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Orogenic Displacement of Protein from the Oil/Water Interface Alan R. Mackie,* A. Patrick Gunning, Peter J. Wilde, and Victor J. Morris Department of Food Biophysics, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, NR4 7UA, UK Received June 4, 1999 Orogenic displacement has been shown to be a mechanism by which protein can be removed from an interface by small surfactant molecules. This paper describes the progressive displacement of two different proteins from an oil/water interface by a nonionic surfactant. The process has been visualized by atomic force microscopy (AFM). Measurement of surface tension and AFM imaging of Langmuir-Blodgett (LB) films formed on mica are used to demonstrate the mechanism of protein desorption from the interface. This paper extends previous work which demonstrated a new orogenic mechanism of protein displacement from an air/water interface. The two proteins used in the present study were β-casein, a largely random coil protein, and β-lactoglobulin, a globular protein. The proteins were displaced from both spread and coadsorbed films using the water-soluble nonionic surfactant Tween 20. The AFM images also provide direct evidence for the formation of a heterogeneous protein layer at the interface. The heterogeneity of the protein film is important in allowing the initial adsorption of the surfactant onto the interface. These nucleated surfactant sites then expand, compressing the protein network, which initially increases in density without increasing in thickness. Once a certain critical density is reached, further compression of the protein layer results in the thickness increasing in order that protein film volume is maintained constant as the surfactant domains expand. At sufficiently high surface pressures, the network fails, releasing proteins which then desorb from the interface.
Introduction Proteins are an important stabilizer of emulsions and foams, particularly in the food industry. It has been known for some time that small molecular weight surfactants, emulsifiers, and lipids destabilize protein emulsions and foams by competing for interfacial area with the proteins. Until recently the exact mechanism of protein displacement by surfactant was still unclear, although various measurements on the displacement of proteins by nonionic surfactants have shown some interesting features, which have helped. Horne and co-workers1 used neutron reflectance to show that at the point of displacement from the air/water interface by nonionic surfactant the thickness of the mixed surfactant protein layer increased. Similarly Miller et al.2 showed by ellipsometry that for both β-casein and β-lactoglobulin the adsorbed layer thickness increased at the point of displacement by nonionic surfactant. Competitive adsorption has also been studied at the oil/water interface in emulsified systems.3-5 Specifically, a study on the displacement of β-lactoglobulin by Tween 206 showed that the interfacial rheology was being altered at much lower bulk surfactant/protein ratios than any displacement of the protein. The coalescence stability of emulsions was also shown to be severely * To whom correspondence should be addressed. Tel: 44 1603 255261. Fax: 44 1603 507723. E-mail:
[email protected]. (1) Horne, D. S.; Atkinson, P. J.; Dickinson, E.; Pinfield, V. J.; Richardson, R. M. Int. Dairy J. 1998, 8, 73. (2) Miller, R.; Fainerman, V. B.; Makievski, A. V.; Grigoriev, D. O.; Wilde, P. J.; Kragel, J. In Food Emulsions and Foams. Interfaces, Interactions and Stability; Dickinson, E., Rodriguez Patino, J. M., Eds., 1999; p 207. (3) Courthaudon, J.-L.; Dickinson, E.; Dalgleish, D. G. J. Colloid Interface Sci. 1991, 145, 390. (4) Chen, J.; Dickinson E. J. Sci. Food Agric. 1993, 62, 283. (5) Dickinson, E.; Ritzoulis, C.; Povey, M. J. W. J. Colloid Interface Sci. 1999, 212, 466. (6) Courthaudon, J.-L.; Dickinson, E.; Matsumura, Y. Colloids Surf. 1991, 56, 293.
affected at low surfactant/protein ratios,7 which was linked with interfacial rheological behavior. Similar effects have been seen in the rheological behavior of emulsions stabilized by caseins.8 It is clear, therefore, that surfactants are capable of disrupting an adsorbed protein layer, prior to displacing any protein. However, a definitive explanation of these observations has yet to be provided. Atomic force microscopy (AFM) has been extensively used to look at various biological molecules,9 including Langmuir-Blodgett films.10 However, these studies have tended to look at the adsorption of protein alone or protein lipid mixtures.11,12 This has meant that measurements have either only been made in dilute regimes, where protein interactions are not significant, or on more packed layers, where images become unclear and height measurements are less reliable. This is the third in a series of papers looking at the precise mechanism by which proteins are competitively displaced from interfaces by surfactants. Previously, we have looked at protein displacement from an air/water interface13 and from a solid/liquid interface.14 It has been found that the displacement was a three-stage mechanism, which has been called an orogenic displacement process. In the first stage the surfactant adsorbs into small defects in the protein film, forming small pools of surfactant, which increases the surface pressure of the film. As surfactant (7) Dickinson, E.; Owusu, R. K.; Williams, A. J. Chem. Soc., Faraday Trans. 1993, 89, 865. (8) Kragel, J.; Wustneck, R.; Husband, F.; Wilde, P. J.; Makievski, A. V.; Grigoriev, D. O.; Li, J. B. Colloids Surf. B-Biointerfaces, 1999, 12, 399. (9) You, H.-X.; Lowe, C. R. Curr. Opin. Biotechnol. 1996, 7, 78 1996. (10) Peachey, N. M.; Eckhardt, C. J. Micron, 1994, 25, 271. (11) Mori, O.; Imae, T., Colloids Surf. B, 1997, 9, 31. (12) Fare, T. L.; Palmer, C. A.; Silvestre, C. G.; Cribbs, D. H.; Turner, D. C.; Brandow, S. L.; Gaber, B. P. Langmuir 1992, 8, 3116. (13) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris V. J. J. Colloid Interface Sci., 1999, 210, 157. (14) Gunning, A. P.; Mackie, A. R.; Wilde, P. J.; Morris, V. J. Langmuir In press.
10.1021/la990711e CCC: $19.00 © 2000 American Chemical Society Published on Web 12/30/1999
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adsorption continues, the surfactant domains grow, but the thickness of the protein layer is unaffected. This is the compression phase. In stage two, the surfactant domains continue to grow, but the decrease in area of the protein film is compensated for by a corresponding increase in film thickness, and thus, the volume of adsorbed protein remains constant. This is the collapse phase. Finally, in stage three, when the surface pressure is sufficiently high, the protein network breaks down, allowing a continuous surfactant phase to be formed, and the protein is totally displaced from the interface, probably in the form of aggregates. This work extends the notion of orogenic displacement to the oil/water interface and looks at the importance of the structure of the initial, adsorbed protein layer. In this study spread and coadsorbed films of the individual proteins β-casein and β-lactoglobulin formed at an oil/water interface were displaced using the surfactant Tween 20. The adsorption of the protein was monitored by surface tension measurements. The protein films were imaged by atomic force microscopy (AFM) using Langmuir-Blodgett (LB) methods to transfer films from the oil/water interface onto mica. Both surface tension and AFM methods have then been used to study the effect of added Tween 20 on the protein films. This was done in order to test the general applicability of the orogenic displacement process to the removal of proteins from interfaces.
Langmuir, Vol. 16, No. 5, 2000 2243 were made as necessary to gradually increase the surface pressure. The surface tension was monitored throughout the period of the experiment. In the case of both the spread and adsorbed experiments this was between 3 and 5 h. LB films were produced by lowering a freshly cleaved piece of mica sheet down through the interface and then pulling it back out again. The mounted piece of mica was driven at a constant rate of 8.4 mm min-1. Surface tension was monitored during the during the dip and showed that the film only transferred to the mica on the upward stroke. The sample was then transferred to the AFM and imaged under butanol. Subsequently both the residual n-tetradecane and the adsorbed Tween 20 dissolved into the butanol in the imaging cell, which accounts for the good contrast in the images and also permitted accurate measurement of protein layer thickness. The precise details of the AFM and the imaging technique are given elsewhere.15 It is however worth briefly describing the methods used to calculate the protein film thickness and area. The protein area was calculated using thresholding to make a binary image which could be used to sum the active pixels. The active pixel sum was then converted to a percentage of the overall number of pixels in the image. This value was the percentage surface area covered by protein. All the AFM images used 256 gray levels to represent heights between 0 and a maximum thickness (typically tens of nanometers). The protein layer thickness was measured using a histogram of the gray levels within an image. Because of the nature of the experiment, the histograms were generally bimodal, with a lower peak representing the mica and an upper peak representing the adsorbed protein. The mean protein film thickness in an image was taken as the difference in gray levels between the two peaks of the histogram.
Materials and Methods
Results
The milk proteins used in this study were β-casein (C-6905, Lot 12H9550) and β-lactoglobulin (L-0130, Lot 91H7005) from Sigma Chemicals (Poole, UK). Samples were initially prepared at 2 mg mL-1 in phosphate-buffered saline, PBS (Sigma Chemicals), at pH 7.0. The water used in this study was surface pure (γ0 ) 72.6 mN m-1 at 20 °C), cleaned using an Elga Elgastat UHQ water purification system, and the oil was n-tetradecane (Sigma Chemicals). The Tween 20 (polyoxyethylene sorbitan monolaurate) was obtained as a 10% solution (Surfact-Amps 20) from Pierce (Rockford, IL). Surface tension measurements were made using a platinum “Du Nouy” ring and a glass trough. The area of the trough was 26 300 mm2. All experiments were performed at room temperature (22 °C) with distilled water as the subphase. Each protein was applied to the surface by either spreading or adsorption. The two proteins used in the study, β-casein and β-lactoglobulin, were spread by carefully adding the required volume of stock solution just above the oil/water interface. Surface tension measurements were made as a function of time for 30 min while the monolayer equilibrated. After this period of equilibration, Tween 20 was injected into the subphase to bring the concentration up to the required level. Care was taken that none of the surfactant was allowed to spread at the interface. This was achieved by adding all the material to the subphase via a glass tube, which was permanently dipped through the oil phase into the subphase. Langmuir-Blodgett (LB) films were then formed at periodic intervals on a hydrophilic freshly cleaved mica substrate. Further additions of Tween 20 to the subphase were made as necessary in order to reach the required surface pressure for data collection. The adsorbed films were produced from solutions containing 1 µM protein and 0.5-1.0 µM Tween 20, which were poured directly into the trough and then covered with 175 mL of n-tetradecane. The sample was allowed to adsorb for 30 min before the subphase was perfused with 2 L of the relevant solution of Tween 20. The perfusion was necessary because of the problem of protein adsorbing directly onto the mica from bulk solution when LB films were transferred. The irreversible nature of protein adsorption to the interface meant that, provided the Tween 20 was kept in equilibrium, the interfacial protein film was maintained despite removal of protein from the bulk phase.13 Once the majority of the protein had been removed from the subphase, LB films were drawn at periodic intervals. Again further additions of Tween 20 to the subphase
The surface tension of the n-tetradecane/water interface was found to be 51 mN m-1. The addition of 66 µg of β-lactoglobulin to the interface reduced the surface tension after 30 min to 35.4 mN m-1, a surface pressure of 15.6 mN m-1. The addition of 36 µg of β-casein to the interface reduced the surface tension to 35.2 mN m-1. These two baseline levels of protein were chosen because they give similar surface pressures after a period of 30 min. After the spread proteins had equilibrated for 30 min, the subphase was brought up to a Tween 20 concentration of 0.2 µM and LB films were transferred onto mica for imaging. Further additions of Tween 20 were made as necessary to increase the surface pressure. Measurements were also made on films coadsorbed from solutions of 1 µM protein and 0.5 µM Tween 20, which were left to adsorb for 30 min before the subphase was perfused as described in the methods section. Again further Tween 20 was added to the subphase as necessary. The first step in any displacement experiment is the formation of the primary (in this case protein) monolayer. Figure 1a shows a high-resolution AFM image of a spread β-lactoglobulin monolayer transferred from the air/water interface at a surface pressure of 12.6 mN m-1. The mean area occupied by the protein molecules was 10.8 nm2. At the scan size shown (100 nm × 100 nm) there should be approximately 930 molecules visible in the image. The mean measured thickness of the layer shown was 0.5 nm, whereas a monolayer thickness should be about 1 nm. This suggests that the dark areas of the image do not represent the AFM tip interacting with the mica surface. This is to be expected because of the problem of convolution of the image by the tip geometry. Convolution (probe broadening) is caused by the fact that the sharpened levers used in this experiment have a radius of curvature of 1020 nm. Clearly, a tip of these dimensions cannot penetrate very far into a defect that is laterally only some 3 nm in (15) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Morris, V. J. Biopolymers 1998, 46, 245.
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Figure 1. AFM image of LB films of β-lactoglobulin (a) transferred at a surface pressure of 12.6 mN m-1 (image is 100 nm × 100 nm) and (b) transferred at a surface pressure of 20 mN m-1 (image is 150 nm × 150 nm).
size. From previous work, we have shown that at the surface pressure at which the protein layer was transferred one would expect a real thickness of about 1 nm. Thus, it seems plausible that defects, with a measured depth of some 0.5 nm from the mean, represent holes in the protein layer. The image in Figure 1a shows the local density fluctuations, which were readily apparent in nearly all the protein films studied on this length scale. Analysis of this and other similar images shows that the defects are no bigger than single protein molecules, although there is some evidence of line defects. The heterogeneity may well be the factor that initially drives the phase separation of protein and surfactant, the small areas of low protein density allowing the surfactant to adsorb to the interface. It seems likely that the surfactant is relatively free to adsorb into these regions of low density until the local surface tension is reduced to that of the surrounding protein layer. Previous studies have shown that there are differences between spread and adsorbed protein layers both in terms of displacement13 and surface rheology.16 For this reason, we have also looked at an adsorbed protein (16) Kragel, J.; Grigoriev, D. O.; Makievski, A. V.; Miller, R.; Fainerman, V. B.; Wilde, P. J.; Wustneck, R. Colloids Surf. B, 1999, 12, 391.
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layer. Figure 1b shows a β-lactoglobulin monolayer adsorbed from a 0.5 µM solution. The monolayer was transferred onto mica from the air/water interface at a surface pressure of 20 mN m-1. The figure again shows the heterogeneity present in Figure 1a, but the mean spread of heights is less. This apparent decrease in heterogeneity is consistent with the idea that defects are removed from the film as the surface pressure increases. After the introduction of surfactant into the subphase, displacement can begin. Figure 2 shows three images of the progressive displacement of β-casein from the oil/water interface taken over a range of surface pressures from 25 to 31 mN m-1. The first of these images (Figure 2a) shows the displacement of protein from a film spread at the oil/ water interface. The monolayer was transferred onto mica at a surface pressure of 25.5 mN m-1. At this early stage, protein still covered 84% of the interface. The holes made in the protein film by the competing surfactant were initially small. As they grew, they became diverse in both size and shape. These holes gradually increased in size as the surface pressure was increased by the adsorbing surfactant. As the surface pressure rose above 30 mN m-1, the situation reached that shown in Figure 2b. This image is of a spread film, transferred at a surface pressure of 30.5 mN m-1, which shows protein that occupied an area of 23%. In this image, although the surfactant pools are very asymmetric in shape, they still have a slightly rounded look to them. The third image in the series (Figure 2c) is of a coadsorbed film transferred at a surface pressure of 28.7 mN m-1. This shows that the protein occupied a similar area to the previous image, namely 32%, indicating that there was little difference between the structures of the spread and coadsorbed films. The other protein used in this study, β-lactoglobulin, shows slightly different behavior. Figure 3 shows three images of the displacement of β-lactoglobulin. The first image (Figure 3a) is for a spread film transferred at a surface pressure of 21.6 mN m-1. This image shows a protein coverage of 80%, which is similar to the stage of displacement shown in Figure 2a. At this early stage in the displacement, the holes in the protein monolayer made by the surfactant look similar for both proteins. For comparison Figure 3b shows a coadsorbed film transferred at a surface pressure of 32 mN m-1 in which the protein occupies an area of 79%. Again the displacement patterns for the spread and adsorbed films look similar to each other but different than those for β-casein. The β-casein domains appear smoother and more rounded than those for β-lactoglobulin. The third image (Figure 3c) shows a coadsorbed film in the last stages of displacement with less than 16% of the surface occupied by protein. This film was transferred at a surface pressure of 33.3 mN m-1 and shows the tenuous “lacelike” structures typical of the later stages of β-lactoglobulin displacement. As in previous work on protein displacement from the air/water interface,13 all the AFM images have been analyzed in order to determine protein surface coverage (percent area) and where possible the film thickness (nanometers). Figure 4 shows the displacement curves for the two proteins in terms of the surface area occupied (%) as a function of surface pressure. The first thing to note is that the curves for the β-casein films formed by both spreading and adsorption are essentially the same. The onset in displacement occurs at about 25 mN m-1, and displacement continues until a surface pressure of about 32 mN m-1, at which point virtually all the protein has been removed from the surface. This is in contrast to the β-lactoglobulin, where the coadsorbed and the spread films were displaced at very different surface pressures.
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Figure 2. AFM images of LB films of β-casein drawn at different surface pressures (a) 1.6 µm × 1.6 µm, Π ) 22.5 mN m-1, spread; (b) 6.4 µm × 6.4 µm, Π ) 30.5 mN m-1, spread; and (c) 6.4 µm × 6.4 µm, Π ) 28.7 mN m-1, adsorbed.
Figure 3. AFM images of LB films of β-lactoglobulin drawn at different surface pressures (a) 1.0 µm × 1.0 µm, Π ) 21.7 mN m-1, spread; (b) 3.2 µm × 3.2 µm, Π ) 32.0 mN m-1, adsorbed; and (c) 6.0 µm × 6.0 µm, Π ) 33.3 mN m-1, adsorbed.
The spread film was displaced at 22 mN m-1 and the coadsorbed film displaced at 33 mN m-1, corresponding to a difference in surface pressure of 11 mN m-1. In addition, both β-lactoglobulin films were displaced over
a much narrower range of surface pressures; i.e., the change in protein area from 100% to 0% occurs in a surface pressure increment of 1 mN m-1. The two proteins were also different in terms of the variation in film thickness
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Figure 4. The displacement of protein by Tween 20 is shown in terms of the proportion of the surface area occupied by the protein as a function of surface pressure. The films were formed either by adsorption of β-casein (curve 1) or β-lactoglobulin (curve 2) or by spreading of β-casein (curve 3) or β-lactoglobulin (curve 4).
Figure 5. The thickness of the films formed by the different proteins as measured by AFM is shown. The square symbols is for β-casein and the crosses for β-lactoglobulin.
as a function of surface pressure, shown in Figure 5. It is clear from the figure that the β-casein film became thicker as it was compressed into a smaller area by the adsorbing surfactant. In fact, the thickness increased quite dramatically from 0.7 nm at a surface pressure of 25 mN m-1 to 3.8 nm at 32 mN m-1. This would be equivalent to a layer some five molecules thick at the point where the protein was finally displaced from the interface if it had the original density. However, it is known that β-casein forms a “hairy” adsorbed layer17 the diffuse outer part of which extends some 10 nm. It is possible that as the protein layer is compressed, the diffuse region starts to be detected by the AFM tip. Thus the increase in layer thickness may be due, at least in part, to molecular deformation rather than rearrangement. In contrast to this, in the case of β-lactoglobulin it has not been possible to observe a change in the layer thickness as a function of surface pressure, (17) Mackie, A. R.; Mingins, J.; North, A. N. J. Chem. Soc., Faraday Trans. 1991, 87 (18), 3043.
particularly in the case of the adsorbed film. However, the thickness values for the adsorbed β-lactoglobulin at the oil/water interface suggest a protein layer several molecules thick when compared to data from the air/water interface. Thus at surface pressures of 32 mN m-1 the layer has already started to collapse. Discussion The precursor to any displacement is the adsorption of surfactant to the interface through the protein layer. In a protein film the large number of interactions means that individual protein molecules do not self-diffuse, although they will flow if sufficient external force is applied. Because of this, the film is not homogeneous but varies in surface density. This fact is clearly demonstrated in Figure 1. The location of surfactant adsorption sites is largely dictated by the heterogeneity of the protein film. These weak points allow the surfactant to penetrate the protein film and adsorb to the interface, which in turn
Orogenic Displacement of Protein
raises the local surface pressure. As more surfactant adsorbs at these nucleation points, the local surface pressure rises and the surrounding protein film is compressed, particularly at weaker points in the network. The AFM images of protein displacement (Figures 2 and 3) show similar patterns to those observed at the air/water interface,13 suggesting that the same orogenic mechanism is responsible. The main difference is that the β-casein displacement domains are not circular, as seen at the air/water interface. This difference seems to indicate that the β-casein network was stronger at the oil/water interface. Further evidence for this is the higher surface pressure that was required to displace the β-casein from the oil-water interface. This may arise from two possible causes. First, the β-casein may have a stronger affinity for the oil/water interface than the air/water interface; this may well be the case purely on the grounds of the increase in density of the upper phase. Alternatively, the protein network may be stronger and thus more difficult to break up. The increase in the β-casein layer thickness as the protein is displaced from the oil/water interface suggests that the latter is the dominant factor. However, there is not sufficient evidence to provide a molecular interpretation for this. The results shown here match the main features of orogenic displacement. First, there is nucleation of the surfactant domains at the weak points in the protein film. If this displacement takes place at relatively low surface pressures, then this may also be a compressional phase in which the area occupied by the protein decreases but the thickness of the layer remains constant. However, if it takes place at higher surface pressures, greater than the protein monolayer collapse point, then the protein layer will thicken as it is compressed. From Figures 4 and 5 it is clear that β-casein was in a compressional phase up to a surface pressure of 25.5 mN m-1. The results for the β-lactoglobulin are much less clearly defined in that the curves for the spread and adsorbed represent essentially two different layer thicknesses. The reason for this may be that the range of pressure over which the respective films were displaced was so small (∼1 mN m-1) that the thickness did not change. It may also indicate that once past the initial compression phase, the protein layer thickness was simply a function of surface pressure. The second and most important stage in orogenic displacement is the growth of the surfactant domains, causing the protein film to increase in thickness. Generally the area occupied by the protein decreases and the protein layer volume remains approximately constant. In the case of the β-casein films, this does appear to be what happened.
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As the area covered by protein decreased, there was a concomitant increase in protein layer thickness. However, in the case of β-lactoglobulin there was essentially no change in thickness as the adsorbed protein layer was displaced. The layer thickness results for the β-lactoglobulin films suggest that at all the surface coverages measured for the coadsorbed films the protein layer was already at least two molecules thick. This must mean that the protein film was already in an advanced state of collapse as the first holes appeared. Thus the film was probably past the region where the volume of the film remains constant. The final stage of orogenic displacement is the breakup of the protein network and the subsequent desorption of the protein into the subphase. In the case of both proteins the final stage of displacement involved the formation of tenuous “lacelike” structures which break up and desorb, leaving only islands of aggregated protein. These aggregates also desorbed, leaving a surface occupied solely by surfactant. The evidence shows that the two proteins studied were indeed displaced from the interface by the mechanism of orogeny, although, in the case of the β-lactoglobulin, the three phases of displacement were compressed into a very limited range of surface pressure. The fact that this mechanism has been shown to occur at the gas/liquid, liquid/liquid, and solid/liquid interfaces suggests that this must be the principle mechanism, at least for displacement of protein by nonionic surfactants, although it remains to be established whether the same mechanism applies to displacement of protein by ionic surfactants. The elucidation of this mechanism clearly has implications for interpreting much of the work done on competitive adsorption using nonionic surfactants over the past few years. This mechanism would seem to provide an explanation for the increase in film thickness at the point of displacement seen by others1,2 and also for the changes in surface rheology6 and emulsion stability7 at low ratios of surfactant to protein. The decrease in surface modulus before any protein is removed from the interface is quite consistent with orogenic displacement, where most of the surface area is occupied by surfactant and all the protein is still attached in thick regions. Acknowledgment. The present research described in the above article was funded by the BBSRC through the core strategic grant to the Institute. LA990711E
