Langmuir 1997, 13, 6219-6225
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Forces between Adsorbed Layers of β-Casein Tommy Nylander*,† and N. Magnus Wahlgren‡ Physical Chemistry 1, Lund University, Center for Chemistry and Chemical Engineering, P. O. Box 124, S-221 00 Lund, Sweden, and Swedish Meat Research Institute, Box 504, S-244 24 Ka¨ vlinge, Sweden Received May 20, 1997. In Final Form: August 27, 1997X The forces between β-casein layers adsorbed onto mica surfaces have been measured as a function of surface separation by using the interferometric surface force apparatus. Both hydrophilic pure mica and a mica surface, which has been made hydrophobic by Langmuir-Blodgett deposition of dimethyldioctadecylammonium bromide, were used. A long-range repulsive force, most probably of electrostatic origin, was observed between β-casein layers adsorbed on hydrobized mica. The results suggest that β-casein forms a monolayer on this surface, where the outer part is less densely packed and protrudes far out into the solution. This open brushlike structure can readily be compressed on which an attractive force arises. The portion of the monolayer closest to the hydrophobized surface is much more compact and has low compressibility. On the pure hydrophilic surface a bilayer structure is more likely, with the a compact inner layer and an outer layer which has a similar structure to the monolayer formed on a hydrophobic surface.
Introduction The interactions between protein-coated surfaces are of particular importance for the stabilization of emulsions and dispersions, where proteins are frequently used as emulsifiers and protective colloids. These interactive forces can be directly measured by the surface force apparatus (SFA), and from the experimental results, information about the structure and the dimensions of the adsorbed protein layer can also be obtained.1 Caseins, which are milk proteins with mostly random secondary structure, are frequently used in various food products because of their excellent properties as emulsifiers and foaming agents.2 β-Casein is one of the four major caseins and has the biological function of stabilizing the colloidal form of calcium phosphate in milk and thereby inhibit crystal growth in the secretary cells (glands).2-5 The amino acid sequence of β-casein is divided into one hydrophilic and one hydrophobic domain which gives the molecule a strong amphiphilic character.6 The protein somewhat resembles a simple ionic surfactant in that it forms monodisperse aggregates above a certain concentration, which is about 0.5 mg/mL at room temperature.7 In parallel with ionic surfactants, the monomer aggregation is a balance between hydrophobic interactions and electrostatic repulsion between the different domains.7,8 The amphiphilic character of β-casein suggests that the molecule will orient on a hydrophobic surface in such a * To whom correspondence should be addressed: e-mail,
[email protected]; phone, Int + 46 46 2228158; fax, Int + 46 46 2224413. † Physical Chemistry 1, Lund University. ‡ Swedish Meat Research Institute. X Abstract published in Advance ACS Abstracts, October 1, 1997. (1) Claesson, P. M.; Blomberg, E.; Fro¨berg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161-227. (2) Walstra, P.; Jenness, R. Dairy chemistry and physics; John Wiley & Sons: New York, 1984. (3) Holt, C.; Sawyer, L. Protein Eng. 1988, 1, 251. (4) Holt, C, In Developments in Dairy Chemistry; Fox, P. F., Ed.; Elsevier Applied Science Publishers: London, 1985; Vol. 3, pp 143181. (5) Holt, C.; Wahlgren, M. N.; Drakenberg, T. Biochem. J. 1996, 314, 1035-1039. (6) Eigel, W. N.; Butler, J. E.; Ernstrom, C. A.; Farrel, H. M., Jr.; Harwalkar, V. R.; Whitney, R. M. J. Dairy Sci. 1984, 67, 1599. (7) Schmidt, D. G.; Payens, T. A. J. J. Colloid Interface Sci. 1972, 39, 655. (8) Payens, T. A.; Markwijk, B. W. Biochim. Biophys. Acta 1963, 71, 517.
S0743-7463(97)00503-9 CCC: $14.00
way that the hydrophobic protein domains are anchored at the interface, whereas the hydrophilic parts pertrude into the aqueous environment. The results from NMR (nuclear magnetic resonance) relaxation measurements show that the molecules adopt a similar conformation and have a similar mobility at the oil/water interface in an emulsion as it has in the micelle-like aggregates in aqueous solution.9 A number of studies using light scattering on particles and emulsion droplets,10-13 X-ray scattering of polystyrene spheres,13 and neutron reflectivity at the oil/ water,14 air/water,14,15 and solid/water16 interfaces have been devoted to the structure of adsorbed layers of β-casein. All these studies demonstrate that β-casein orients at hydrophobic surfaces in such a way that the hydrophilic part of the molecule pertrudes extensively into the aqueous phase to form a thick interfacial layer. The structure of this layer has been found to be dependent on the properties of the hydrophobic surface.11 In our previous work,17,18 we used endoproteinase Asp-N, a proteolytic enzyme which cleaves specifically at the N-terminal part of aspartic amino acid residues, to test whether these residues (two out of totally four) in the hydrophilic part of β-casein are accessible for the enzyme. A reduction in the amount of β-casein on a hydrophobic surface was observed and found to correspond to an enzymatic cleavage at the hydrophilic cleavage sites. This further confirms that the hydrophilic part of the β-casein forms a open brushlike structure when the protein is adsorbed at an interface. Such a structure suggests that steric forces should act when two β-casein(9) ter Beek, L. C.; Ketelaars, M.; McCain, D. C.; Smulders, P. E. A.; Walstra, P.; Hemminga, M. A. Biophys. J. 1996, 70, 2396-2402. (10) Dalgleish, D. G.; Leaver, J. J. Colloid Interface Sci. 1991, 141, 288. (11) Leaver, J.; Dalgleish, J. J. Colloid Interface Sci. 1992, 149, 4955. (12) Brooksbank, D. V.; Davidson, C. M.; Horne, D. S.; Leaver, J. J. Chem. Soc., Faraday Trans. 1993, 89, 3419-3425. (13) Mackie, A. R.; Mingins, J.; North, A. N. J. Chem. Soc., Faraday. Trans. 1991, 87, 3043. (14) Dickinson, E.; Horne, D. S.; Phipps, J. S.; Richardson, R. M. Langmuir 1993, 9, 242-248. (15) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 2847-2854. (16) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657-660. (17) Nylander, T.; Wahlgren, N. M. J. Colloid Interface Sci. 1994, 162, 151-162. (18) Kull, T.; Nylander, T.; Tiberg, F.; Wahlgren, M. N. Langmuir 1997, 13, 5141.
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covered surfaces are brought together and therefore be important for β-casein-stabilized emulsions and dispersions. However, the study by Brooksbank et al.12 suggests flocculation of β-casein-covered hydrophobic particles is prevented not only by steric but also by electrostatic repulsive forces. The present study was undertaken to reveal the forces acting when two β-casein-covered surfaces are brought together. Direct force versus distance measurements between β-casein layers adsorbed onto both hydrophobic and hydrophilic surfaces have been made. We will discuss the experimental findings with respect to the structure of the adsorbed layer and its consequences for stabilization of emulsions and dispersions. Materials and Methods Materials. β-Casein A1 was prepared from milk obtained from a homozygote cow (genotype Rs1-B, β-A1, κ-A) at the Swedish University of Agricultural Science, Alnarp, Sweden, as described earlier.17 Dimethyldioctadecylammonium bromide (DDOAB) was purchased from TCI, Tokyo and recrystallized two times from ethyl acetate. The water used was ion-exchanged, distilled, and passed through a Milli-Q water purification system (Millipore Corp.), giving water with a conductivity of 0.7 µS/cm and showing no bubble persistence. All glassware was cleaned in a mixture of concentrated sulfuric and nitric acid, 1:1 (v/v), and thoroughly rinsed with pure water. All chemicals used were of analytical grade, and the sodium chloride was further purified by roasting for 4 h at 600 °C. Methods. Preparation of Hydrophobic Surfaces. The mica surfaces were made hydrophobic by depositing DDOAB according to the Langmuir-Blodgett procedure.19,20 A KSV 5000 Langmuir-Blodgett film balance system (KSV Instruments Ltd., Finland) was used, where the surface tension was measured by the Wilhelmy plate method, using a platinum plate, cleaned by glowing it in a flame, of 2 cm width. The Teflon trough (maximum area 150 × 675 mm2) was thoroughly cleaned with hexane, ethanol, and water. After water had been added (about 2 L) and the temperature was equilibrated to 25 °C, the surface was cleaned by sweeping it with the Teflon barrier. Any surface active components were removed by aspiration at maximum compression. This was repeated until the measured surface pressure was below 0.1 mN/m on complete compression of the film. To prevent recontamination, the barrier was moved to maximum surface area just before the silica disks were lowered below the air/water interface. The DDOAB solution (200 µL of 0.5 mg/mL of DDOAB in a mixture of 95 mL of hexane and 5 mL of ethanol) was carefully applied at the interface by means of a microsyringe. The solvent was allowed to evaporate for 10 min before the surface film was compressed to 25 mN/m at a barrier speed of 20 mm/min. While keeping the surface pressure constant, the mica sheets (glued onto silica disks) were slowly raised out of the water at a speed of 2 mm/min. The hydrophobicity of the surfaces was checked by placing a small droplet of water on the surfaces. The backside of the silica disks were carefully rinsed under water and blown dry with nitrogen, before the disks were mounted in the SFA. Surface Force Measurements. The interferometric surface force apparatus (SFA) was used to measure the interaction between adsorbed layers of β-casein. The technique is described in more detail elsewhere21,22 as well as the particular version of the apparatus (Mark IV) used in this study.22 The force is measured between two curved mica surfaces (mean radius of curvature, R, of about 1-2 cm) in a crossed cylinder configuration. The two mica sheets, supported on half-cylindrical silica disks, are mounted on a double canterleaver spring and on a piezoelectric crystal, respectively. The surface separation, D, between the two surfaces is controlled by the piezoelectric crystal and measured by an interferometric technique with an accuracy of (19) Blodgett, K. B. J. Am. Chem. Soc. 1935, 57, 1007. (20) Blodgett, K. B.; Langmuir, I. Phys. Rev. 1937, 964. (21) Israelachvili, J. N.; Adams, G. E. J. Chem. Soc., Faraday Trans. 1 1978, 74, 975. (22) Parker, J. L.; Christenson, H. K.; Ninham, B. W. Rev. Sci. Instrum. 1989, 60, 3135.
Nylander and Wahlgren 2 Å. The magnitude of the force, F, can be determined from the measured spring deflection down to about 10-7N and is given normalized with the mean radius of curvature. The SFA, equipped with the small volume chamber of 25 mL, was dismantled, and all inner parts were thoroughly clean with hot water, rinsed with water and ethanol, and finally blown dry with ultrapure nitrogen. All operations were performed in a laminar flow cabinet under essentially dust-free conditions. Ruby mica (Associated Commodity Corporation Ltd., New York) was cleaved into molecularly smooth sheets, cut in about 1 × 1 cm pieces and put down on a freshly cleaved mica backing sheet. An about 560 Å thick silver layer was evaporated onto the mica surface, to make them reflective. The mica pieces were then glued with the silvered side down onto optically polished halfcylindrical silica disks. The disks were mounted in the SFA and the mica-mica contact position was measured in air. If the contact was found to be adhesive and contamination free, 1 mM sodium chloride was injected and the contact position and at least two reproducible force curves were recorded. The experiment was continued only if the experimental force curves corresponded to the expected ones. When hydrophobic surfaces were used, the piezo and top lid of the SFA, with the spring attached, chamber were removed. The disks were dismantled from the piezo and the spring and mounted in the dipping device on the Langmuir trough. The DDOAB deposition was carried out as described above and the disks were then mounted in the SFA. The chamber was thoroughly rinsed with fresh salt solution and the air/liquid interface was aspirated several times to remove surface active contaminants. The top lid with the spring and the lower disk was immediately mounted onto the chamber, ensuring that the surface of the disk passed the air/solution interface as quickly as possible. The interface was aspirated once more and the piezo with upper disk was put in place in the same way. The surfaces were brought into close contact, after which a force curve was recorded and the contact position was measured. This was done within a couple of hours to avoid damage and contamination of the surfaces. The surfaces were then separated by 0.2 mm and the chamber was emptied, leaving a drop of solution between the surfaces. The proteins solution, which was prepared by dissolving the β-casein at 0.1 mg/mL in 1 mM NaCl solution and adjusting pH to 7.0 by using 0.1 M NaOH or HCl, was injected into the chamber. The surfaces were separated and brought together several times to ensure that the liquid residing between the surfaces was properly mixed with the bulk. The same procedure was used when the experimental conditions were changed during an experiment, e.g., replacing the protein with sodium chloride solution. At least two reproducible force curves were recorded under the same conditions. All experiments were performed at 22.0 ( 0.2 °C.
Results and Discussion Interaction between β-Casein Layers on Hydrophobized Mica Surfaces. As shown in Figure 1A, a long range attractive force is observed between DDOAB surfaces in 1 mM NaCl. Note, that zero surface separation for the force curves using hydrophobic surfaces is defined as the contact between DDOAB covered surfaces. The thickness of the DDOAB layer, estimated from the contact position relative to pure mica surfaces, was found to be about 20 Å. Both the range of the force and the thickness of the layer are in agreement with earlier reports.23,24 It should be noted that in order to avoid contamination and possible deterioration of the hydrophobic surface, which has been reported to occur at high ionic strength,25 the force curves between the hydrophobic surfaces were recorded immediately after preparation. The presence of a long-range attractive force was observed, which together with the decrease in water wettability of the surface (contact angle >90°) and determined DDOAB layer (23) Christenson, H. K.; Claesson, P. M. Science 1988, 239, 390392. (24) Claesson, P. M.; Christenson, H. K. J. Phys. Chem. 1988, 92, 1650-1655. (25) Claesson, P. M.; Blom, C. E.; Herder, P. C.; Ninham, B. W. J. Colloid Interface Sci. 1986, 114, 234-242.
Forces between Adsorbed Layers on β-Casein
Figure 1. Normalized force measured between hydrophobized mica surfaces coated with an adsorbed β-casein layer in a solution containing 0.1 mg/mL β-casein in 1 mM NaCl (pH ) 7.0) (2, 4) and after dilution in 1 mM NaCl (b, O). Filled and unfilled symbols represent the force on compression and decompression, respectively. The force recorded when approaching the pure hydrophobic surfaces in 1 mM NaCl is also inserted (+). Note, that the surface separation is given relative the contact between the DDOAB covered surfaces. The thickness of the DDOAB layer was ≈20 Å. The arrows indicate jump due to instability of the spring. Part A shows the compression in detail and the solid line represents a DLVO fit, where the plane of charge and origin of the van der Waals forces are placed at the onset of the steric wall (surface separation 82 Å), the Debye length is 96 Å, and the surface potential is -48 mV. Part B shows the decompression curve, where a pull-off force of about 2.4 mN/m was observed.
thickness of 20 Å demonstrate that the Langmuir deposition was successful and the mica surface became hydrophobic. The long-range attractive forces observed between hydrophobic surfaces are believed to be nonequilibrium forces,23,24 but it is beyond the scope of this study to investigate the observed attractive force in detail. It should be noted that the origin of the long-range attractive forces between hydrophobic surfaces is a matter of controversy, but the occurrence of theses long-range forces has been related to instability of the deposited monolayers.26-29 The introduction of β-casein drastically changes the interaction to a long-range repulsive force, most probably of electrostatic origin (Figure 1A). As the surfaces are brought closer into contact, the repulsive force is at about 250 Å surface separation overcome by an attractive force which causes the protein-covered surfaces to slide into (26) Wood, J.; Sharma, R. Langmuir 1995, 11, 4797. (27) Yaminsky, V. V.; Ninham, B. V.; Christenson, H. K.; Pashley, R. M. Langmuir 1996, 12, 1936. (28) Yaminsky, V. V.; Nylander, T.; Ninham, B. V. Langmuir 1997, 13, 1746. (29) Eriksson, L. G. T.; Claesson, P. M.; Ohnishi, S.; Hato, M. Thin Solid Films, in press.
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contact. Further compression was not found to significantly change the surface separation, 82 Å, relative to the contact position between hydrophobized mica surfaces. The force curve recorded on separation (Figure 1B) shows that the contact is adhesive, with a measured pull-off force of about 2.4 mN/m. It should also be noted that the increase of surface separation by about 50 Å observed upon decompression before the surfaces jump apart indicates a considerable expansion of the β-casein layer. This suggests that the adsorbed layer is compressed, but no significant desorption occurs. A second approach of the surfaces gives the same force curve, indicating that the integrity of the adsorbed film is maintained. When the β-casein solution is replaced with pure 1 mM NaCl, almost the same force curve was observed. However, it is noteworthy that a slight decrease in the magnitude of the double-layer force was observed. This is probably associated with a decrease in the surface charge, which in turn is due to desorption of β-casein during the rinsing step. If the plane of charge and origin of the van der Waals forces are placed at the onset of the steric wall, that is at a surface separation of 82 Å, the repulsive part is consistent with what is expected by the DLVO theory at the given ionic strength. Thus, we observed a Debye length of 96 Å and a surface potential of -48 mV. Several studies have been devoted to the structure of adsorbed β-casein layers, among these light scattering measurements of adsorbed layer thickness on particles and emulsion droplets,10-13 where the results indicate that β-casein extends some 100 Å out from the interface. Considering the primary structure of the protein, one would expect that the molecule would orient in such a way that the hydrophobic moieties are anchored at the (hydrophobic) interface, with the hydrophilic parts pertruding into the solution. In fact small-angle X-ray scattering data, for layers adsorbed at the air/water interface, suggest that the bulk of the protein is confined within about 20 Å of the surface and that a segment of about 40 amino acid residues, which roughly corresponds to the hydrophilic part, penetrates out into the solution reaching about 100 Å out from the interface.13 The inner, more dense layer, is composed of the hydrophobic segments of the protein and the outer more hydrophilic layer has a more open and flexible structure as depicted in Figure 4A. This layered structure of the adsorbed β-casein film has been confirmed by neutron reflectivity measurements of β-casein adsorbed at the air/water,14,15 hexane/water,14 and solid/water16 interface. In the latter study, which is most relevant to ours, the adsorption of β-casein to a hydrophobic self-assembled monolayer, formed on a silicon surface from octadecyltrichlorosilane solution, was reported.16 The thickness of the inner layer was found to be 23 Å, which should be compared with an outer layer thickness of 35 Å. It is noteworthy that while the protein in the inner layer occupied 61% of the volume, the volume fraction of protein in the outer layer was only about 12%. This means that the hydrophilic segments can well interpenetrate when the surface are brought into contact. There might even be enough room for other macromolecules to enter in between the hydrophilic tails. In fact our earlier studies show that the hydrophilic part of β-casein is accessible for attack by endoproteinase Asp-N when the protein is adsorbed at a surface.17,18 On the basis of our force measurements and the studies cited above, we now can give a plausible picture of what is likely to happen when two β-casein layers, adsorbed on hydrophobic surfaces, approach each other (parts A and B of Figure 4). Initially an electrostatic double-layer force prevails (Figure 4A) until the surface separation reaches
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about 250 Å. At this separation the hydrophilic tails come in close contact and we obtained a marked deviation from the double-layer force, which indicates that the layer thickness is about 125 Å. The segment density of the hydrophilic tails is low enough to allow entanglement and bridging, which gives rise to an attractive force. Thus the surfaces slide into contact. Further compression is not possible as quite a compact layer is formed (Figure 4B). This layer is thicker than the values obtained for the protein inner layer from for instance neutron reflectivity measurements (23 Å). However, during the compression in the surface force measurement we also have to the take into account the space occupied by the compressed hydrophilic protein segments. When we subsequently tried to separate the surfaces, an attractive force was apparent, which, at least partially, depends on entanglement and/or bridging of the polypeptide chains. The β-casein layer thickness of 125 Å observed at the onset of the attractive force is larger than the values obtained by in particular neutron reflectivity14-16 and ellipsometry18 measurements, and it might therefore be rewarding to discuss the differences between methods used to measure layer thickness. Leermakers et al.30 used selfconsistent-field modeling to study the interfacial behavior of a β-casein look-alike at the solid/liquid interface. The total segment density profile φ(z), where z is the distance from the interface, was found to fall off in a featureless fashion from very high values (≈0.95) close to the interface to values approaching the “protein” bulk concentration for z > 50 Å. A very dilute tail region (φ(z) < 0.01), corresponding to the N-terminal hydrophilic sequence of ≈40 segments (amino acid residues), was found to extend out from z ) 30-70 Å to z ≈ 200 Å, depending on pH, ionic strength, and protein bulk concentration. The layer thicknesses obtained from these calculations are closer to the values obtained in the present study and light scattering measurements10-13 than the significantly thinner layers determined by the neutron reflectivity14-16 and ellipsometry.18 The reason for this has to do with how well the technique can detect a layer with low segment density.30 The local volume fraction of protein in the periphery of the adsorbed layer is probably too low to be detected by neutron reflectivity or ellipsometry measurements. Furthermore, with ellipsometry it is usually not possible to get enough data to determine a segment density profile. Hence, a homogeneous layer has to be assumed, which gives an average layer thickness. This can be substantially smaller than the actual physical thickness, depending on the actual density profile. Both the model study30 and the reflectivity measurements15 clearly demonstrate that electrostatic interactions are significant in determining the adsorbed layer thickness. Thus, a thicker layer is observed when the protein net charge is reduced, that is at pH closer to the isoelectric pH or when increasing the ionic strength. A more closely packed monolayer, where the polypeptide chain is more stretched out, or the formation of a second layer on top of the inner layer can both account for this increase in thickness.15 The calculations, using the self-consistent field model, give a potential about -45 mV for z ≈ 45 Å, which is comparable with the surface potential of -48 mV estimated from the surface force measurements, where the plane of charge is set to the onset of the steric wall (layer thickness of 41 Å). To our knowledge, the forces between adsorbed layers of β-casein have not been studied previously. However, in a similar study the interaction between layers of proteoheparan sulfate, also a protein with strong am(30) Leermakers, F. A. M.; Atkinson, P. J.; Dickinson, E.; Horne, D. S. J. Colloid Interface Sci. 1996, 178, 681-693.
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phiphilic character, adsorbed on hydrophobic surfaces was studied by Malmsten et al.31 This molecule consists of a protein part, which in nature interacts with endothelial cell membranes, and a glycosaminoglycan chain, which protrudes into the extracellular solution. The long range repulsive force between the adsorbed layers was found to have about the same decay length as expected from a double layer force. However, if a divalent ion, Ca2+, was added, the measured decay length is no longer the one expected from a double layer force, which suggests that steric forces contribute significantly under these conditions. In the present study an inward jump was observed when the contact between the protein-covered surfaces is approached. This was not observed for the interaction between proteoheparan sulfate layers, where instead a corresponding plateau, with a less repulsive force, is evident in the force curves. Thus proteoheparan sulfate is likely to give a denser (outer) layer than β-casein on the hydrophobic surface. Similar to the interaction between β-casein layers, an expansion of the adsorbed proteoheparan sulfate layer was observed upon decompression, just before the surfaces jump apart. Interaction between β-Casein Layers on Pure Hydrophilic Mica Surfaces. Clearly, a β-casein molecule adsorbed to a hydrophobic surface is highly oriented, with its hydrophilic part penetrating into the solution. Thus the surface properties are bound to affect the structure of the interfacial layer. It was therefore natural to extend our study to include force measurements of β-casein layers on hydrophilic surfaces. The force curve recorded between the pure surfaces in the presence of 1 mM sodium chloride (Figure 2A) can be fitted to the DLVO theory, which, in agreement with previous studies,32,33 gives a decay length of 96 Å and a surface potential of -125 mV. As expected an inward jump occurs due to van der Waals attractive forces, and an adhesion is evident when the surfaces are separated. The effect of adding 0.1 mg/mL of β-casein is also shown in Figure 2A, where the force curves recorded after 2 and 17 h are given. Beyond 17 h no changes in the surface force versus distance curves were observed. The interaction between the surfaces changes completely after the addition of β-casein, and the force is now entirely repulsive, that is, the same force was observed upon decompression as on compression (data not shown). The force curves are plotted in a logarithmic scale in Figure 2B, and the ones recorded in the presence of β-casein parallel the force recorded in pure sodium chloride solution, except at small surface separations. This indicates that double-layer forces strongly contribute to the repulsive force between β-casein layers on hydrophilic surfaces. The force curves are of course shifted outward as the adsorbed layer is built up. At surface separations close to 300 Å, a slightly lower force than expected for a pure double layer force is observed (Figure 2B). Compared to the interaction between β-casein adsorbed to hydrophobic surfaces the electrostatic double layer force is much stronger and the separation at contact is more distant. This shows that much more of β-casein is adsorbed on the pure mica than on the hydrophobic surface. The same trend was observed when comparing the amounts of β-casein adsorbed on hydrophobized with those on pure, hydrophilic silica surfaces.18 Figure 3A demonstrates that the repulsive force is reduced by order of magnitude when the β-casein solution is replaced with protein-free 1 mM sodium chloride solution. This is probably due to protein (31) Malmsten, M.; Claesson, P.; Siegel, G. Langmuir 1994, 10, 12741280. (32) Pashley, R. M. J. Colloid Interface Sci. 1981, 83, 531-546. (33) Shubin, V. E.; Ke´kicheff, P. J. Colloid Interface Sci. 1993, 155, 108-123.
Forces between Adsorbed Layers on β-Casein
Figure 2. Force between two pure hydrophilic mica surfaces as a function of surface separation in the presence of 1 mM NaCl (×) and 0.1 mg/mL β-casein in 1 mM NaCl (pH ) 7.0) after 2 h ([) and 17 h (2, 9) equilibration time. Two consecutive surface force runs were performed after 17 h: (2) and (9), respectively. Note, that the curves recorded on decompression in the presence of β-casein coincide with those recorded on compression (data not shown). The solid line shows a DLVOfit, where the upper curve and lower curves are calculated assuming constant surface potential and surface charge, respectively. A surface potential of -125 mV and a Debye length of 96 Å were used. Part A shows the force curve in a linear plot and (B) the same data in logarithmic scale.
desorption during the rinsing step. After desorption the long range force can be fitted to the DLVO theory provided that the onset of the force is shifted 130 Å outward relative the mica-mica contact (Figure 3B). However, the magnitude of the repulsive force is substantially lower than that observed for the interaction between the pure mica surfaces, and consequently the calculated surface potential is much lower (-64 mV compared to -125 mV). One might not expect that a protein with net negative charge, such as β-casein, adsorbs to a negatively charged mica surface and reduces the apparent surface charge. It should, however, be borne in mind that even if β-casein has a net negative charge, it still carries about 19 positive charges per molecule at neutral pH. This means that the charge repulsion can be reduced by proper orientation of the protein at the interface. Furthermore, the dissociation of ionizable groups on the protein might be less favorable in the proximity of the mica-aqueous interface. This can partly be due to the adsorption of the protein which results in a lowering of the dielectric constant and thus will affect the charging at the mica surface. Comparison between the β-Casein Layers on the Hydrophilic and Hydrophobic Surfaces. As discussed above, a drastic change in the force versus distance profile is observed for the interaction between β-casein layers adsorbed on hydrophilic surface, when the β-casein solution is replaced with pure buffer. Not only is the
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Figure 3. (A) The normalized force measured between pure hydrophilic mica surfaces coated with an adsorbed β-casein layer in a solution containing 0.1 mg/mL β-casein and 1 mM NaCl (pH ) 7.0) (9) and after dilution in 1 mM NaCl ([, ]). The unfilled symbol (]) indicates the curve recorded during decompression in 1 mM NaCl. (B) The forces between β-casein layers adsorbed onto hydrophilic ([, ]) and hydrophobized (b, O) mica after the protein solution has been removed and replaced with pure 1 mM NaCl. The unfilled symbols indicate the curves recorded during decompression. To facilitate comparison, the force curve for β-casein on hydrophobized has been shifted 47 Å outward. The dashed and solid line is the calculated DLVO curve for constant surface charge and potential, respectively. The parameters used for the calculations are a surface potential of -64 mV and a Debye length of 96 Å and the onset of the forces is located at the steric wall 130 Å from the mica-mica contact.
magnitude of the double-layer force reduced, but the interaction is no longer entirely repulsive as an attractive force is apparent at surface separations below about 300 Å (Figure 3A). This causes the surfaces to spontaneously move toward each other until they come to the steric force wall, located about 130 Å out from the mica-mica contact (Figure 3B). This value is not very much different from the one observed before replacing the β-casein solution with 1 mM sodium chloride, which is notable when considering the large drop in the repulsive force. Most remarkable, however, is the fact that the force curve after rinsing very much resembles the one recorded between β-casein layers on hydrophobized mica although the steric wall seems to be located some 47 Å out. In fact, if we shift the force curve recorded for β-casein on hydrophobized mica 47 Å out and place it on the same graph as the curve obtained for β-casein on pure mica after rinse, they almost coincide (Figure 3B). The resulting β-casein layers exposed to the aqueous solution apparently look similar on the two types of surfaces. It is therefore tempting to consider formation of a β-casein bilayer on the hydrophilic surface as depicted in parts C and D of Figure 4. This has been suggested to occur on hydrophobic surfaces15 at low pH and high salt and protein concentration as discussed above. The protein in the layer closest to the hydrophilic
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Figure 4. Schematic figure, which shows the plausible structures of the adsorbed layers of β-casein on hydrophobized mica (A, B) and pure hydrophilic mica (C, D). The structure at large separations, where electrostatic repulsive forces dominate (A, C), and at small surface separations, where steric interactions dominate (B, D), are shown. On hydrophobic surfaces a monolayer is assumed, with the hydrophilic chain (shown in gray) protruding into the solution and the hydrophobic chain (black) anchored at the surface) (A, B). A bilayer is assumed on the hydrophilic surface, where the inner layer is more dense and makes the surface more hydrophobic, which leads to the adsorption of a second layer with basically the same structure as the protein monolayer on the hydrophobic surface (C, D).
mica surface is suggested to adsorb in a more or less flat conformation, reducing the charge density of the mica. This will give a more hydrophobic surface to which β-casein molecules can adsorb in basically the same conformation as on a pure hydrophobic surface (Figure 4C). The steric force observed on compressing a β-casein layer on the hydrophilic surface in the presence 1 mM NaCl is similar to the corresponding layer on the hydrophobic surface. Thus, the outer layer is likely to have a similar structure to the monolayer on the hydrophobic surface also if a high compression force is applied, as indicated in parts D and B of Figure 4, respectively. This is also confirmed by the presence of a similar adhesive force upon decompression (Figure 3A). The outer layer on the hydrophilic surface is likely to be less firmly anchored at the interface, and parts of it are desorbed when the surrounding β-casein solution is replaced with 1 mM sodium chloride solution. However, the outer layer could not be removed by applying a high compressive force. The results from the present study are largely parallel to the ones found in our adsorption studies using ellipsometry.17,18 As ellipsometry cannot give much specific information about the structure of the adsorbed layer, but merely physical parameters like the layer thickness, refractive index, and amount adsorbed, we used a specific proteolytic enzyme endoproteinase Asp-N in those studies. This enzyme can cleave at four different sites on β-casein. Two are located in the more hydrophilic part (residues 43 and 47) and two in the hydrophobic part (residues 129 and 184). By following the enzyme action, by means of measuring changes of the adsorbed layer, we were able to get information about the accessibility of the cleavage
Nylander and Wahlgren
sites and, hence, the orientation of β-casein at the two types of interface. Also these studies support the idea that the adsorbed layer of β-casein on a hydrophobic surface can be described as a monolayer with an inner dense region comprising the relatively large hydrophobic portions of the protein molecules and an outer region of the highly charged N-terminal portions pertruding into the aqueous phase. However, as pointed out by Leaver and Dalgleish the type of hydrophobic surface matters.11 They found, by studying the products of trypsin hydrolyses of β-casein adsorbed to oil droplets, that the structure of the layer was different if the oil was pure tetradecane or if a triglyceride (soya) oil was used. As in the present study, where mica is used as the hydrophilic surface, the ellipsometry study showed a larger and more reversible (larger desorption upon rinsing with pure buffer) adsorption on hydrophilic silica surface compared to the adsorption on hydrophobized silica.18 Also, the reduction in surface excess of β-casein upon addition of endoproteinase Asp-N confirmed the difference in structure of the adsorbed layer between the two types of silica surfaces. Besides the bilayer structure, the adsorption of β-casein aggregates at a hydrophilic surface is also feasible. As discussed above β-casein forms aggregates, “micelles” with a cmc of about 0.5 mg/mL.7 Even if we in the present work and in the earlier ellipsometry study18 use a lower protein concentration, it is quite possible that aggregates can form in the proximity of the interface. Contrary to the present study, the thickness of the β-casein layer adsorbed on the hydrophilic silica, obtained from the ellipsometry measurements, was the same or less than the one on the hydrophobized silica. From those measurements we could not conclude whether the protein adsorbed as a bilayer on the hydrophilic silica surface or not. It should be noted that although both mica and silica carry a negative charge in the pH range studied, their interfacial structures are quite different. For silica, the surface is charged by ionization of specific hydroxyl groups, whereas the negative charge of mica comes from dissociation and exchange of potassium ions in the mica lattice with the solution.34,35 Furthermore, as opposed to the ellipsometry study, the surface force measurements were carried out without stirring of the solution during the measurements. However, the most likely reason, which was discussed above, is that ellipsometry and surface force measurements give different types of thickness. It is thus possible that the average thickness of the adsorbed layer on the hydrophilic surface, determined by ellipsometry, corresponds to much larger “real” thickness if the proper segment density profile would have been taken into account. Conclusions In this study we have demonstrated that the interacting forces between adsorbed layers of β-casein layers are both electrostatic and steric in nature. Basically the same type and magnitude of forces exist between these layers in pure salt solution independent of whether β-casein was adsorbed on a pure hydrophilic or on a hydrophobized mica surface. This implies that β-casein might be used not only to stabilize hydrophobic “particles” like oil droplets but also to have a large potential for stabilizing particle suspensions which are hydrophilic in nature. In both cases the contribution of electrostatic forces are important, which implies that for real colloidal systems the type of added electrolyte can have considerable impact on the stability of the dispersion or emulsion. Furthermore, we (34) Gaines, G. L.; Tabor, D. Nature 1956, 178, 1304. (35) Gaines, G. L. J. Phys. Chem. 1957, 61, 1408.
Forces between Adsorbed Layers on β-Casein
have demonstrated that surface force measurements can give important information about not only the nature and magnitude of the stabilizing forces, but they can also provide insight into the structure of the adsorbed layer of the dispersing agent, in this case β-casein. The results from this study are relevant to explain the role of β-casein in its natural environment, the casein micelle, as well as the formation of monodisperse β-casein aggregates as described in the introduction. The force measurements show that electrostatic repulsive forces between the hydrophilic domains of the protein are important for orientation of the molecule on a hydrophobic surface. In parallel to this the hydrophobic domain of the molecule may act as a hydrophobic interface when β-casein aggregates are formed. This is confirmed by the behavior at the hydrophilic mica where a bilayer was formed and the inner β-casein layer creates a hydrophobic surface to which the outer layer is adsorbed. It has been suggested that the calcium phosphate nanoclusters of casein micelles are stabilized by interaction with the phosphorylated
Langmuir, Vol. 13, No. 23, 1997 6225
sequences of the calcium sensitive caseins.3 Our present results further suggest that part of this interaction could be a β-casein bilayer type interaction which effectively cross-links the calcium phosphate nanoclusters through an interaction between the hydrophobic parts of two or more β-casein molecules. This suggestion for cross-linking is in addition to the possibility for cross-linking provided by the Rs1- and Rs2-caseins, which unlike β-caseins, contains two or more sequences rich in phosphorylated residues and hence could directly link the nanoclusters through a single peptide chain.
Acknowledgment. We are indebted to Dr. Per Claesson, Dr. Hugo Christenson, Dr Carl Holt, and Professor Frank Blum for fruitful discussions. The financial support from the Swedish Council for Forestry and Agricultural Research is acknowledged. LA970503R
