. In the presence of a sufficient number of contrasts, the volume fraction of each component can be calculated for each interfacial layer using eq 6. This is particularly complicated at the oil/water interface where several components are present (in this case hexadecane, water, and BSA). Such an approach has already been undertaken to estimate the distribution of DSPC lipids at the hexadecane/water interface,35 where the interface was divided into discrete layers with no interfacial roughness. When the interlayer roughness is used, the diffuse nature of the interface may add complications, and the use of such an approach is discouraged.18 However, results obtained from contrast CM2.2 suggest that the oil, unlike water, does not extend throughout the adsorbed layer but remains confined to the hydrophobic side of the interface. Thus, it is safe to assume that at [BSA] = 1 mg mL−1 no oil penetrates further down to the secondary layer (layer 3). Only H2O and BSA are therefore present there, meaning that the volume fraction of BSA in the secondary layer can be readily estimated from the Nb of the layer using a derivation of eq 2 Nb layer = NbH 2OΦH 2O + NbBSA ΦBSA

Table 3. Total Adsorbed Amount (Γ) and Adsorbed Amount in the Primary Monolayer (Γ1) at the Air/Water28 and Oil/ Water Interfacesa

1 NA Γ1

Γtotal/mg m−2

Γ1/mg m−2

APM in primary monolayer/Å2

oil/water

0.1 1.0

2.16 ± 0.12 3.23 ± 0.10

2.16 ± 0.12 2.73 ± 0.10

4130 ± 240 3270 ± 120

air/ water28

0.05 1.0

2.10 ± 0.20 2.90 ± 0.20

2.10 ± 0.20 2.35 ± 0.20

5000 ± 350 3800 ± 300

a

The area per molecule in the primary monolayer is also shown. Note that at lower concentration both at the air/water and oil/water interfaces Γ1 and Γtotal are the same because no underlying structure was detected.

Figure 6. Area per molecule in the primary monolayer at the air/water (•, ref 28) and oil/water (□) interfaces. Values are reported in Table 3.

Given the roughly cylindrical shape with dimensions of 40 × 40 × 140 Å,40 the limiting APM for BSA for the sideway adsorption is 5600 Å2. At the air/water interface the limiting area per molecule upon adsorption was encountered at concentrations of around 0.05 mg mL−1.28 When the concentration increased to 1.0 mg mL−1, the APM in the primary monolayer decreased to 3800 Å2: this value is much smaller than the 5600 Å2 limit, indicating some degree of structural deformation. We assumed that for steric reasons no more material could henceforth be incorporated into the monolayer and BSA molecules started forming a secondary layer in contact with the primary monolayer. This transition happened at an APM of between 5000 and 3800 Å2. At the oil/water interface the APM is already significantly smaller than the limiting value at [BSA] = 0.1 mg mL−1 (4130 ± 240 Å2). As the concentration increases to 1 mg mL−1, the APM decreases to 3270 ± 120 Å2, which is significantly smaller than the value of 3800 ± 300 Å2 found at the air/water interface. It has been discussed how the presence of oil induces partial changes in the secondary structures of the protein, thus part of the denatured fraction is now accommodated in the oil phase. This is responsible for the broadening of the primary monolayer and reduces the steric repulsion between adsorbed BSA molecules, hence the reduction observed in APM. It is noteworthy that the transition between monolayer and primary monolayer + secondary layer, which takes place at 5000 < APM

(6)

where Φ is the volume fraction and Nblayer is the experimentally determined scattering-length density of layer 3. The equation can be solved by applying the constraint that the sum of all volume fractions is 1 (ΦH2O + ΦBSA = 1). The volume fraction of BSA in the diffuse region is 0.12, identical to what was reported for the air/water interface. Therefore, the amount of protein in the secondary layer can be subtracted from the total adsorbed amount Γ to give the amount of protein in the primary monolayer (Γ1). It must be stressed that the amount of BSA immersed in the oil layer (layer 1) cannot be separated from Γ1 and, for all intents and purposes, must be considered to be part of the primary monolayer itself. Namely, the primary monolayer refers to the amount of protein contained in both layers 1 and 2. The area per molecule in the primary monolayer (APM) is then calculated using eq 7 APM =

[BSA]/mg mL−1

system

(7)

where NA is Avogadro’s constant. The values for APM in the primary monolayer are given in Table 3, together with Γ1 and total Γ. Figure 6 shows the APM in the primary monolayer at the oil/water interface, enabling a comparison with the results obtained at the air/water interface.28 5620

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Langmuir < 3800 Å2 at the air/water interface, is now shifted to 4130 < APM < 3270 Å2. It is worth briefly discussing the BSA conformation upon adsorption in the secondary layer. The neutron reflectivity technique does not allow sensitivity to changes in protein structure but only to changes in the neutron refractive index. This means that it is not possible to distinguish whether the secondary layer contains elongated protein molecules from the primary monolayer or if there is a distinct second protein layer extending toward the aqueous phase. However, the latter seems to be most likely for at least two reasons. First, it has been discussed how the protein denaturation is triggered by the presence of the hexadecane molecules upon adsorption at the interface. Because no oil was found to extend as far as the secondary layer, it would be hard to justify the presence of denatured protein in the absence of hexadecane molecules on the aqueous side. Second, if the presence of the secondary layer were to be attributed to the reorientation of elongated protein molecules extending toward the aqueous phase, this would inevitably go along with a further disruption of the protein structure. However, this would be in disagreement with the observation of only partial loss of secondary structure upon adsorption by Day et al.23 Therefore, the secondary layer is expected to be composed of interfacially adsorbed BSA molecules in their native structure. The results obtained are schematically represented in Figure 7, where the proposed structure of BSA at the oil/water

explain why at higher BSA concentration (Figure 7d) a slight increase in the surface adsorbed amount was observed rather than at the air/water interface. The increased adsorbed amount is in fact not accommodated in the secondary layer, which contains the same protein volume fraction as at the air/water interface and must therefore reside in the primary monolayer.

■

CONCLUSIONS This work reports a novel methodology to perform systematic studies of protein adsorption at the oil/water interface using neutron reflectivity. BSA was chosen as a model protein for its extensive characterization. Results show that the protein forms a monolayer at the interface over a low protein concentration range and a secondary layer then forms underneath the primary layer when the concentration is above 0.1 mg mL−1. This feature is rather similar to what was observed at the air/water interface. However, the oil causes denaturation, resulting in a broader primary layer than that formed at the air/water interface. The major structural changes upon adsorption facilitate the interfacial packing, allowing a higher adsorbed amount as the concentration of the protein increases. These results agree with those of Day et al.,23 who recently described the changes in secondary structures undergone by BSA upon adsorption at the oil/water interface. This study opens a route to the detailed exploration of protein adsorption at the liquid/liquid interface. The ability to link protein molecular structures to adsorption patterns at interfaces will in fact enable a fuller understanding and an optimization of the emulsification processes, with important implications in all areas where protein-stabilized emulsions are required.

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ASSOCIATED CONTENT

S Supporting Information *

Thick film approximation and the test for the absence of fatty acid in BSA. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.langmuir.5b00646.

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Figure 7. Schematic representation of the interfacial area at both the air/water (a, b) and oil/water (c, d) interfaces. The figure is a simple sketch and does not aim to represent the true spatial arrangement of the protein molecules upon adsorption.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

interface (right) is compared to that observed at the air/water system. At low concentration, BSA molecules adsorb at the air/ water interface (Figure 7a) with their major axis parallel to the surface. No denaturation occurs, and the molecules retain their globular conformation. As the concentration of BSA increases, the primary monolayer achieves full coverage and a secondary monolayer appears, extending toward the aqueous phase (Figure 7b). At the oil/water interface the presence of hexadecane promotes changes in BSA’s secondary structure. The denatured fraction forms a layer extending toward the oil phase that reaches saturation already at low protein concentration (Figure 7c). The molecules in the primary monolayer have lost their tertiary structure, thus enabling better interfacial packing. Therefore, more protein molecules can be accommodated in the primary monolayer when full coverage is achieved than at the air/water interface. This observation is in fact in agreement with the conclusions drawn by Day et al.,23 who reported a major structural rearrangement of BSA upon adsorption at the hexadecane/water interface. These results also

The authors declare no competing financial interest.

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REFERENCES

(1) McClements, D. J. Protein-stabilized emulsions. Curr. Opin. Colloid Interface 2004, 9, 305−313. (2) McClements, D. J. Food Emulsions, 2nd ed.; CRC Press: Boca Raton, FL, 2005. (3) Damodaran, S. Protein Stabilization of Emulsions and Foams. J. Food Sci. 2005, 70, R54−R66. (4) Dickinson, E. Food Emulsions and Foams; Woodhead Publishing Ltd: Abingdon, U.K., 1987. (5) Teglia, A.; Secchi, G. Principles of Polymer Science and Technology in Cosmetics and Personal Care; Marcel Dekker: New York, 1999. (6) Nik, A. M.; Wright, A. J.; Corredig, M. Interfacial design of protein-stabilized emulsions for optimal delivery of nutrients. Food Funct. 2010, 1, 141−148. (7) Dickinson, E. Proteins at interfaces and in emulsions stability, rheology and interactions. J. Chem. Soc., Faraday Trans. 1998, 94, 1657−1669. 5621

DOI: 10.1021/acs.langmuir.5b00646 Langmuir 2015, 31, 5614−5622

Article

Langmuir (8) Wilde, P.; Mackie, A.; Husband, F.; Gunning, P.; Morris, V. Proteins and emulsifiers at liquid interfaces. Adv. Colloid Interface Sci. 2004, 108−109, 63−71. (9) Dickinson, E. Interfacial structure and stability of food emulsions as affected by protein−polysaccharide interactions. Soft Matter 2008, 4, 932−942. (10) Nnanna, I. A.; Xia, J. Protein-Based Surfactants: Synthesis: Physicochemical Properties, and Applications; Marcel Dekker: New York, 2001; Chapter 1. (11) Zhai, J. L.; Day, L.; Aguilar, M. I.; Wooster, T. J. Protein folding at emulsion oil/water interfaces. Curr. Opin. Colloid Interface 2013, 18, 257−271. (12) Dickinson, E.; Horne, D. S.; Phipps, J. S.; Richardson, R. M. A neutron reflectivity study of the adsorption of β-casein at fluid interfaces. Langmuir 1993, 9, 242−248. (13) Zarbakhsh, A.; Bowers, J.; Webster, J. R. P. A new approach for measuring neutron reflection from a liquid/liquid interface. Meas. Sci. Technol. 1999, 10, 738−743. (14) Zarbakhsh, A.; Querol, A.; Bowers, J.; Webster, J. R. P. Structural studies of amphiphiles adsorbed at liquid−liquid interfaces using neutron reflectometry. Faraday Discuss. 2005, 129, 155−167. (15) Zarbakhsh, A.; Querol, A.; Bowers, J.; Yaseen, M.; Lu, J. R.; Webster, J. R. P. Neutron Reflection from the Liquid−Liquid Interface: Adsorption of Hexadecylphosphorylcholine to the Hexadecane−Aqueous Solution Interface. Langmuir 2005, 21, 11704− 11709. (16) Campana, M.; Webster, J. R. P.; Zarbakhsh, A. Structural Studies of Nonionic Dodecanol Ethoxylates at the Oil−Water Interface: Effect of Increasing Head Group Size. Langmuir 2014, 30, 10241−10247. (17) Fitter, J.; Gutberlet, T.; Katsaras, J.; Timmins, P. Neutron Scattering in Biology: Techniques and Applications Springer: Berlin, 2006; Chapter 5. (18) Campana, M.; Webster, J. R. P.; Gutberlet, T.; Wojciechowski, K.; Zarbakhsh, A. Surfactant mixtures at the oil−water interface. J. Colloid Interface Sci. 2013, 398, 126−133. (19) Farrell, H. M., Jr; Jimenez-Flores, R.; Bleck, G. T.; Brown, E. M.; Butler, J. E.; Creamer, L. K.; Hicks, C. L.; Hollar, C. M.; Ng-KwaiHang, K. F.; Swaisgood, H. E. Nomenclature of the Proteins of Cows’ MilkSixth Revision. J. Dairy Sci. 2004, 87, 1641−1674. (20) Carter, D. C.; Ho, J. X. Structure of Serum Albumin. Adv. Protein Chem. 1994, 45, 153−203. (21) Peters, T. J. Serum albumin: recent progress in the understanding of its structure and biosynthesis. Clin. Chem. 1977, 23, 5−12. (22) Damodaran, S. Food Proteins and Their Applications; Marcel Dekker: New York, 1997. (23) Day, L.; Zhai, J.; Xu, M.; Jones, N. C.; Hoffmann, S. V.; Wooster, T. J. Conformational changes of globular proteins adsorbed at oil-in-water emulsion interfaces examined by synchrotron radiation circular dichroism. Food Hydrocolloids 2014, 34, 78−87. (24) Wei, X.; Liu, H. Relationship between foaming properties and solution properties of protein/nonionic surfactant mixtures. J. Surfact. Deterg. 2000, 3, 491−495. (25) Haque, Z.; Kinsella, J. E. Relative Emulsifying Activity of Bovine Serum Albumin and Casein as Assessed by Three Different Methods. J. Food Sci. 1989, 54, 1341−1344. (26) Lad, M. D.; Birembaut, F.; Mathew, J. M.; Frazier, R. A.; Green, R. J. The adsorbed conformation of globular proteins at the air/water interface. Phys. Chem. Chem. Phys. 2006, 8, 2179−2186. (27) Noskov, B. A.; Mikhailovskaya, A. A.; Lin, S. Y.; Loglio, G.; Miller, R. Bovine Serum Albumin Unfolding at the Air/Water Interface as Studied by Dilational Surface Rheology. Langmuir 2010, 26, 17225−17231. (28) 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. 1999, 213, 426−437. (29) Jorgensen, L.; Van De Weert, M.; Vermehren, C.; Bjerregaard, S.; Frokjaer, S. Probing structural changes of proteins incorporated into water-in-oil emulsions. J. Pharm. Sci. 2004, 93, 1847−1859.

(30) Lu, J. R.; Li, Z. X.; Thomas, R. K.; Penfold, J. Structure of hydrocarbon chains in surfactant monolayers at the air/water interface: neutron reflection from dodecyl trimethylammonium bromide. J. Chem. Soc., Faraday Trans. 1996, 92, 403−408. (31) Knock, M. M.Monolayers of Cationic Surfactants at the Air-Water and Oil-Water Interfaces. D.Phil. Thesis, University of Oxford, 2002. (32) Webster, J. R. P.; Langridge, S.; Dalgliesh, R. M.; Charlton, T. R. Reflectometry techniques on the Second Target Station at ISIS: Methods and science. Eur. Phys. J. Plus 2011, 126, 112. (33) Heavens, O. S. Optical Properties of Thin Solid Films; Butterworths Scientific Publications: London, 1955. (34) Meilleur, F.; Weiss, K. L.; Myles, D. A. A. Micro and Nano Technologies in Bioanalysis: Methods and Protocols; Humana Press: New York, 2009. (35) Campana, M.; Webster, J. R. P.; Lawrence, M. J.; Zarbakhsh, A. Structural conformation of lipids at the oil−water interface. Soft Matter 2012, 8, 8904−8910. (36) Fischer, H.; Polikarpov, I.; Craievich, A. F. Average protein density is a molecular-weight-dependent function. Protein Sci. 2004, 13, 2825−2828. (37) Nagarajan, R.; Wasan, D. T. Measurement of Dynamic Interfacial Tension by an Expanding Drop Tensiometer. J. Colloid Interface Sci. 1993, 159, 164−173. (38) Mitrinovic, D. M.; Tikhonov, A. M.; Li, M.; Huang, Z.; Schlossman, M. L. Noncapillary-Wave Structure at the Water-Alkane Interface. Phys. Rev. Lett. 2000, 85, 582−585. (39) Zarbakhsh, A.; Bowers, J.; Webster, J. R. P. Width of the Hexadecane−Water Interface: A Discrepancy Resolved. Langmuir 2005, 21, 11596−11598. (40) Peters, T., Jr. Serum Albumin. Adv. Protein Chem. 1985, 37, 161−245.

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