Protein Adsorption at the Electrified Air–Water Interface: Implications

Apr 24, 2012 - BSA covered air–water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins...
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Protein Adsorption at the Electrified Air−Water Interface: Implications on Foam Stability Kathrin Engelhardt,† Armin Rumpel,†,‡ Johannes Walter,† Jannika Dombrowski,§ Ulrich Kulozik,§ Björn Braunschweig,† and Wolfgang Peukert*,†,‡ †

Institute of Particle Technology (LFG), University of Erlangen-Nuremberg, Cauerstrasse 4, 91058 Erlangen, Germany Erlangen Graduate School in Advanced Optical Technologies (SAOT), University of Erlangen-Nuremberg, Paul-Gordan-Strasse 6, 91052 Erlangen, Germany § Chair for Food Process Engineering and Dairy Technology, Research Center for Nutrition and Food Sciences (ZIEL) − Department Technology, Technische Universität München, Freising-Weihenstephan, Germany ‡

ABSTRACT: The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air−water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H2O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H2O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte−air interface for the first time. BSA covered air−water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.

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INTRODUCTION Foams are materials of particular importance since they are applied in a broad range of applications such as metal foams for lightweight structures,1 polymer foams for thermal insulation or foams in food products, just to mention a few. Although the chemical composition of these foams is largely different, they share common similarities for foam formation and stabilization. The latter is controlled by adsorption processes at the interface between the gaseous and the surrounding solid or liquid phase. Therefore, it is of great importance to understand foam stabilization processes at interfaces in order to design advanced materials with tunable properties.2 Stabilization of protein foams is dominated by a molecular layer at the interface which can be controlled experimentally.3−6 For that reason, protein foams represent a model system for mechanistic studies of foams and investigations on the origin that causes a liquid to foam. Hence, hierarchical studies of the relationship between molecular structure and interactions, interface design and macroscopic properties, has become an important part of current research in this field.7−9 A detailed molecular level understanding of the surface chemistry of proteins at liquid interfaces has, however, not been established. © 2012 American Chemical Society

In order to reveal the interactions of proteins at interfaces, information on the interfacial composition, for example, the arrangement of ions and water molecules in the adjacent electrolyte subphase, possible protein unfolding processes, and the formation of single or multilayers, is imperative.10−13 The physical and chemical stability of proteins is influenced by different factors such as temperature, chemical composition of the electrolyte, and the pH of the bulk electrolyte.14 While it seems to be accepted that structural rearrangement of proteins due to the adsorption to the interface can occur,15−18 the extent of unfolding or surface aggregation and a possible reversibility of this process are still a matter of considerable debate.9,19,20 The lack of molecular level information is mainly due to a lack of suitable experimental techniques that can actually reveal both composition as well as conformation of protein adlayers and other interfacial molecules such as H2O. In previous studies, it was already shown that sum-frequency generation (SFG) is a powerful optical probe for the investigation of Received: April 3, 2012 Revised: April 23, 2012 Published: April 24, 2012 7780

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protein interfaces.21−26 Wang et al.27 reported the pH dependent charging of bovine serum albumin (BSA) at the air−liquid interface where the influence of pH on the SFG spectra in the region of methyl and OH stretching vibrations was attributed to a charge reversal of interfacial BSA. As the surface charge of the proteins is affected by the electrolyte pH, it leads to a strong electric field and, consequently, to the formation of an electric double layer at the protein surface. The local electric field of proteins and its effects on the surrounding electrolyte layer is minimized at a pH where the net charge of the protein with bound ions is zero.28 Obviously, this point is of great scientific interest as it is protein specific and is a function of the amino acid sequence at the protein surface. Although the isoelectric point of proteins in the bulk electrolyte can be determined by zeta potential measurements, the conditions at electrolyte interfaces can be dramatically different from those in the bulk. The concentrations of proteins, ions, and water molecules as well as their lateral interactions may be modified significantly at an interface. Consequently, it is a priori not clear if the isoelectric points of proteins at interfaces and in bulk electrolytes are identical. In order to reveal the intriguing relationship between pH dependent charging of a protein, protein adsorption, interfacial molecular structure, and macroscopic properties such as foam stability, we have studied the model protein BSA at the electrified air−water interface with broadband SFG, ellipsometry, and a macroscopic analysis of the foam stability. This hierarchical approach has enabled us to determine the interfacial isoelectric point as well as the structure of BSA layers adsorbed to the electrolyte−air interface. New information on the link between microscopic properties of BSA at interfaces and macroscopic properties such as the stability of foams from BSA solutions is provided.

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rapid acquisitions of experimental data compared to the conventional nulling technique.29 For each experiment, 15 μM BSA sample solution was poured into a Petri dish with a diameter of 10 cm and was allowed to equilibrate for about 30 min. Angle scans between 51° and 55° versus the surface normal were performed with a step width of 0.5°. In order to ensure reproducibility, at least six measurements were recorded and averaged for every pH value. Angle-resolved data from ellipsometry were fitted under the assumption of a three layer model with refractive indices of 1.33, 1.40,12 and 1.00 for the electrolyte subphase, the protein layer, and air, respectively. In general two parameters are unknown in this three layer system: the thickness of the adsorbed protein layer and the corresponding refractive index. Due to the fact that these parameters cannot be determined independently, one of them, for example, the refractive index, has to be chosen as a fixed input parameter for all model calculations. Since the refractive index of BSA at an interface is unknown, the latter assumption causes a systematic error of the layer thickness that depends on the deviation of the assumed refractive index from its actual value. However, since we only compare relative changes in the layer thickness as a function of the electrolyte pH, interpretations in this respect are not impaired. Vibrational Sum-Frequency Generation (SFG). SFG is a second-order nonlinear optical technique30 where two laser beams are overlapped temporally and spatially at the interface of interest and generate photons with the sum frequency of the two impinging laser beams. One laser has a frequency ωVIS in the visible region (800 nm), and the other laser is tunable in the infrared region with frequencies ωIR. The intensity of sum-frequency output I(ω) depends on the intensities of the impinging laser beams IVIS and IIR as well as on the (2) nonresonant χ(2) NR and resonant χk parts of the second-order nonlinear susceptibility χ(2) as follows: 2 (2) I(ω) ∝ χNR +

∑ χk(2) k

IVISIIR

with

χk(2) =

Ak exp(iφk ) ω k − ω + i Γk (1)

depends on the amplitude Ak = The resonant contribution χ(2) k N⟨αkμk⟩, the relative phase φk, the resonance frequency ωk, and the bandwidth Γk of the vibrational mode k. The amplitude Ak is a function of the molecular number density N at the interface and an orientational average of the Raman polarizability αk and the dynamic dipole moment μk. The latter dependence of Ak on the orientation of molecules at interfaces has far reaching consequences: SFG is not allowed in materials with centrosymmetry or isotropic materials without long-range order, that is, liquids and gases when the positions of molecules are averaged over time. At interfaces, the bulk symmetry is necessarily broken and nonzero components of χ(2) solely from the interface exist and give rise to surface sensitive SFG. A perfectly polarordered adlayer results in the highest amplitude and SFG intensity, while a layer with identical coverage, but randomly oriented interfacial molecules, has negligible SFG intensity. Hence, SFG combines the advantages of optical techniques with intrinsic surface/symmetry sensitivity and is a very powerful and highly versatile spectroscopic tool for studies of surfaces and interfaces. However, most studies of proteins at the air−water interface were limited to the methyl and water stretching region27,31−34 which yields information about the interfacial water structure but not about the protein amide I band around 1650 cm−1. For our SFG measurements a home built broadband SFG setup was applied, as described elsewhere.35 The setup enables us to tune the IR frequency and record the SFG intensity for IR frequencies which are within the bandwidth of a broadband IR pulse (200 cm−1). All spectra were recorded with s-polarized sumfrequency, s-polarized visible and p-polarized IR beams (ssp). The presented spectra were normalized to a reference spectrum of a polycrystalline Au sample that was previously subjected to oxygen plasma. The 15 μM BSA samples were poured in a Petri dish, and SFG spectra were collected. Each spectrum was measured by scanning the broadband IR beam with a step width of 130 cm−1 and total acquisition times of 7 and 8 min for 2800−3800 and 1300−1800 cm−1 spectral regions, respectively.

EXPERIMENTAL SECTION

Sample Preparation. BSA (essentially acid free) was purchased from Sigma Aldrich (A7030) and was used as-received. BSA solutions (15 μM for spectroscopic measurements, 150 μM for zeta potential measurements) were prepared by dissolving the dry protein in ultrapure water (18.2 MΩ cm−1; total oxidizable carbon < 10 ppb). The pH was adjusted by adding either HCl (Merck; Suprapur grade) or NaOH (99.99%; Alfa Aesar) and measured with an InLab Micro Pro pH electrode (Mettler Toledo). In order to remove possible contaminations, the necessary glassware was soaked in a mixture of concentrated sulfuric acid (98%; analytical grade) and NOCHROMIX for at least 24 h, thoroughly rinsed with ultrapure water, and subsequently cleaned in boiling ultrapure water. All measurements were performed at a temperature of 24 °C. Zeta Potential Measurements. Zeta potentials were measured with a commercial Zetasizer Nano ZS instrument (Malvern Instruments). The pH of 150 μM BSA aqueous solutions was adjusted by adding either acid or base. The samples were filtered using 0.2 μm cellulose acetate filters (VWR 514-0060) and thoroughly cleaned glass syringes before transferring them into the cuvette. For every data point, at least four measurements with four different cuvettes were performed. For good and reproducible zeta potential measurements, a minimum BSA concentration of approximately 150 μM is needed. For that reason, 10-fold higher BSA concentrations have been chosen for measurements of the bulk zeta potential compared to ellipsometry and SFG measurements at air−water interfaces. Ellipsometry. The thickness of adsorbed protein layers was determined with a phase modulated ellipsometer (Picometer Ellipsometer; Beaglehole Instruments) that was operated with a wavelength of 632.8 nm. Phase modulated ellipsometry offers the possibility to record data near or at the Brewster angle (∼55°) of the studied system which increases the sensitivity considerably and enables 7781

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Foam Preparation and Characterization. Foaming experiments were carried out with a commercial dynamic foam analyzer DFA 100 (Krüss GmbH, Germany). Foams were produced in a glass column of 0.25 m length and 0.04 m thickness by a stream of air that was introduced into the protein dilutions through a porous glass filter (pore size: 9−16 μm) with a constant flow rate of 5 mL/s. Experiments were performed in triplicates. According to Glaser et al.,36 stability and capacity are key parameters which can be used to characterize macroscopic foam properties. The foam capacity can be determined by the volume increase due to the foaming of the initial BSA dilution with a volume Vi of 40 mL. In our experiments the foam capacity is given by the maximum (foam) volume (Vf) that is reached after 10 s of gas flow through the BSA dilution. Foam capacity [%] = (Vf − Vi)/Vi × 100. Subsequent to the formation, foam degradation given by the Foam stability [%] = (Vt=300s/Vt=0) × 100 was monitored for an additional 300 s.

behavior of surface adsorbed BSA layers in the following sections. Ellipsometry. The use of ellipsometry enables us to investigate the pH effects on the thickness of adsorbed BSA layers and to estimate the interfacial number density N of BSA. In Figure 2, the thickness of BSA layers adsorbed to the air−

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RESULTS Determination of the Bulk Zeta Potential. The zeta potential is often used as stability parameter in colloidal chemistry whereby a potential larger than |30 mV| leads to stable suspensions.37 In contrast to this, the zeta potential of proteins is determined to be mostly lower than |40 mV|28,38 and is often used to determine the isoelectric point.39 The isoelectric point is defined as the point where the zeta potential is equal to zero. At the isoelectric point, the protein carries no net charge, while there is an excess of positive or negative charge for pH values higher and lower than the pH of the isoelectric point, respectively. Previous studies of the BSA’s isoelectric point have shown that its exact determination is impaired by the applied experimental method and the background electrolyte. For that reason it is not surprising that isoelectric points between pH 4.7 and 5.6 have been reported.28 Figure 1 shows the zeta potential of BSA in a bulk solution as a function of the electrolyte pH. From a close inspection of

Figure 2. Thickness of adsorbed BSA layers at the air−water interface as a function of the electrolyte pH. As explained in the text, the thickness was determined by ellipsometry under the assumption of a simple water−BSA−air layer model. The dashed line is a guide to the eye.

water interface is presented as a function of electrolyte pH. For acidic conditions, the thickness first increases with increasing pH, reaches a pronounced maximum at pH ∼5.5, and decreases subsequently for higher pH values. Obviously, pH values near the bulk isoelectric point (see previous section) lead to much thicker BSA layers as compared to more acidic or alkaline conditions. Assignment of Vibrational Bands in SFG Spectra. To gain further insight in the molecular structure of the interface, vibrational SFG spectra were recorded in the entire spectral region of 1000−3700 cm−1. To the best of our knowledge these are the first vibrational SFG spectra of a protein that were measured in such a broad spectral region. This approach allows us to identify vibrational fingerprints of BSA and interfacial water molecules and to select specific regions of interest where changes with the electrolyte pH are most pronounced. Figure 3 shows vibrational SFG spectra of BSA adsorbed to the air−water interface for pH values of 8.2 and 4.3. A close comparison of the spectra reveals that they are dominated by

Figure 1. Zeta potential of BSA as a function of the electrolyte pH. Lines are a guide to the eye.

Figure 1, the pH of the isoelectric point of BSA can be determined to 5.2 ± 0.1. Although the isoelectric point of BSA can be established in the bulk electrolyte, it is not a priori known whether the isoelectric points of bulk and surface regions are identical. In fact, the small electric charge of BSA near or at the isoelectric points leads to very small repulsion between individual proteins and therefore to the tendency of agglomeration and possibly to precipitation of BSA. Consequently, it is not clear if and how stable BSA layers can actually form at the air−water interface under these conditions. To study this effect further, we will show the pH dependent

Figure 3. Vibrational SFG spectra of BSA at air−water interfaces for pH 8.2 and 4.3. Solid lines are fits to the experimental data as explained in the text. Details to the spectral regions I−III can be found in the text. 7782

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CH aromatic bending vibrations.58 Since substantial changes as a function of the bulk pH are observed for CH, OH, amide I, and carboxylate vibrations (Figure 3), we have focused further pH dependent studies to the latter vibrations. An overview of the SFG active BSA resonances relevant to the further discussion and their attribution to specific molecular vibrations can be found in Table 1.

vibrational bands in unique spectral regions where substantial changes as a function of the electrolyte pH can be observed: (I) Functional groups of adsorbed BSA give rise to strong vibrational bands at ∼2875 and ∼2936 cm−1 that are attributable to CH3 symmetric stretching vibrations and to the CH3 Fermi resonance, respectively. Furthermore, CH3 asymmetric, symmetric CH2, and aromatic CH stretching vibrations give rise to bands at 2964, 2850, and 3060 cm−1, respectively.27,31−33 Strong bands between 3100 and 3800 cm−1 originate from OH stretching vibrations of interfacial water molecules.34,40,41 (II) In a second spectral region, additional vibrational bands of interfacial BSA at ∼1654 and 1410 cm−1 are observed and can be attributed to amide I and to carboxylate (R−COO−) symmetric stretching vibrations, respectively.42,43 The amide I band originates from R−CO carbonyl vibrations of molecular groups in the protein backbone44−48 where they can form hydrogen bonds to neighboring amide groups49,50 (Figure 4).

Table 1. Assignment of Vibrational Bands in SFG Spectra of BSA Adsorbed to the Air−Water Interfacea band

[cm−1]

ref

amide III COO− (ss) amide II amide I CH2 (ss) CH3 (ss) CH3 (F) arom. CH OH (ss) OH (ss)

1250 1410 1550 1650 2850 2875 2936 3060 3200 3450

57 43 50 48, 53 31−33 31−33 31−33 31−33 40, 41 40, 41

a

(F), (ss), and (as) stand for Fermi resonance, symmetric, and antisymmetric stretching vibrations, respectively.

pH Dependence of the Interfacial Molecular Structure. In order to reveal changes in the interfacial molecular structure, SFG spectra of BSA adsorbed to the air−water interface were recorded and analyzed for pH values of 3−10. Figure 5 shows representative SFG spectra in this pH range. OH, CH, and amide I bands have strong pH dependencies. In particular, for the OH stretching bands of interfacial water molecules, we observe dramatic changes in the SFG intensity (Figure 5b). At a pH of 5.5, the SFG intensity of the H2O bands is negligible, but increases substantially for lower and higher pH values than 5.5. Similar but less pronounced behavior is observed for the SFG intensity in the CH stretching region. Compared to the latter bands, BSA amide I and carboxylate modes show much weaker changes in SFG intensity with the pH (Figure 5a). In order to analyze changes of SFG bands as a function of pH, we have fitted our spectra with model functions according to eq 1 and determined the amplitude of the vibrational bands in our SFG spectra. We have used the amplitude Ak, the resonance frequencies ωk, and the nonresonant contribution χNR(2) as adjustable parameters in our fitting procedures. Here, the overview spectra in Figure 3 are extremely helpful, since the nonresonant contribution and its influence on the dispersion of the vibrational resonances can be easily estimated. In Figure 6a, b, and c, the results of our fitting procedures are presented for the amplitudes of amide I, carboxylate (R− COO−), and OH vibrations, respectively. The pH dependence of the three amplitudes shows a local minimum around pH 5. While the amplitudes of amide I and carboxylate bands have a narrow minimum at pH 5 and are only slightly smaller for more alkaline than for acidic pH values, the amplitude of OH vibrations is also minimal at pH 5, but varies in a much broader pH range. Nearly all carboxyl groups with a pK of 4.3 from the amino acid side chains of BSA59 are deprotonated in the studied pH range and exist as carboxylates. This causes the amplitudes of the R−COO− vibration to stay nearly constant. However, since the density of these groups at the interface

Figure 4. Schematic representation of amide groups and their local orientation in a protein with an α-helix secondary structure.

The strength of the hydrogen bonds influences the frequency of the amide I band greatly and is, therefore, strongly dependent on the secondary structure of the protein. In previous IR studies, changes in the position of this band were often referred to denaturation, unfolding, or aggregation processes.51,52 Since BSA consists mainly of an α-helical structure, unfolding would lead to a blue shift in the amide I frequency. As was previously shown, the spectral frequencies of amide I vibrations can be attributed to different secondary structures42,53 and the kinetics of conformational changes was also resolved with SFG.54 At this point, it should be noted that the so-called amide II band at ∼1550 cm−1 that can be observed with linear IR spectroscopy50,55 contributes only weakly to the SFG intensity. Prerequisite for an SFG active mode is both Raman as well as IR activity. Since the amide II mode does lead to weak Raman resonances only,56 weak resonant SFG contributions from this band are also likely. Furthermore, the amide II band is a combination of C−N stretch and N−H bending vibrations and has a dynamic dipole moment that is perpendicular to the dipole moment of the amide I band (Figure 4). As a result, the intensity of this band is additionally weakened due to weaker excitations with p polarized IR light. (III) In the spectral region below 1400 cm−1, a band centered at ∼1250 cm−1 is observed and has been attributed to amide III vibrations.56,57 Three weak bands at ∼1140, ∼1070, and ∼1020 cm−1 are also observed in our spectra, which we relate to −C−O− stretching modes, N−H deformation, and 7783

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Figure 5. Vibrational SFG spectra of BSA at air−water interfaces as a function of the electrolyte pH: (a) Symmetric carboxylate (R-COO−) and amide I bands and (b) CH and OH stretching bands as explained in the text (Table 1). The pH was as indicated in the figure. Solid lines are fits to the experimental data according to eq 1. The color scale represents schematically the interfacial charge density as a function of the pH.

Figure 6. pH dependence of the amide I (a) and carboxylate R− COO− (b) vibrational amplitude in arbitrary units. (c) Amplitude of OH stretching vibrations at 3200 cm−1 (black solid square) and 3400 cm−1 (red solid triangle). Dotted Lines are guide to the eye.

increases (Figure 2), the observed change in SFG amplitude is a signature of a decrease in interfacial polar order that will be discussed in more detail in the following section. The vibrational band of interfacial H2O shows a dramatic decrease in amplitude for pH 3−5 with a subsequent dramatic increase of almost 1 order of magnitude (Figure 6c). However, not only the amplitudes of the vibrational bands change, but there is also a notable change in the polarity of the band at ∼3060 cm−1 due to aromatic CH stretching vibrations. At pH < 5, this band appears as a positive going feature, while for pH > 5 a negative going feature is observed (Figure 5b). These changes are not directly related to the ∼3060 cm−1 band but to a change in the orientation of the interfacial water molecules. The phases of the broad OH stretching bands are rotated by 180°, and according to eq 1 the spectral interference of H2O and CH bands is altered. The change in the average orientation of the interfacial water molecules points to a charge reversal of the interface and, consequently, to a reversal of the electric field that causes the polar ordering of H2O at the interface. This result confirms previous SFG studies of BSA adsorption by Wang et al.27

DISCUSSION We will now compare the observed pH effects of adsorbed BSA adlayers from ellipsometry with SFG measurements and the zeta potential of BSA in the bulk electrolyte. It is obvious that the bulk isoelectric point of BSA at pH 5.2 with a zeta potential equal to zero (Figure 1) clearly corresponds to a minimum in SFG amplitudes of the BSA and interfacial H2O related bands (Figure 6), but to a maximum in the thickness of adsorbed BSA adlayers (Figure 2). In fact, our ellipsometry results indicate the presence of multilayers for pH values near the bulk isoelectric point and are in agreement with previous neutron reflection studies of BSA at air−water interfaces. In their report, Lu et al. have estimated the adsorbate thickness to approximately 7 nm and a reduced thickness of approximately 4 nm for pH values higher or lower than the bulk isoelectric point.11 Modeling the geometric dimensions of BSA by a simple incompressible ellipsoid, thicknesses of ∼4 nm in Figure 2 point to a monolayer of adsorbed BSA proteins with their long axis oriented parallel to the interface.12 Threefold thicker layers at a pH near 5 are also observed (Figure 2) and suggest the formation of multilayers. Although we observe a seemingly

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can be rationalized in terms of hydrophilic−hydrophobic interactions, where the hydrophobic parts of the BSA proteins tend to protrude into the gas phase. In contrast to the protein layer, the interfacial water molecules are highly disordered and lead to negligible SFG amplitudes (Figure 6c). Having established the interfacial molecular structure, we can now deduce (to some extent) macroscopic properties such as foam stability and foam capacity. Prerequisite for good foam formation is a fast diffusion of the proteins to the interface, where they can build a viscoelastic adsorbed layer around the gas bubbles and prevent destabilization of the foam lamella.62 As we have shown, at the interfacial isoelectric point of BSA, a network of agglomerated proteins is formed and held together by weak attractive forces, while at a pH more alkaline or more acidic than the isoelectric point BSA forms monolayers with repulsive interactions. It is therefore likely that producing BSA foams at the isoelectric point will lead to foams with higher stability. We have tested this hypothesis with macroscopic foams from BSA dilutions. The results for foam capacity, which describes the ability of a protein solution to enclose air, and foam stability measurements are shown in Figure 7a and b, respectively. Both

opposite behavior of SFG amplitudes and adsorbate thickness, it can be concluded that the isoelectric points of bulk and interfacial BSA proteins are identical. pH values at the isoelectric point of the interface lead to a highly disordered electrolyte subphase and BSA (multi)layers with a low degree of order: We recall that the SFG amplitude Ak is dependent on both the number density of interfacial molecules and their molecular order. In the present case, the contribution of the number density to the SFG amplitudes is only minor otherwise the amplitude should reach a maximum at a pH where the thickness of the BSA adlayer is also maximal. Since we observe the opposite, signals in SFG spectra must be dominated by the interfacial molecular order. At a pH near the bulk isoelectric point, the net charge of BSA proteins is negligible and a macroscopic electric field at the interface is absent. As the pH increases or decreases with respect to the isoelectric point of BSA, the charge density and, consequently, the electric field of the interface increase. As a result, polar ordering of interfacial water molecules and BSA is induced by the interfacial electric field. Further support for this hypothesis comes from SFG experiments at electrified oxide interfaces where electric fieldinduced polar ordering of interfacial water molecules was shown.40,41,60,61 Observations of field induced polar ordering in vibrational SFG are, consequently, directly related to the strength of the interfacial electric field and allow an estimate of the isoelectric point of the interface. At this point, it should be noted that electrokinetic zeta potential measurements and SFG spectroscopy actually probe different physical properties which, however, have their physical origin both in the charge distribution at the surface. The zeta potential refers to the electric potential at the hydrodynamic shear (or slipping) plane. The slipping plane separates ions (if present) and solvent molecules which travel with the migrating protein from those in the diffuse layer that do not travel with the charged protein in an external electric field. In contrast, SFG probes the average orientation of molecules within the interfacial electric field. Since the amide I band originates from molecular groups in the interior of the protein (see above), pH effects on polar ordering must be related to the net charge of the protein surface which is also determined by the charge of possible bound counterions in the adjacent Stern layer. The physical origins of polar ordering as seen in SFG and in the zeta potential are, thus, identical. The remarkable resemblance of amide I, carboxylate (R−COO−), and OH amplitudes in Figure 6 also signifies a similar origin for the latter bands. For pH values near the isoelectric point, BSA adsorption is controlled by a gain in entropy and the formation of attractive noncovalent interactions such as van der Waals forces and hydrogen bonds of the BSA hydration shell. The situation is different for pH values of 6.7 where electrostatic forces dominate and lead to a polar ordered BSA monolayer. In order to establish multiple BSA layers at the interface, the lateral interactions between individual BSA proteins have to be attractive. In fact, the absence of a strong electric charge at the BSA surface, as it is suggested by the zeta potential of BSA in the bulk, leads to weak repulsive electrostatic interactions and consequently to an agglomerated BSA adlayer. At the isoelectric point, the orientation of adsorbed BSA is, however, not completely random since the nonzero SFG amplitudes of amide I and carboxylate vibrations indicate that to some extent a preferential orientation of BSA is maintained. This observation

Figure 7. Foam capacity (a) and stability (b) as a function of pH. Lines are a guide to the eye.

the foam capacity and stability show a clear maximum around pH 5 (Figure 7). Obviously, a network of agglomerated protein multilayers can encapsulate the air and prevent the foam from drainage much more efficiently than ordered protein monolayers with repulsive interactions.

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SUMMARY AND CONCLUSION New information on molecular processes at interfaces and macroscopic phenomena of soft matter is provided. For that purpose, we have addressed the surface chemistry of the protein bovine serum albumin (BSA) at the air−water interface and the effect of pH. Combining ellipsometry and broadband sumfrequency generation (SFG) has allowed us to reveal the molecular composition and molecular order of BSA adlayers and the electrolyte subphase in unprecedented detail. pH dependent charging of BSA leads to the formation of electrified interfaces and to polar ordering of interfacial BSA and H2O. Using the latter as a measure of the electric field at the interface, 7785

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we have deduced the isoelectric point of BSA at air−water interfaces to pH ∼ 5, which is close to that of bulk BSA. The molecular level information presented in this study can explain the high foam stability around pH 5, which was determined in additional experiments: Around pH 5, disordered multilayers are present at the interface and form an agglomerated network of BSA proteins that can be used to form macroscopic foams with excellent stability. Here, BSA agglomerates stabilize gas bubbles at the ubiquitous air−water interface extremely efficiently and prevent the foam from drainage. For pH values smaller or larger than the isoelectric point, BSA monolayers with repulsive interactions are formed and lead to a decreased stability of BSA foams.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge funding of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) and by the German National Science Foundation (DFG) through the Leibniz program and project PE427/21-1. B.B. is grateful for support by the Alexander von Humboldt foundation and a Feodor Lynen fellowship. J.D. is grateful for support by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e. V., Bonn), project AiF 17124 N.

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