Article pubs.acs.org/Langmuir
Equilibrium and Dynamic Interfacial Properties of Protein/IonicLiquid-Type Surfactant Solutions at the Decane/Water Interface Chong Cao,† Jinmei Lei,† Lu Zhang,‡ and Feng-Pei Du*,† †
Department of Applied Chemistry, College of Science, China Agriculture University, 2 Yuanmingyuan Xilu, Haidian Dist., Beijing 100193, China ‡ Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhongguancun East Road, Beijing 100190, China ABSTRACT: The interfacial behavior of β-casein and lysozyme solutions has been investigated in the presence of an ionic liquid-type imidazolium surfactant ([C16mim]Br) at the decane/water interface. The dynamic dilational properties of the protein/surfactant solutions are investigated by the oscillating drop method and interfacial tension relaxation method. The interfacial tension isotherms for the mixed adsorption layers indicate that the increased addition of [C16mim]Br to a pure protein changes the properties of the complex formed at the decane/water interface. Whereas the interfacial tension data of the protein/surfactant mixed layers do not clearly show differences with changing bulk composition, the dilational rheology provides undoubted evidence that the structure and, in particular, the dynamics of the adsorbed layers depend on the bulk surfactant concentration. The experiment data for β-casein/ [C16mim]Br solutions indicate that at higher bulk [C16mim]Br concentrations, β-casein in the interfacial layer is subject to conformational changes, where it gives space to [C16mim]Br molecules in the form of coadsorb rather than replacement; in contrast, in lysozyme/[C16mim]Br solutions some lysozyme molecules desorb from the interface due to the competitive adsorption of free [C16mim]Br molecules. Experimental results related to the interfacial dilational properties of the protein/ surfactant solutions show that the dilational modulus turns out to be more sensitive to the conformation of protein/surfactant mixture at the liquid interface than the interfacial tension. different models,7,12−14 only a few investigations have been devoted to the adsorption of protein/surfactant mixtures at the oil/water interface.15,16 A description of the interfacial phenomena at the oil/water interface involving these compounds is quite complex and is not yet fully understood.15 The interest in interfacial rheology has increased in recent years.15,17,18 The interfacial rheological properties are based on the thermodynamics and kinetics of the adsorption layers, which not only plays a significant role in determining the properties of the adsorption layer, such as molecular orientation, molecular interaction and characteristics of mixed surfactant/macromolecule films, but also has been proven to be related to the stability of foams and emulsions.9,19,20 The dilational dynamic elasticity turns out to be more sensitive to the conformation of macromolecules at the liquid interface than the interfacial tension. Additionally, the dilational rheology reflects not only the molecular state of the interfacial layer but also that of the subinterfacial layer.9,20 Until recently, most of the investigations on protein/ surfactant mixtures were focused on conventional surfactants, such as sodium dodecyl sulfate (SDS),21,22 cetyltrimethylammonium bromide (CTAB),23 and other nonionic surfactants,9
1. INTRODUCTION As we know, the protein/surfactant system is very important in the formation and stabilization of foams and emulsions in many fields, such as in food processing, in the cosmetic and pharmaceutical industry, and in the manufacture of pesticide formulations.1−4 The protein/surfactant complexes formed due to different physical interactions modify the properties of the liquid/liquid interface, resulting in a decrease in the interfacial tension and a change in the viscoelastic properties.5−7 In addition, some proteins denature at the liquid/liquid interface in the absence of surfactants. The surfactant concentration strongly affects the complexation process and the characteristics of the complex.7,8 Moreover, the different physical interactions, which can be electrostatic or hydrophobic interactions, can modify and stabilize the complex.9 In the case of electrostatic interactions, increasing the surfactant concentration may cause a net charge inversion, and the free surfactant molecules can gradually replace the protein molecules from the interface film due to competitive adsorption.7,9 The structure of the protein plays an important role in the characteristics of the interfacial adsorption layer. For globular proteins, the adsorption and conformational rearrangement in the interfacial layer are extremely slow, and the adsorption layer is more rigid compared with flexible proteins.10,11 While the adsorption of a protein/surfactant mixture at the water/air interface has been widely investigated with various methods and described by © 2014 American Chemical Society
Received: April 1, 2014 Revised: September 11, 2014 Published: October 30, 2014 13744
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using different methods, including potentiometry, fluorescence, dynamic light scattering, and AFM.2,24,25 The properties of protein/imidazolium surfactants, have not been adequately explored. Ionic liquid-type imidazolium surfactants constitute a new generation of amphiphilic molecules.26 Compared with conventional ammonium surfactants, they display a significantly stronger tendency toward self-aggregation and a stronger attraction toward aromatic rings through π−π interactions due to the existence of imidazolium head groups.27,28 Because of these advantages, it is meaningful to investigate their interaction with proteins, which has potential applications in areas such as sterilization, medicine, oil-field development, and so on. To the best of our knowledge, there have been few investigations on the interfacial dilational rheology properties of protein/imidazolium surfactant solutions.26,29,30 In the present work, we have investigated the interfacial rheological properties of adsorbed films at the decane/water interface formed by the combination of different proteins (lysozyme and β-casein) with an ionic liquid-type imidazolium surfactant ([C16mim]Br) by means of interfacial dilational rheological methods. The aim is to understand the various interactions between proteins and ionic-liquid-type imidazolium surfactant and to obtain information that would be useful for the practical applications.
isotropic interfacial stress. The dilatational relaxation modulus, E(t), is related to the dilatational modulus, ε, via a Fourier transform. The static modulus of irreversibly adsorbed proteins, E∞, is directly determined from the experimental data for E(t → ∞).10,11
3. MATERIALS AND METHODS Materials. The proteins studied were hen egg-white lysozyme and β-casein with molecular weights of 14.3 and 24 kDa, respectively, both purchased from Sigma-Aldrich (Germany) and used as received. Ionicliquid-type imidazolium surfactant [C16mim]Br was obtained from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences. The purity of the sample was checked by 1H NMR spectroscopy, and the structure is displayed in Figure 1. Decane was purchased from
Figure 1. Chemical structure of the ionic-liquid-type imidazolium surfactant [C16mim]Br. Sigma Chemical (Germany) and used without further purification. All solutions were prepared in a sodium phosphate buffer (Sinopharm Chemical Reagent Beijing) using appropriate amounts. The pH values of the solutions before and after the experiments were in the range of 6.8 to 7.0. The protein/surfactant mixed solutions were prepared at constant protein concentrations (hen egg-white lysozyme at 7 × 10−7 mol/L and β-casein at 8 × 10−8 mol/L) and varying [C16mim]Br concentrations in the range of 1 × 10−8 to 1 × 10−5 mol/L. All solutions were prepared using Milli-Q water. Methods. Interfacial tension and the interfacial rheological parameters were measured by an oscillating drop accessory ODG-20 obtained from Data Physics Instruments, Germany. Theoretically, the evolution of interfacial tension is the result of adsorption of the protein molecules from the bulk and unfolding at the liquid/liquid interface. The driving force for adsorption is the difference between the current interfacial tension and the equilibrium interfacial tension. Thus, the evolution rate of interfacial tension is proportional to such a driving force.33 To measure the dilational viscoelasticity of the adsorbed films at the decane/water interface, we syringed the water phase into a thermostated optical glass cuvette containing the oil phase (decane). The image of the drop was capture by a CCD camera and transferred to the data acquisition computer, where it was recorded by digitizing and analyzing the profile of the droplet fitted by the Young−Laplace equation to its coordinates. (Yeung34,35 demonstrated that the Young−Laplace equation assumed implicitly that the interfacial tension is a scalar quantity and it may exhibit different values in different surface directions, so the interfacial tension should be termed the apparent interfacial tension.) At the end of the experiment, the software retrieved the images, performed a Fourier transform analysis, and determined the dilational modulus (ε) and the phase angle (θ). For the experiments described in this article, the drop oscillations at a frequency of 0.1 Hz were carried out during the process of equilibrium. Then, the oscillation frequencies were changed, which were varied between 0.005 and 0.1 Hz, and the oscillation amplitude was 10% (ΔA/A) in sine mode. In the interfacial tension relaxation measurement, the film was expanded ∼15% in area by a sudden expansion in 0.5 s. All experiments were carried out at 25 ± 0.1 °C. All experiments were repeated three times, and the error was within 3%.
2. THEORETICAL BACKGROUND In sinusoidal interfacial compression and expansion experiments, the surface dilational modulus, ε, which provides a measure of the interfacial resistance to changes in area, is defined as the change in interfacial tension, γ, for a small relative change in interfacial area, A.31,32 ε = dγ /dA
(1)
Alternately, the dilational modulus can also be defined as ε = εd + iωηd
(2)
where ω is the oscillating frequency, the real part εd is called the storage modulus or dilational elasticity, representing the elastic energy stored at the interface and is known as dilational elasticity, and ωηd is dilational viscosity. The imaginary part (loss modulus) accounting for the energy dissipated in the relaxation process and is expressed in terms of the interfacial dilational viscosity modulus. The phase angle, θ, resulting from the change in the dynamic interfacial tension, γ, is derived from a small change in interfacial area, A; therefore ε′ = |ε| cos θ
(3)
ε″ = (|ε| /ω) sin θ
(4)
Interfacial tension relaxation experiments are a reliable way to obtain interfacial dilational parameters.10,11 To determine the dilatational relaxation modulus, E(t), we measure the stress relaxation after a sudden strain displacement. In these experiments, the interface is instantaneously deformed and then held constant while the interfacial stress is measured. The dilatational relaxation modulus is calculated as a function of time from the following expression E(t) = Δγ(t )
A0 ΔA
4. RESULTS AND DISCUSSION Dynamic Interfacial Tension of the Protein/Surfactant Mixed Solutions at the Decane/Water Interface. The dynamic interfacial tensions of β-casein/[C16mim]Br and lysozyme/[C16mim]Br mixed solutions at the decane/water
(5)
In eq 5, Δγ is the difference between the static interfacial tension prior to interfacial deformation and the measured 13745
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Figure 2. Dynamic interfacial tension of β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
Figure 3. Equilibrium interfacial tension as a function of surfactant concentration for β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
molecules in each case, leading to an alteration of the interfacial state and the dynamic dependencies. Interfacial Tension of β-Casein/[C16mim]Br and Lysozyme/[C16mim]Br at the Decane/Water Interface. The equilibrium interfacial tension of β-casein/[C16mim]Br and lysozyme/[C16mim]Br at the decane/water interface is presented in Figures 3a,b, respectively. The values of the interfacial tension were assumed to represent equilibrium and were measured at 3 h after interface formation. The interfacial tensions of the two proteins are shown by a straight solid line in both panels. To make comparisons with pure [C16mim]Br solutions, we also present the concentration dependence of pure [C16mim]Br solution interfacial tension. The two mixed protein/surfactant solutions exhibit a similar trend for interfacial tension. At low [C16mim]Br concentrations (5 × 10−6 mol/L), the interfacial tension of the mixtures decreases below that of both the pure protein and surfactant solutions. From the interfacial tension data, one can see that the interfacial layer of the protein/surfactant mixture has contributions from both the protein and the surfactant. β-Casein has no tertiary structure, is disordered and flexible, and is a random coil protein with only 1−10% of the chain in an α-helix and 1 × 10−6 mol/L) compared with the values obtained at lower [C16mim]Br concentrations. However, the lysozyme/[C16mim]Br solutions exhibit the opposite trend. The same results are also observed while measuring the dynamic interfacial dilational modulus of protein/surfactant solutions at various concentrations of [C16mim]Br as a function of adsorption layer age in Figure 4. This may be due to different interfacial behaviors of the protein/[C16mim]Br mixed solutions at higher [C16mim]Br concentrations; to study this reason, we have studied the effect of the surfactant concentration on the interfacial dilational rheology properties.
strong electrostatic interactions between the [C16mim]+ ions and the negatively charged N-terminal groups of β-casein. As a result, the formation of loops and tails becomes more difficult, leading to the shifts in the location of the maxima in the direction of longer interface lifetime compared with pure βcasein solutions. In addition, the dilational modulus of βcasein/[C16mim]Br solutions increases when β-casein is combined with [C16mim]Br. This indicates that [C16mim]Br can enhance the strength of the protein film at the decane/ water interface. As for pure lysozyme solutions, the observed late-time decrease in the dilatational modulus is somewhat unexpected because skin formation is usually associated with a slow continued increase in the value of dilatational modulus.11,44,45 However, for lysozyme/[C16mim]Br solutions, the interfacial dilational modulus increases monotonically with interface age approaching high values at ∼2 h after interface formation. The high interfacial dilational modulus is typical for solutions of globular proteins.21 While at low [C16mim]Br concentrations (in the range of 1 × 10−8 to 1 × 10−6 mol/L) the dilational moduli are higher for lysozyme/[C16mim]Br solutions than those for pure lysozyme solutions, at [C16mim]Br concentrations higher than a critical value of 1 × 10−6 mol/L, the dilational moduli are lower for lysozyme/[C16mim]Br solutions than those obtained for pure lysozyme solution. These results indicate that the concentration of [C16mim]Br at the interface concentration is a key factor determining dilational properties. Figure 5a,b shows the dynamic interfacial dilational modulus of β-casein/[C16mim]Br and lysozyme/[C16mim]Br solutions as a function of interfacial pressure at the decane/water interface. The profile of the curves agrees with the general trend observed for interfacial dilational modulus measured as a function of lifetime of complex formation layer. Moreover, the curve of dynamic interfacial dilational modulus as a function of interfacial pressure makes the interfacial dilational modulus with lifetime of the complex formation more evident. It can be seen that the local maximum of the interfacial dilational modulus for β-casein/[C16mim]Br solutions occurs at an interfacial pressure close to 14 mN/m for all concentrations of [C16mim]Br lower than 1 × 10−6 mol/L. This characteristic value corresponds to the beginning of the formation of the distal region of the interface layer.43 However, for lysozyme/ [C16mim]Br solutions, the interfacial dilational modulus increases monotonically with the interfacial pressure approaching high values at ∼70 mN/m, which is slightly higher compared with values obtained for the pure protein (∼65 mN/ m). 13748
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Figure 6. Influence of concentrations of [C16mim]Br on the dilational modulus (a) and phase angle (b) of β-casein/[C16mim]Br at the water/ decane interface.
Figure 7. Influence of concentration of [C16mim]Br on the dilational modulus (a) and the phase angle (b) of lysozyme/[C16mim]Br at the decane/ water interface.
protein concentration is 8 × 10−8 M, so the charge equilibrium concentration = 8 × 10−8 M × 15e = 1.2 × 10−6 M. At low [C16mim]Br concentrations (below 1 × 10−6 mol/L), the βcasein/[C16mim]Br complexes are formed due to electrostatic interactions, where the charges of β-casein are gradually neutralized by the oppositely charged headgroup of [C16mim]Br. Therefore, in this concentration region, the βcasein/[C16mim]Br complex formed by electrostatic interactions dominates the adsorption interfacial layer. Our experimental results show that the dilational viscous component is close to zero, and the dilational elasticity exhibits a plateau in this concentration regime. As a result, the phase angle remains at a constant value of approximately zero. This result indicates that in this range of [C16mim]Br concentration the interfacial layer is almost elastic. At higher [C16mim]Br concentrations (from 1 × 10−6 to 1 × 10−5 mol/L), the hydrophobic interactions between β-casein and [C16mim]Br come into play. Basak47 found that the casein adopted more ordered conformations in the presence of CTAB at diluted (∼10−6 M) concentrations, and the protein itself wraps around micellar aggregates of CTAB that have cationic head groups association with its negatively charged/polar residues. As the total concentration of [C16mim]Br increases, the dilational modulus increases due to an increasing amount of bound surfactant and the folding of the protein to reach a maximum value at a concentration of 1 × 10−5 mol/L. Subsequent increase in the concentration of [C16mim]Br above 1 × 10−5 mol/L results in a decrease in the dilational modulus due to the exchange of surfactant molecules between the interface and the bulk. Note that at surfactant concentrations lower than 1 × 10−6 mol/L, where complete protein charge neutralization is assumed, the phase angle increases rapidly.
Dependence of the Interfacial Dilational Properties of Protein/Surfactant Solutions on the Surfactant Concentration at the Water/Decane Interface. Figure 6 shows the influence of [C16mim]Br concentrations on the dilational rheology properties of β-casein/[C16mim]Br solutions at the decane/water interface measured at different frequencies (from 0.005 to 0.1 Hz). While the dilational modulus of β-casein/[C16mim]Br is independent of concentration at values below 1 × 10−6 mol/L, it starts increasing to a concentration value of 1 × 10−5 mol/L, beyond which it starts decreasing rapidly. In addition, the phase angle shows little change with [C16mim]Br concentration up to 1 × 10−6 mol/L and increases rapidly above this value. The concentration and the nature of the surfactant strongly affect the protein/surfactant complexation process and the characteristics of the protein/surfactant complex.9 In the case of electrostatic interactions between the protein and the surfactant, a net charge inversion can take place with increasing surfactant concentrations.46 Upon further increase in the surfactant concentration, hydrophobic interactions become more important. As a result, the adsorption layer composition and the interfacial properties become highly concentrationdependent. In addition, with an increase in the surfactant concentration, there is an increase in the amount of free surfactant molecules and the extent of competitive adsorption.7,9 At pH 7, β-casein bears a net negative charge of −15e, and charge neutralization occurs around a [C16mim]Br concentration of 1 × 10−6 mol/L. As previously mentioned, at pH 7, the charge of β-casein is −15e; also, the negative charge belongs to the hydrophilic segment N-terminal, yet the relatively hydrophobic portion of the molecule is almost neutral and the 13749
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Figure 8. Slope of linear fits of log ε−log ω versus different [C16mim]Br concentrations for β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
Figure 9. Static modulus, E∞, as a function of [C16mim]Br concentrations for β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
the surfactant in the proximal region. It revealed that the dilational modulus of lysozyme/[C16mim]Br mixture has been stored in the sublayer of the interfacial film. Moreover, the influence of increasing surfactant concentration on dilational viscosity is similar to that observed for dilational elasticity, and the phase angle remains nearly constant. Subsequent increase in the [C16mim]Br concentration (>1 × 10−6 mol/L) influences the dilational properties of the adsorbed layer in two ways: First, the amount of free [C16mim]Br molecules increases, resulting in an increase in the competitive adsorption between the free [C16mim]Br molecules and the lysozyme/[C16mim]Br complex in interfacial film; as a result, the complex is partially replaced at the interface by the surfactant molecules. Second, increasing the concentration of [C16mim]Br concentration results in a faster exchange between the surfactant molecules restrained on the lysozyme/[C16mim]Br complex and the free [C16mim]Br molecules. Also, Behbehani48 demonstrated that at low concentrations (∼10−6 M) of DTAB the interactions of DTAB/lysozyme are mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme, and these initial interactions cause some protein unfolding and expose additional hydrophobic sites. All effects result in a decrease in the dilational modulus and an increase in the phase angle. The slope of linear fits of log ε versus log ω, with increasing [C16mim]Br concentrations for the protein/[C16mim]Br solutions at the decane/water interface, is shown in Figure 8. The lower the slope, the more elastic the film appears to be. At low [C16mim]Br concentrations (
Equilibrium and Dynamic Interfacial Properties of Protein/IonicLiquid-Type Surfactant Solutions at the Decane/Water Interface Chong Cao,† Jinmei Lei,† Lu Zhang,‡ and Feng-Pei Du*,† †
Department of Applied Chemistry, College of Science, China Agriculture University, 2 Yuanmingyuan Xilu, Haidian Dist., Beijing 100193, China ‡ Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhongguancun East Road, Beijing 100190, China ABSTRACT: The interfacial behavior of β-casein and lysozyme solutions has been investigated in the presence of an ionic liquid-type imidazolium surfactant ([C16mim]Br) at the decane/water interface. The dynamic dilational properties of the protein/surfactant solutions are investigated by the oscillating drop method and interfacial tension relaxation method. The interfacial tension isotherms for the mixed adsorption layers indicate that the increased addition of [C16mim]Br to a pure protein changes the properties of the complex formed at the decane/water interface. Whereas the interfacial tension data of the protein/surfactant mixed layers do not clearly show differences with changing bulk composition, the dilational rheology provides undoubted evidence that the structure and, in particular, the dynamics of the adsorbed layers depend on the bulk surfactant concentration. The experiment data for β-casein/ [C16mim]Br solutions indicate that at higher bulk [C16mim]Br concentrations, β-casein in the interfacial layer is subject to conformational changes, where it gives space to [C16mim]Br molecules in the form of coadsorb rather than replacement; in contrast, in lysozyme/[C16mim]Br solutions some lysozyme molecules desorb from the interface due to the competitive adsorption of free [C16mim]Br molecules. Experimental results related to the interfacial dilational properties of the protein/ surfactant solutions show that the dilational modulus turns out to be more sensitive to the conformation of protein/surfactant mixture at the liquid interface than the interfacial tension. different models,7,12−14 only a few investigations have been devoted to the adsorption of protein/surfactant mixtures at the oil/water interface.15,16 A description of the interfacial phenomena at the oil/water interface involving these compounds is quite complex and is not yet fully understood.15 The interest in interfacial rheology has increased in recent years.15,17,18 The interfacial rheological properties are based on the thermodynamics and kinetics of the adsorption layers, which not only plays a significant role in determining the properties of the adsorption layer, such as molecular orientation, molecular interaction and characteristics of mixed surfactant/macromolecule films, but also has been proven to be related to the stability of foams and emulsions.9,19,20 The dilational dynamic elasticity turns out to be more sensitive to the conformation of macromolecules at the liquid interface than the interfacial tension. Additionally, the dilational rheology reflects not only the molecular state of the interfacial layer but also that of the subinterfacial layer.9,20 Until recently, most of the investigations on protein/ surfactant mixtures were focused on conventional surfactants, such as sodium dodecyl sulfate (SDS),21,22 cetyltrimethylammonium bromide (CTAB),23 and other nonionic surfactants,9
1. INTRODUCTION As we know, the protein/surfactant system is very important in the formation and stabilization of foams and emulsions in many fields, such as in food processing, in the cosmetic and pharmaceutical industry, and in the manufacture of pesticide formulations.1−4 The protein/surfactant complexes formed due to different physical interactions modify the properties of the liquid/liquid interface, resulting in a decrease in the interfacial tension and a change in the viscoelastic properties.5−7 In addition, some proteins denature at the liquid/liquid interface in the absence of surfactants. The surfactant concentration strongly affects the complexation process and the characteristics of the complex.7,8 Moreover, the different physical interactions, which can be electrostatic or hydrophobic interactions, can modify and stabilize the complex.9 In the case of electrostatic interactions, increasing the surfactant concentration may cause a net charge inversion, and the free surfactant molecules can gradually replace the protein molecules from the interface film due to competitive adsorption.7,9 The structure of the protein plays an important role in the characteristics of the interfacial adsorption layer. For globular proteins, the adsorption and conformational rearrangement in the interfacial layer are extremely slow, and the adsorption layer is more rigid compared with flexible proteins.10,11 While the adsorption of a protein/surfactant mixture at the water/air interface has been widely investigated with various methods and described by © 2014 American Chemical Society
Received: April 1, 2014 Revised: September 11, 2014 Published: October 30, 2014 13744
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using different methods, including potentiometry, fluorescence, dynamic light scattering, and AFM.2,24,25 The properties of protein/imidazolium surfactants, have not been adequately explored. Ionic liquid-type imidazolium surfactants constitute a new generation of amphiphilic molecules.26 Compared with conventional ammonium surfactants, they display a significantly stronger tendency toward self-aggregation and a stronger attraction toward aromatic rings through π−π interactions due to the existence of imidazolium head groups.27,28 Because of these advantages, it is meaningful to investigate their interaction with proteins, which has potential applications in areas such as sterilization, medicine, oil-field development, and so on. To the best of our knowledge, there have been few investigations on the interfacial dilational rheology properties of protein/imidazolium surfactant solutions.26,29,30 In the present work, we have investigated the interfacial rheological properties of adsorbed films at the decane/water interface formed by the combination of different proteins (lysozyme and β-casein) with an ionic liquid-type imidazolium surfactant ([C16mim]Br) by means of interfacial dilational rheological methods. The aim is to understand the various interactions between proteins and ionic-liquid-type imidazolium surfactant and to obtain information that would be useful for the practical applications.
isotropic interfacial stress. The dilatational relaxation modulus, E(t), is related to the dilatational modulus, ε, via a Fourier transform. The static modulus of irreversibly adsorbed proteins, E∞, is directly determined from the experimental data for E(t → ∞).10,11
3. MATERIALS AND METHODS Materials. The proteins studied were hen egg-white lysozyme and β-casein with molecular weights of 14.3 and 24 kDa, respectively, both purchased from Sigma-Aldrich (Germany) and used as received. Ionicliquid-type imidazolium surfactant [C16mim]Br was obtained from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences. The purity of the sample was checked by 1H NMR spectroscopy, and the structure is displayed in Figure 1. Decane was purchased from
Figure 1. Chemical structure of the ionic-liquid-type imidazolium surfactant [C16mim]Br. Sigma Chemical (Germany) and used without further purification. All solutions were prepared in a sodium phosphate buffer (Sinopharm Chemical Reagent Beijing) using appropriate amounts. The pH values of the solutions before and after the experiments were in the range of 6.8 to 7.0. The protein/surfactant mixed solutions were prepared at constant protein concentrations (hen egg-white lysozyme at 7 × 10−7 mol/L and β-casein at 8 × 10−8 mol/L) and varying [C16mim]Br concentrations in the range of 1 × 10−8 to 1 × 10−5 mol/L. All solutions were prepared using Milli-Q water. Methods. Interfacial tension and the interfacial rheological parameters were measured by an oscillating drop accessory ODG-20 obtained from Data Physics Instruments, Germany. Theoretically, the evolution of interfacial tension is the result of adsorption of the protein molecules from the bulk and unfolding at the liquid/liquid interface. The driving force for adsorption is the difference between the current interfacial tension and the equilibrium interfacial tension. Thus, the evolution rate of interfacial tension is proportional to such a driving force.33 To measure the dilational viscoelasticity of the adsorbed films at the decane/water interface, we syringed the water phase into a thermostated optical glass cuvette containing the oil phase (decane). The image of the drop was capture by a CCD camera and transferred to the data acquisition computer, where it was recorded by digitizing and analyzing the profile of the droplet fitted by the Young−Laplace equation to its coordinates. (Yeung34,35 demonstrated that the Young−Laplace equation assumed implicitly that the interfacial tension is a scalar quantity and it may exhibit different values in different surface directions, so the interfacial tension should be termed the apparent interfacial tension.) At the end of the experiment, the software retrieved the images, performed a Fourier transform analysis, and determined the dilational modulus (ε) and the phase angle (θ). For the experiments described in this article, the drop oscillations at a frequency of 0.1 Hz were carried out during the process of equilibrium. Then, the oscillation frequencies were changed, which were varied between 0.005 and 0.1 Hz, and the oscillation amplitude was 10% (ΔA/A) in sine mode. In the interfacial tension relaxation measurement, the film was expanded ∼15% in area by a sudden expansion in 0.5 s. All experiments were carried out at 25 ± 0.1 °C. All experiments were repeated three times, and the error was within 3%.
2. THEORETICAL BACKGROUND In sinusoidal interfacial compression and expansion experiments, the surface dilational modulus, ε, which provides a measure of the interfacial resistance to changes in area, is defined as the change in interfacial tension, γ, for a small relative change in interfacial area, A.31,32 ε = dγ /dA
(1)
Alternately, the dilational modulus can also be defined as ε = εd + iωηd
(2)
where ω is the oscillating frequency, the real part εd is called the storage modulus or dilational elasticity, representing the elastic energy stored at the interface and is known as dilational elasticity, and ωηd is dilational viscosity. The imaginary part (loss modulus) accounting for the energy dissipated in the relaxation process and is expressed in terms of the interfacial dilational viscosity modulus. The phase angle, θ, resulting from the change in the dynamic interfacial tension, γ, is derived from a small change in interfacial area, A; therefore ε′ = |ε| cos θ
(3)
ε″ = (|ε| /ω) sin θ
(4)
Interfacial tension relaxation experiments are a reliable way to obtain interfacial dilational parameters.10,11 To determine the dilatational relaxation modulus, E(t), we measure the stress relaxation after a sudden strain displacement. In these experiments, the interface is instantaneously deformed and then held constant while the interfacial stress is measured. The dilatational relaxation modulus is calculated as a function of time from the following expression E(t) = Δγ(t )
A0 ΔA
4. RESULTS AND DISCUSSION Dynamic Interfacial Tension of the Protein/Surfactant Mixed Solutions at the Decane/Water Interface. The dynamic interfacial tensions of β-casein/[C16mim]Br and lysozyme/[C16mim]Br mixed solutions at the decane/water
(5)
In eq 5, Δγ is the difference between the static interfacial tension prior to interfacial deformation and the measured 13745
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Figure 2. Dynamic interfacial tension of β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
Figure 3. Equilibrium interfacial tension as a function of surfactant concentration for β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
molecules in each case, leading to an alteration of the interfacial state and the dynamic dependencies. Interfacial Tension of β-Casein/[C16mim]Br and Lysozyme/[C16mim]Br at the Decane/Water Interface. The equilibrium interfacial tension of β-casein/[C16mim]Br and lysozyme/[C16mim]Br at the decane/water interface is presented in Figures 3a,b, respectively. The values of the interfacial tension were assumed to represent equilibrium and were measured at 3 h after interface formation. The interfacial tensions of the two proteins are shown by a straight solid line in both panels. To make comparisons with pure [C16mim]Br solutions, we also present the concentration dependence of pure [C16mim]Br solution interfacial tension. The two mixed protein/surfactant solutions exhibit a similar trend for interfacial tension. At low [C16mim]Br concentrations (5 × 10−6 mol/L), the interfacial tension of the mixtures decreases below that of both the pure protein and surfactant solutions. From the interfacial tension data, one can see that the interfacial layer of the protein/surfactant mixture has contributions from both the protein and the surfactant. β-Casein has no tertiary structure, is disordered and flexible, and is a random coil protein with only 1−10% of the chain in an α-helix and 1 × 10−6 mol/L) compared with the values obtained at lower [C16mim]Br concentrations. However, the lysozyme/[C16mim]Br solutions exhibit the opposite trend. The same results are also observed while measuring the dynamic interfacial dilational modulus of protein/surfactant solutions at various concentrations of [C16mim]Br as a function of adsorption layer age in Figure 4. This may be due to different interfacial behaviors of the protein/[C16mim]Br mixed solutions at higher [C16mim]Br concentrations; to study this reason, we have studied the effect of the surfactant concentration on the interfacial dilational rheology properties.
strong electrostatic interactions between the [C16mim]+ ions and the negatively charged N-terminal groups of β-casein. As a result, the formation of loops and tails becomes more difficult, leading to the shifts in the location of the maxima in the direction of longer interface lifetime compared with pure βcasein solutions. In addition, the dilational modulus of βcasein/[C16mim]Br solutions increases when β-casein is combined with [C16mim]Br. This indicates that [C16mim]Br can enhance the strength of the protein film at the decane/ water interface. As for pure lysozyme solutions, the observed late-time decrease in the dilatational modulus is somewhat unexpected because skin formation is usually associated with a slow continued increase in the value of dilatational modulus.11,44,45 However, for lysozyme/[C16mim]Br solutions, the interfacial dilational modulus increases monotonically with interface age approaching high values at ∼2 h after interface formation. The high interfacial dilational modulus is typical for solutions of globular proteins.21 While at low [C16mim]Br concentrations (in the range of 1 × 10−8 to 1 × 10−6 mol/L) the dilational moduli are higher for lysozyme/[C16mim]Br solutions than those for pure lysozyme solutions, at [C16mim]Br concentrations higher than a critical value of 1 × 10−6 mol/L, the dilational moduli are lower for lysozyme/[C16mim]Br solutions than those obtained for pure lysozyme solution. These results indicate that the concentration of [C16mim]Br at the interface concentration is a key factor determining dilational properties. Figure 5a,b shows the dynamic interfacial dilational modulus of β-casein/[C16mim]Br and lysozyme/[C16mim]Br solutions as a function of interfacial pressure at the decane/water interface. The profile of the curves agrees with the general trend observed for interfacial dilational modulus measured as a function of lifetime of complex formation layer. Moreover, the curve of dynamic interfacial dilational modulus as a function of interfacial pressure makes the interfacial dilational modulus with lifetime of the complex formation more evident. It can be seen that the local maximum of the interfacial dilational modulus for β-casein/[C16mim]Br solutions occurs at an interfacial pressure close to 14 mN/m for all concentrations of [C16mim]Br lower than 1 × 10−6 mol/L. This characteristic value corresponds to the beginning of the formation of the distal region of the interface layer.43 However, for lysozyme/ [C16mim]Br solutions, the interfacial dilational modulus increases monotonically with the interfacial pressure approaching high values at ∼70 mN/m, which is slightly higher compared with values obtained for the pure protein (∼65 mN/ m). 13748
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Figure 6. Influence of concentrations of [C16mim]Br on the dilational modulus (a) and phase angle (b) of β-casein/[C16mim]Br at the water/ decane interface.
Figure 7. Influence of concentration of [C16mim]Br on the dilational modulus (a) and the phase angle (b) of lysozyme/[C16mim]Br at the decane/ water interface.
protein concentration is 8 × 10−8 M, so the charge equilibrium concentration = 8 × 10−8 M × 15e = 1.2 × 10−6 M. At low [C16mim]Br concentrations (below 1 × 10−6 mol/L), the βcasein/[C16mim]Br complexes are formed due to electrostatic interactions, where the charges of β-casein are gradually neutralized by the oppositely charged headgroup of [C16mim]Br. Therefore, in this concentration region, the βcasein/[C16mim]Br complex formed by electrostatic interactions dominates the adsorption interfacial layer. Our experimental results show that the dilational viscous component is close to zero, and the dilational elasticity exhibits a plateau in this concentration regime. As a result, the phase angle remains at a constant value of approximately zero. This result indicates that in this range of [C16mim]Br concentration the interfacial layer is almost elastic. At higher [C16mim]Br concentrations (from 1 × 10−6 to 1 × 10−5 mol/L), the hydrophobic interactions between β-casein and [C16mim]Br come into play. Basak47 found that the casein adopted more ordered conformations in the presence of CTAB at diluted (∼10−6 M) concentrations, and the protein itself wraps around micellar aggregates of CTAB that have cationic head groups association with its negatively charged/polar residues. As the total concentration of [C16mim]Br increases, the dilational modulus increases due to an increasing amount of bound surfactant and the folding of the protein to reach a maximum value at a concentration of 1 × 10−5 mol/L. Subsequent increase in the concentration of [C16mim]Br above 1 × 10−5 mol/L results in a decrease in the dilational modulus due to the exchange of surfactant molecules between the interface and the bulk. Note that at surfactant concentrations lower than 1 × 10−6 mol/L, where complete protein charge neutralization is assumed, the phase angle increases rapidly.
Dependence of the Interfacial Dilational Properties of Protein/Surfactant Solutions on the Surfactant Concentration at the Water/Decane Interface. Figure 6 shows the influence of [C16mim]Br concentrations on the dilational rheology properties of β-casein/[C16mim]Br solutions at the decane/water interface measured at different frequencies (from 0.005 to 0.1 Hz). While the dilational modulus of β-casein/[C16mim]Br is independent of concentration at values below 1 × 10−6 mol/L, it starts increasing to a concentration value of 1 × 10−5 mol/L, beyond which it starts decreasing rapidly. In addition, the phase angle shows little change with [C16mim]Br concentration up to 1 × 10−6 mol/L and increases rapidly above this value. The concentration and the nature of the surfactant strongly affect the protein/surfactant complexation process and the characteristics of the protein/surfactant complex.9 In the case of electrostatic interactions between the protein and the surfactant, a net charge inversion can take place with increasing surfactant concentrations.46 Upon further increase in the surfactant concentration, hydrophobic interactions become more important. As a result, the adsorption layer composition and the interfacial properties become highly concentrationdependent. In addition, with an increase in the surfactant concentration, there is an increase in the amount of free surfactant molecules and the extent of competitive adsorption.7,9 At pH 7, β-casein bears a net negative charge of −15e, and charge neutralization occurs around a [C16mim]Br concentration of 1 × 10−6 mol/L. As previously mentioned, at pH 7, the charge of β-casein is −15e; also, the negative charge belongs to the hydrophilic segment N-terminal, yet the relatively hydrophobic portion of the molecule is almost neutral and the 13749
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Figure 8. Slope of linear fits of log ε−log ω versus different [C16mim]Br concentrations for β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
Figure 9. Static modulus, E∞, as a function of [C16mim]Br concentrations for β-casein/[C16mim]Br (a) and lysozyme/[C16mim]Br (b) solutions at the decane/water interface.
the surfactant in the proximal region. It revealed that the dilational modulus of lysozyme/[C16mim]Br mixture has been stored in the sublayer of the interfacial film. Moreover, the influence of increasing surfactant concentration on dilational viscosity is similar to that observed for dilational elasticity, and the phase angle remains nearly constant. Subsequent increase in the [C16mim]Br concentration (>1 × 10−6 mol/L) influences the dilational properties of the adsorbed layer in two ways: First, the amount of free [C16mim]Br molecules increases, resulting in an increase in the competitive adsorption between the free [C16mim]Br molecules and the lysozyme/[C16mim]Br complex in interfacial film; as a result, the complex is partially replaced at the interface by the surfactant molecules. Second, increasing the concentration of [C16mim]Br concentration results in a faster exchange between the surfactant molecules restrained on the lysozyme/[C16mim]Br complex and the free [C16mim]Br molecules. Also, Behbehani48 demonstrated that at low concentrations (∼10−6 M) of DTAB the interactions of DTAB/lysozyme are mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme, and these initial interactions cause some protein unfolding and expose additional hydrophobic sites. All effects result in a decrease in the dilational modulus and an increase in the phase angle. The slope of linear fits of log ε versus log ω, with increasing [C16mim]Br concentrations for the protein/[C16mim]Br solutions at the decane/water interface, is shown in Figure 8. The lower the slope, the more elastic the film appears to be. At low [C16mim]Br concentrations (
