J. Phys. Chem. C 2007, 111, 16895-16901
16895
Dynamic Surface Elasticity of β-Casein Solutions during Adsorption B. A. Noskov,† A. V. Latnikova,† S.-Y. Lin,‡ G. Loglio,§ and R. Miller*,| Chemical Faculty, St. Petersburg State UniVersity, UniVersitetsky pr. 2, 198904 St. Petersburg, Russia, Chemical Engineering Department, National Taiwan UniVersity of Science and Technology, 43 Keelung Road, Section 4, Taipei, 106 Taiwan, Dipartimento di Chimica Organica, UniVersita degli Studi di Firenze, Via della Lastruccia 13, 50019 Sesto Fiorentino, Firenze, Italy, and MPI fu¨r Kolloid- und Grenzfla¨chenforschung, D14476 Golm, Germany ReceiVed: May 17, 2007; In Final Form: August 17, 2007
The nonmonotonic kinetic dependencies of the dynamic elasticity of adsorbed and spread β-casein layers at the liquid-gas interface have been determined by the oscillating barrier method. While two local maxima in the surface elasticity versus concentration dependence are well documented in literature, these features have not been reported for kinetic curve. The surface elasticity in the time range of the second maximum depended on the β-casein bulk concentration and deviated from the elasticity of spread β-casein layers at the same surface pressures. In parallel to the surface viscoelasticity of PEO-PPO-PEO block copolymers, the experimental findings for β-casein can be explained by a separation of relatively hydrophobic groups of the polypeptide chain during the slow process of protein adsorption.
Introduction The broad interest in surface properties of aqueous β-casein solutions is mainly caused by its high surface activity and the resulting large variety of applications as stabilizer of disperse systems. Unlike most other milk proteins, β-casein has no tertiary structure in its dilute solutions and insignificant secondary structure. As a result it does not form compact globules in aqueous solutions but forms a random coil. These peculiarities are probably the reason why this protein is frequently used as a model substance in studies of interrelations between protein structure and surface properties of their aqueous solutions. β-casein molecules contain both positive and negative charges, and it is a polyampholyte like other proteins. The charge distribution in the molecule is nonhomogeneous, and one-third of the whole charge belongs to 50 amino acid groups at the N-terminal. Therefore the molecule consists of a hydrophilic tail and a more hydrophobic main part with lower charge density. Thus, a diblock copolymer with hydrophilic and hydrophobic blocks can be seen as a rough physical model of β-casein.1,2 A more advanced model is a multiblock copolymer containing a few hydrophilic and hydrophobic blocks.2-6 Because of its amphiphilic nature, β-casein self-organizes in aqueous solutions into micelles, which resemble aggregates of synthetic block copolymers.7,8 The central hydrophobic and relatively dense part of the micelle is surrounded by a looser shell consisting of charged and more hydrophilic parts of β-casein molecules. The adsorbed and spread layers of β-casein at the interface between a liquid and a fluid phase have been studied by surface tensiometry,1,4-24 radiotracer methods,13 ellipsometry,6,13,25,26 the methods of X-ray17 and neutron2,4,17 reflection, infrared reflection-absorption spectroscopy,27,28 atomic force microscopy,28,29 * To whom correspondence should be addressed. † St. Petersburg State University. ‡ National Taiwan University of Science and Technology. § Universita degli Studi di Firenze. | MPI fu ¨ r Kolloid- und Grenzfla¨chenforschung.
Brewster angle microscopy,18,29 surface shear10,30-32 and dilational4-6,10,11,15,16,19-24,31,33-37 rheology. The dilational viscoelasticity of β-casein surface layers probably was investigated to the greatest extent as compared with other polymeric systems. Note that studies of the dilational surface viscoelasticity of solutions of synthetic nonionic polymers37-38 and polyelectrolytes41,42 have been started only recently. A special interest in the dilational elasticity of β-casein layers is caused on the one hand by its important role in the formation and destruction of foams and emulsions,10,36 and on the other hand by its high sensitivity to macromolecular conformations in the surface layer.39 For example, the adsorption of main conformers of bovine serum albumin at the aqueous solution-air interface at different pH leads to approximately the same surface tensions while the corresponding dynamic surface elasticity differs significantly.36 The dependencies of the dynamic and static surface elasticities on the β-casein concentration or the surface pressure are nonmonotonic. Different authors discovered one5,6,15 or two22,24,33,37,38 local maxima of the corresponding experimental curves for the aqueous solution-air interface. The formation of the first maximum is usually explained by a conformational transition from all trains conformation to trains with tails or loops. A bilayer formation is also assumed sometimes.24,26 There is presently no detailed interpretation of surface elasticity versus concentration dependencies particularly due to significant differences between the various experimental data. The position, number, and absolute values of these maxima are often different in these studies. Cicuta and Hopkinson20 showed that these differences could be explained at least partly by the high sensitivity of the dilational surface elasticity to the ionic strength and pH of buffer solutions. Significant discrepancies also exist in the conclusions about the influence of the way of surface layer formation on its dilational elasticity and on the relationship between static and dynamic elasticity values. Graham and Phillips discovered differences in the static elasticities for spread and adsorbed
10.1021/jp073813j CCC: $37.00 © 2007 American Chemical Society Published on Web 10/12/2007
16896 J. Phys. Chem. C, Vol. 111, No. 45, 2007 β-casein layers14 and also between the static and dynamic elasticities of these systems.34 Subsequent studies, however, showed agreement between static and dynamic properties of the two kinds of surface layers15 and the lack of noticeable influence of its formation history.16 At the same time Cicuta and Hopkinson observed large differences between the static and dynamic (at a frequency of about 10 kHz) elasticities of spread layers and explained them with the influence of the segment exchange between different zones of the surface layer.20 Recently Maldonado-Valderama et al. have noticed that the agreement between the dilational surface elasticities can be improved after a few expansion and compression cycles of spread protein layers.22 This behavior indicates a difference in the protein conformations in these two types of surface layers. Distinctions between the conformations of some proteins in spread and adsorbed layers and also conformation changes during the adsorption process have been discovered recently by infrared reflection-absorption spectroscopy.27 At the same time, structure formation in adsorption layers has rarely been studied up to now by measurements of the kinetic dependency of the dynamic surface elasticity. To the best of our knowledge the kinetic dependency of the dynamic surface elasticity of β-casein solutions was determined only at high concentration (1 g/l) when only slow processes of surface gel formation can be observed.35 Mellema et al. described the kinetic dependency of the dynamic surface elasticity of β-casein solutions only qualitatively.16 Recently preformed investigations of the dynamic surface elasticity provided new information on the conformations of nonionic homopolymers,39-41 polyelectrolytes,42,43 and block copolymers44,45 at liquid surfaces. In the present study, we apply this approach to β-casein solutions. Special attention is paid to the regions of local maxima, which correspond to conformational transitions. The comparison with new results for solutions of block copolymers with hydrophobic and hydrophilic parts45 and also for spread β-casein layers is used for the interpretation of experimental kinetic data. Materials and Methods β-casein, 90%, from Sigma Aldrich Co. (Germany) was used as received. Solutions of β-casein were prepared by dilution of a 0.25 g/l stock solution, which was stored at a temperature below 5 °C for no longer than one week. Most of the solutions were prepared in phosphate buffer with an ionic strength of 0.075 (pH 7), prepared from Na2HPO4 and NaH2PO4 (from Sigma Aldrich). Fresh triple-distilled water was used for the preparation of the solutions. An all-Pyrex apparatus and alkaline permanganate were employed in the second and third stages of distillation. The surface tension of the buffer solution without protein was 72.8 mN/m at 20 °C. The β-casein layers were formed onto the buffer solution surface at pH 7 in a poly(tetrafluoroethylene) (PTFE) Langmuir trough (120 × 160 × 200 mm) by the Trurnit method46 using a β-casein solution of 0.1 mg/mL for spreading. The protein concentration in the spread layers was changed by successive additions of small aliquots or by continuous compression of the surface layer with a compression of less than 1% of the liquid surface area per minute. All measurements were carried out at 20 °C ( 1 °C. The surface tension was measured by the Wilhelmy plate method using a roughened glass plate. The complex dynamic surface elasticity was measured by the oscillating barrier method as described in detail elsewhere.39-43 In brief, the low-frequency oscillations of the liquid surface area
Noskov et al.
Figure 1. Surface pressure vs surface concentration of β-casein layers spread by successive additions of small aliquots of a concentrated protein solution onto a buffer subphase at constant surface area (circles) or by slow compression of the area at a fixed protein amount (squares).
in the Langmuir trough were induced by a moving PTFE barrier. A mechanical generator transformed the rotation of an electromotor to the translational motion with reversion and gave the possibility to control frequency and amplitude. The moving part of the generator was connected to the PTFE barrier by a steel rod. In operation, the barrier glided back and forth along the polished brims of the Langmuir trough and produced oscillations of the liquid surface area with relative amplitudes between 0.4 and 4%. The corresponding surface tension oscillations were measured by the Wilhelmy plate method. The dynamic surface elasticity could be determined from the oscillations of the surface tension γ and surface area S using the relation
(ω) ) r + ii ) δγ/δ ln S where , δγ and δS are complex quantities. The elasticity modulus was determined from the amplitude ratio of the oscillations of surface tension and surface area, while the phase shift between the oscillations of the two parameters (surface tension and surface area) determined the phase angle of the dynamic surface elasticity. At frequencies less than about 0.2 Hz, the length of surface longitudinal waves far exceeded the length of the Langmuir trough and they did not influence the surface tension oscillations in the trough.36,39,40 Nevertheless, the surface tension can change slightly from point to point in the trough probably because of the influence of the trough walls on the liquid flow. If the Wilhelmy plate was not fixed at a given position relative to the brims, this effect could lead to an insufficient accuracy of the surface elasticity measurements (about ( 10%). Therefore, all results in this work correspond to a fixed Wilhelmy plate position in the center of the Langmuir trough. This configuration allowed a significant reduction of the relative experimental error. The surface shear viscosity of β-casein layers is negligible and cannot influence measurements of surface dilational properties.30 Results Figure 1 shows the surface pressure of spread β-casein layers as a function of the surface concentration Γ. The surface layers were formed by successive spreading of small aliquots of the concentrated protein solution onto the surface of a buffer solution either at a constant surface area or by slow compression
Dynamic Surface Elasticity of β-Casein Solutions
Figure 2. Modulus of the dynamic surface elasticity vs surface tension of spread β-casein layers. The line is a guide for the eye.
of the area at a fixed protein amount. The way of formation of the surface layer did not influence the surface pressure isotherm remarkably and both sets of experimental data are in agreement and close to the results of preceding studies.5,15,20,22 The dynamic surface elasticity at a frequency of 0.14 Hz was determined simultaneously with the surface pressure measurements of the spread β-casein layers. The real part of the surface elasticity was 1 order of magnitude higher than the imaginary part, which did not exceed 5 mN/m even in the region of the surface elasticity local maximum. The β-casein surface layers proved to be essentially elastic. Therefore, the figures below contain only data on the modulus of the dynamic surface elasticity, which is close to the real part of the elasticity. One can observe a local maximum of the surface elasticity modulus as a function of surface pressure at π ≈ 5 mN/m, which corresponds to Γ ≈ 1.3 mg/m2 (Figure 2). The elasticity maximum in this range of surface pressure was noticed in all published works on the dilational surface elasticity of spread and adsorbed β-casein layers, and its position and shape are not very sensitive to the properties of the aqueous subphase.4-6,11,15,16,18-20,22,24,33-36 At the same time the isotherms of the dynamic surface elasticity at higher surface pressure do not agree in between the different studies. The data in Figure 2 are close to the results of the quasi-static surface elasticity measured by the Langmuir balance technique.16 Beyond the surface elasticity maximum the surface elasticity drops fast and remains almost constant over the surface tension range from 60 to 63 mN/m. The elasticity decreases again at γ < 60 mN/m and remains almost constant down to γ < 57 mN/m. Rodriguez Nin˜o et al.19 and Maldorado-Valderrama et al.22 report another pattern of (π) dependency at γ < 63 mN/m for spread β-casein layers: the quasi-static and dynamic elasticities go through a local minimum or a minimum and maximum in this range of surface pressures. The second dynamic surface elasticity maximum at γ < 57 mN/m was also observed by other authors.4,24,33 Mellema et al.16 and Cicuta and Hopkinson20 showed that at pH 7, the second maximum arose for spread layers only at high ionic strength I. While most of the authors following Graham and Phillips34 explain the first maximum by the formation of tails and loops, the interpretation of the second maximum remains unclear. Cicuta and Hopkinson assumed that the first maximum corresponded to a transition into the subphase of only the tails from the N-terminal end, while the looping of the second region of the molecule begins in the range of the second maximum.20 However, this assumption does not agree
J. Phys. Chem. C, Vol. 111, No. 45, 2007 16897
Figure 3. Dynamic surface pressure of β-casein solutions at concentrations 0.001 (crosses), 0.0015 (asterisks), 0.002 (circles), 0.005 (squares), and 0.01 mg/mL (triangles).
with data from X-ray reflectivity for adsorbed layers, which indicate the preferential formation of loops even at low β-casein concentrations.17 Figure 2 shows that the second maximum does not arise at pH 7 and I ) 0.075 but the dependency ||(π) becomes less smooth in the surface tension range between 57 mN/m and 63 mN/m. Unlike the case of spread layers, one can measure the properties of adsorbed layers as a function of surface age t from the very early stages of surface layer formation corresponding to almost zero surface pressure up to the state where the proximal region of the surface layer is close to saturation and the surface pressure takes maximum possible values. In this case, one can observe in principle the whole succession of conformational transitions in the layer after a new surface creation at the initial moment t ) 0. Figure 3 shows the surface pressure kinetic curves of β-casein solutions in the concentration range from 0.001 to 0.01 mg/mL. At C > 0.002 mg/mL, the surface pressure starts to increase abruptly during the first minutes after surface formation. One can observe an induction period for more dilute solutions, which is connected with the peculiarities of the surface equation of state.45 At C < 0.005 mg/mL, the surface tension does not reach equilibrium values within a few hours after surface formation. The studies of β-casein adsorption kinetics at the solution-air interface show that the initial steps are determined by protein diffusion from the bulk phase to the surface while the extremely slow surface tension changes at the approach to equilibrium are connected with conformational transitions in the adsorption layer.1,16,47 However, the monotonic kinetic curves of the surface tension provide insufficient information for the investigation of these transition processes. On the contrary, the kinetic dependencies of the dynamic surface elasticity modulus are nonmonotonic (Figure 4). Moreover, two local maxima arise on the kinetic dependencies of the surface elasticity as observed also in the dependencies on the β-casein concentration.4,24,33 The maximum surface elasticity modulus (the first maximum) is about 26 mN/m and is close to the corresponding value in Figure 2. Some deviations from this mean value for the curves in Figure 4, for example, at C ) 0.002 mg/mL, are obviously connected with the fast || changes in the range of the maximum. Slight deviations from the surface age corresponding to the maximum can lead to a surface elasticity much lower than the maximum value. The maxima in the dependency (π) shift toward low surface ages with increasing concentration, and at C g 0.01 mg/mL the first
16898 J. Phys. Chem. C, Vol. 111, No. 45, 2007
Figure 4. Dynamic surface elasticity modulus of β-casein solutions at concentrations 0.001 (crosses), 0.0015 (asterisks), 0.002 (circles), 0.005 (squares), and 0.01 mg/mL (triangles). The lines are guides for the eye.
maximum corresponds to a surface life time, which is beyond the scope of the applied experimental technique. The second surface elasticity maximum arises at higher surface life times and is lower. Nevertheless, the difference between the second maximum || value and the subsequent plateau value exceeds the error limits. The surface elasticity in the range of the second maximum, unlike the first maximum, depends on the β-casein bulk concentration: the maximum value decreases with increasing concentration (Figure 4). To the best of our knowledge no kinetic dependencies with two local maxima of the dynamic surface elasticity have been reported earlier for β-casein solutions. Mellema et al. describe qualitatively only the kinetic curve with a single elasticity maximum.16 One can assume that the maxima in the dependency of dynamic surface elasticity on surface age correspond to conformation transitions arising successively during the adsorption of protein molecules with the continuously increasing surface coverage. One can obtain additional information on the β-casein adsorption layer formation via comparison of results for spread and adsorbed layers. For this aim, the kinetic data in Figure 4 were replotted in coordinates of dynamic surface elasticity modulus on surface pressure (Figure 5). As long as π < 7 mN/ m, the dynamic surface elasticities for solutions of different protein concentrations coincide within the error limits and the dependency (π) coincides with that obtained for spread layers (Figure 2). This is in agreement with the literature4,36 where it is claimed that the dynamic surface elasticities do not differ from static values and represent a single function on surface pressure. On the other hand, this would mean that the conformation of β-casein molecules reached in the initial steps of adsorption is subject to changes as it is the case for spread layers upon compression. At further increase of the surface pressure, however, a second maximum arises in the dependency (π) for adsorption layers (Figure 5), which is not observed for spread layers (Figure 2). The surface elasticity in the range of this second maximum decreases monotonically when the β-casein concentration in the bulk phase is increased (Figure 5). Therefore, in this range the surface elasticity depends not only on surface pressure but also on protein concentration unlike the case of the first maximum where the surface elasticity is a unique function of surface pressure. Note that according to ref 16, the surface pressure of π ≈ 7 mN/m corresponds approximately to the value when conformational transitions start
Noskov et al.
Figure 5. Dynamic surface elasticity modulus vs surface pressure of β-casein solutions at concentrations 0.001 (crosses), 0.0015 (asterisks), 0.002 (circles), 0.005 (squares), and 0.01 mg/mL (triangles). The lines are guides for the eye.
Figure 6. Dynamic surface elasticity modulus of β-casein solutions at pH 5 (squares) and 7 (circles). C ) 0.002 mg/mL. The line is a guide for the eye.
to influence the dynamic surface tensions of β-casein solutions. From this surface, pressure on the dynamic surface elasticity depends on the history of surface layer formation. One can assume that the main factor is the adsorption rate, which increases with the bulk concentration. Finally for π g 17 mN/m the dynamic surface elasticity does not depend once again in the error limits on the bulk concentration for a given surface tension. The influence of the rate of relaxation processes taking place in the studied system on the dynamic surface elasticity can reflect the dependence of the elasticity on oscillation frequency. However, the measurements performed in the frequency range between 0.01 and 0.2 Hz showed only minor changes in the real and imaginary components of the surface elasticity. Probably the main relaxation times correspond to even lower frequencies. This conclusion agrees with data reported in refs 5 and 36. Note, also the solution pH influences strongly the observed kinetic dependencies of the dynamic surface elasticity. The kinetic dependency of (t) at pH 5, close to the point of zero charge of β-casein, has only a single maximum and is increased by about 50% in comparison with pH 7 (Figure 6). Probably the role of hydrophobic interactions between the corresponding
Dynamic Surface Elasticity of β-Casein Solutions
J. Phys. Chem. C, Vol. 111, No. 45, 2007 16899
Figure 7. Dynamic surface elasticity modulus vs surface pressure of surface layers of triblock copolymer PEO76-PPO29-PEO76. The data are replotted from ref 45.
parts of the polypeptide chains increases when approaching the point of zero charge thus leading in particular to a higher stability of the all train conformation at the liquid surface (cf. below). Discussion A comparison of the dynamic surface properties of β-casein solutions with those of other proteins has little sense, as all these are globular and thus have entirely different conformations in the surface layer.33,35,36 At the same time, a comparison is possible with the dilation surface viscoelasticity of solutions of linear synthetic polyelectrolytes with various charge distributions along the polymer chain and at different ionic strengths.42,43 The kinetic dependencies of the dynamic surface elasticity of β-casein solutions under investigation (Figure 4) are entirely different from corresponding results for synthetic polyelectrolytes. In the latter case, the modulus of dynamic surface elasticity deviates from zero only at higher concentrations (>0.1 mg/mL), reaches rather high values (up to 80 mN/m42), and changes extremely slowly and monotonically with surface aging.42,43 These strong discrepancies probably indicate different interactions in the surface layer. One can assume that the surface properties of β-casein solutions cannot be governed by electrostatic repulsions between similarly charged parts of the polypeptide chain, which are only of minor importance. This conclusion was also drawn earlier from studies of surface properties close to the equilibrium state.4-6,23 A possible explanation of the pH effect on surface properties consists in the change of the number of dissociated groups and, consequently, in the number of strongly hydrophilic groups in a macromolecule. One cannot exclude also the influence of attractions between oppositely charged groups. Both the time and concentration dependencies of the dynamic surface elasticity obtained for β-casein remind us to the corresponding results for block copolymers of polyethylene oxide and polypropylene oxide (PEO-PPO-PEO) for which these dependencies are nonmonotonic (Figure 7).44,45 The analogy between the static surface properties of β-casein and PEO-PPO-PEO solutions has been considered already earlier when applying the scaling theory to β-casein surface layers.4-6 Here, we want to use similar considerations for the interpretation of the kinetic dependencies of the dynamic surface elasticity under the assumption that electrostatic interactions are negligible as a first approximation.
Figure 8. Schematic of conformational changes of a single β-casein molecule upon surface pressure increase in the range below the first maximum (a), close to the local minimum (b), and beyond the second local maximum (c). The hydrophobic parts are depicted in blue and the hydrophilic parts in green.
During the initial adsorption steps of flexible nonionic homopolymers or block copolymers the macromolecules are transferred to the surface layer as trains without long loops and tails.39-41,44,45 The corresponding conformations are usually designated as pancakes. The increase of surface concentration upon adsorption layer compression or during the course of adsorption leads to a repulsion of adsorbed segments and, consequently, to an increase in the surface elasticity. If one takes into account that β-casein is a nonglobular protein with a flexible polypeptide chain and contains hydrophobic groups, it is possible to assume that β-casein also forms pancakes initially. This idea is really used in numerous studies and agrees with the scaling description.3-6,14-16,18-22,24,33,36 Figure 8a shows a very simplified scheme of the corresponding conformation where the hydrophobic and hydrophilic parts of the molecule are represented by two pairs of blocks. At further increase of the surface pressure during adsorption, some hydrophilic segments begin to be displaced from the proximal region of the surface layer thus forming tails and loops.39-41,44,45 For PEO76-PPO29-PEO76 copolymers, the PEO segments start to penetrate into the subphase at π ∼ 6 mN/m (Figure 7), and the mechanical relaxation of surface stresses becomes possible at the expense of segment exchange between different regions of the surface layer.44,45 One can describe these processes by means of a combination of the de Gennes reptation theory and the Rouse model.48 The increase of the number of loops and tails in the course of adsorption leads to an acceleration of the relaxation processes and, consequently, to a decrease in dynamic surface elasticity at a given frequency.39
16900 J. Phys. Chem. C, Vol. 111, No. 45, 2007 The dynamic surface elasticity as a function of surface age goes through a local minimum at π ∼ 11 mN/m. The first minimum for the β-casein solutions is also usually explained by the formation of loops and tails.3-6,14-16,20-22,33 Results from neutron and X-ray reflectivity, and also self-consistent field numerical calculations confirm the existence of loops and tails.2,17 Obviously, the loops and tails are formed by the more hydrophilic parts of the molecule, the N-terminal ends first of all (Figure 8b). The decrease of the dynamic surface elasticity for PEOPPO-PEO copolymer solutions proceeds up to the moment when the larger part of PEO blocks transits to the subphase and the more hydrophobic PPO blocks start to interact in the surface layer.44,45 In this case, the dynamic surface elasticity of block copolymer surface layers begins to further increase while it decreases up to almost zero for homopolymer PEO layers.39 One can assume that the increase of the surface elasticity of β-casein solutions beyond the local minimum (Figures 4 and 5) is also connected with interactions between more hydrophobic parts of the protein molecule, which increase up to the second local maximum. More hydrophobic parts also start to penetrate into the subphase at further surface pressure increase in the region of the second elasticity maximum. Recent results indicate that PPO blocks start to be displaced from the surface into the subphase with increasing PEO-PPO-PEO copolymer concentration, and one can observe the second maximum in the surface elasticity versus concentration dependence at π ∼ 18 mN/m (Figure 7).45 The second maximum in the dynamic surface elasticity of β-casein solutions probably can be explained in the same way. Small discrepancies between the values of the surface elasticity modulus corresponding to the second maximum and minimum (∼3 mN/m) indicate slight differences in the extent of hydrophobicity of the two parts of the β-casein molecule, which forms loops and tails in the vicinity of the first and second maxima, respectively. It is probable that the more hydrophobic as well as the more hydrophilic amino acid residues take part in the formation of long loops and tails, and the difference consists only in their relative content (Figure 8c). Moreover, the influence of the total β-casein concentration on the dynamic surface elasticity (Figure 4) indicates that a possible segregation of hydrophobic and hydrophilic regions in the surface layer can depend on the adsorption rate. One can assume that the formation of local relatively hydrophobic regions as a result of hydrophobic interactions is more probable during a slow adsorption. This process hinders the formation of tails and loops at the expense of more hydrophobic parts of the β-casein molecule, which begins only at higher surface pressure beyond the second surface elasticity maximum. It is well known that in a few hours after surface creation the interaction between β-casein molecules in adsorption layers due to hydrophobic effects can lead gradually to the formation of surface gels and aggregates.29,32,35 Probably the formation of the nuclei of surface aggregates starts already during the transition of protein coils to the surface and their subsequent unfolding. This process influences the redistribution of amino acid residues between different regions of the surface layer at surface expansion and compression and, consequently, influences also the dynamic surface elasticity. Note that Figure 8c depicts only a conformation of a single β-casein molecule and does not reflect this cooperative aspect of the adsorption layer formation (hydrophobic interactions between different molecules leading to a separation of hydrophobic regions). Although the solutions studied in this work are well below the CMC, which is at about
Noskov et al. 3 mg/l8, a slow aggregation is possible in the surface layer.28,29 The formation of additional loops and tails leads to the increase of the total adsorbed amount. However, this effect cannot be strong because the influence of loop and tail formation on the dynamic surface elasticity is much higher than on the adsorption.40 We have to take also into account that besides hydrophobic interaction, the formation of intra- or intermolecular hydrogen bonds can also influence the surface layer structure.4 When surface layers are formed by the spreading of concentrated protein solutions on the surface of buffer subphase, the conformation of macromolecules proves to be different from those in adsorption layers, the formation of local hydrophobic regions becomes improbable, and the second elasticity maximum is not observed (Figure 2). Infrared reflection-absorption spectroscopy indicates discrepancies between the conformation of some proteins in spread and adsorbed surface layers.27 Studies of β-casein displacement from the surface layer by low molecular weight surfactants also show differences between adsorbed and spread protein layers.49 Adsorption layers of β-casein are more resistant and are destroyed at higher surface pressure than spread layers. At surface tensions π > 17 mN/m, the dynamic surface elasticity becomes independent of surface pressure and the way of surface layer formation. Then, equivalent to adsorption layers of PEO-PPO-PEO copolymers,45 the different parts of the macromolecules mix in the proximal, middle, and distal regions of the surface layer, and some β-casein molecules probably go entirely into the subphase upon compression. In this range of high surface pressure, one can expect bilayer formation.17 This process probably does not influence noticeably the mechanical surface properties of β-casein solutions, at least in the studied range of γ > 54 mN/m. Conclusions During the initial steps of β-casein adsorption, the dynamic surface elasticity is directly determined by the surface pressure in accordance with an all-trains adsorption model. With increasing surface pressure, a local maximum of the surface elasticity is observed, which is connected with the penetration of tails and loops of relatively hydrophilic parts of β-casein molecules into the subphase in agreement with the preceding studies. It is shown for the first time that the kinetic dependencies of the dynamic surface elasticity exhibits a second maximum. In this range of adsorption times the surface elasticity depends not only on the surface pressure but also on the adsorption rate, and the viscoelastic behavior of the adsorbed β-casein layers becomes different from that of spread layers. The comparison of the dilational viscoelasticity of β-casein with PEO-PPO-PEO surface layers shows that one can connect the second maximum of the surface elasticity as a function of both surface age and surface pressure with a protrusion of the more hydrophobic parts of the β-casein molecules into the subphase as loops and tails. The influence of the adsorption rate (β-casein bulk concentration) on the dynamic surface elasticity probably indicates the cooperative nature of the processes in the surface layer which correspond to the second local maximum of the surface elasticity kinetic dependence. The segregation of more hydrophilic and more hydrophobic parts in the surface layer can proceed during a slow adsorption and probably is more difficult in β-casein surface layers after spreading. This also leads to distinctions between the surface elasticities of spread and adsorbed layers. The same segregation probably leads to surface aggregation and gel formation at significantly longer surface life times.29,32,35
Dynamic Surface Elasticity of β-Casein Solutions Acknowledgment. This work was financially supported by the National Taiwan University of Science and Technology (Project NTUST-2007-R-06), the National Science Council of Taiwan, the Russian Foundation of Basic Research (Joint Project No. 05-03-90580 HHC_a), and the European Space Agency (AO-99-052). References and Notes (1) Wu¨stneck, R.; Kra¨gel, J.; Miller, R.; Fainerman, V. B.; Wilde, P. J.; Sarker, D. K.; Clark, D. C. Food Hydrocolloids 1996, 10, 395. (2) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Leermakers, F. A. M.; Richardson, R. M. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 994. (3) Aguie-Beghin, V.; Leclerc, D.; Daoud, M.; Douillard, R. J. Colloid Interface Sci. 1999, 214, 72. (4) Aschi, A.; Gharbi, A.; Bitri, L.; Calmettes, P.; Daoud, M.; AguieBeghin, V.; Douillard, R. Langmuir 2001, 17, 1896. (5) Hambardzumyan, A.; Aguie-Beghin, V.; Panaitov, I.; Douillard, R. Langmuir 2003, 19, 72. (6) Hambardzumyan, A.; Aguie-Beghin, V.; Daoud, M.; Douillard, R. Langmuir 2004, 20, 756. (7) O’Connell, J. E.; Grinberg, V. Ya.; de Kruif, C. J. J.Colloid Interface Sci. 2003, 258, 33. (8) Portnaya, I.; Cogan, U.; Livney, I. D.; Ramon, O.; Shimoni, K.; Rosenberg, M.; Danino, D. J. Agric. Food Chem. 2006, 54, 5555. (9) Bull, H. B. AdV. Protein Chem. 1947, 3, 95. (10) Protein at liquid interfaces; Mo¨bius, D., Miller R., Eds.; Elsevier: Amsterdam, The Netherlands, 1998. (11) Benjamins, J.; de Feyter, J. A.; Evans, M. T.; Graham, D. E.; Phillips, M. C. Faraday Discuss. Chem. Soc. 1975, 59, 218. (12) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403. (13) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 415. (14) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427. (15) Gau, C-S.; Yu, H.; Zografi, G. J. Colloid Interface Sci. 1994, 162, 214. (16) Mellema, M.; Clark, D. C.; Husband, F. A.; Mackie, A. R. Langmuir 1998, 14, 1753. (17) Harzallah, B.; Aguie-Beghin, V.; Douillard, R.; Bosio, L. Int. J. Biol. Macromol. 1998, 23, 73. (18) Rodriguez Nino, M. R.; Carrera Sanchez, C.; Rodriguez Patino, J. M. Colloids Surf., B 1999, 12, 161. (19) Rodriguez Nino, M. R.; Carrera Sanchez, C.; Rodriguez Patino, J. M. J. Colloid Interface Sci. 2001, 242, 141. (20) Cicuta, P.; Hopkinson, I. J. Chem. Phys. 2001, 114, 8659. (21) Cicuta, P.; Hopkinson, I.; Petrov, P. G. J. Chem. Phys. 2001, 115, 9991. (22) Maldorado-Valderrama, J.; Wege, H. A.; Rodriguez-Valverde, M. A.; Galvez-Ruiz, M. J.; Cabrerizo-Vilchez, M. A. Langmuir 2003, 19, 8436.
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