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Langmuir 1993,9,2102-2108
Effect of Anionic Surfactant on Interactions between Lysozyme Layers Adsorbed on Mica Robert D. Tilton,* Eva Blomberg, and Per M. Claesson The Surface Force Group, The Institute for Surface Chemistry, Box 5607, S-114 86 Stockholm, and Department of Physical Chemistry, Royal Institute of Technology, S-100 44 Stockholm, Sweden Received October 21, 1992. I n Final Form: May 19,1993 Protein-surfactant interactions play a key role in competitive adsorption and desorption of adsorbed proteins by surfactants. The influence of nonmicellar solutions of an anionic surfactant, sodium dodecanesulfonate, on the interaction forces between preadsorbed layers of lysozyme on mica were investigated using the surface force apparatus. In the absence of surfactant, lysozyme adsorbs irreversibly and neutralizes the negative mica surface charge. The force profile indicates that the adsorbed layer is heterogeneous and deformable, consistingof protein monomersand loosely associated dimers. An adhesion force is established upon contact of the adsorbed layers. Surfactant binding increases the interfacial charge and eliminates the adhesion force between opposing surfaces. Large-scale desorption or conformational change in response to surfactant binding is not observed, although partial desorption of the outer members of adsorbed dimers is induced at the highest surfactant concentration investigated.
Introduction Protein-surfactant interactions and competitive adsorption are influential in a number of applications, including solubilization of membrane proteins, protein solubilization in reverse micelles, surface fouling and cleaning, and stabilization of food colloids. Ionic surfactants adsorb to nonpolar surfaces via hydrophobic interactions, and to oppositely charged surfaces or molecules via electrostaticinteractions. Proteins adsorb readily from aqueous solutions to both fluid and solid interfaces in response to a combination of simultaneouslyacting forces. Cases may be identified where adsorption is driven largely by hydrophobic interactions14 or by electrostatic intera c t i o n ~ .Entropic ~~ factors related to surface-induced conformational changes in the protein may also contribute to the adsorption driving f ~ r c e . ~ ~ ~ Protein adsorption is often irreversible with respect to dilution of the bulk phase, but proteins may be desorbed by changing experimental conditions to alter the balance of intermolecular forces, for example, by changing the pH.51~8Proteins may also be desorbed from surfaces by adding surfactants to the bulk phase. Desorption of proteins from surfaces by surfactants has been studied at both solid'?-12 and fluid interfaces,13J4and has even been applied as a probe of time-dependent changes in the binding strength between proteins and solid surfaces.gJ0
* To whom correspondence should be addressed. Current address: Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213-3890. (1) Norde, W. Adu. Colloid Interface Sci. 1986, 25, 267. (2) Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstrdm, I. J. Colloid Interface Sci. 1987, 199, 203. (3) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci.
1988, 124, 28. (4) Tilton, R. D.;Robertaon, C. R.; G u t , A.P. Langmoir 1991,7,2710. (5) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 257. (6) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (7) Bagchi, P.; Birnbaum, S. M. J. ColloidZnterface Sci. 1981,83,460. (8) Hlady, V.; FUredi-Milhofer,H. J. Colloid Interface Sci. 1979,69, 460. (9) Bohnert, J. L.; Horbett, T. A. J.Colloid Interface Sci. 1986,111, 363. (10) Rapoza, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990,136, 480. (11) Wahlgren, M. C.; Arnebrant, T. J. Colloid Interface Sci. 1991, 142, 503. (12) Elwing, H.; Gdlander, C.-G. Adu. Colloid Znterface Sci. 1990,32, 317.
Protein desorption may occur by direct interaction of the surfactantwith the surface to displace adsorbed protein segments. It may also occur by surfactant interaction with the adsorbed protein molecules themselves to create a protein-surfactant complex with decreased surface activity. Surfactant binding modifies the strength of the interaction forces between the protein-surfactant complex and the surface, depending on the mode of surfactant binding. In dilute solutions at pH values below the protein isoelectric point, stoichiometric binding of aniodic surfactants to cationic amino acid residues creates a less hydrophilic c0mp1ex.l~ This may increase its surface activity (favoring adsorption of protein-surfactant complexes). At higher surfactant concentrations, cooperative surfactant binding to proteins occurs via hydrophobic association of the tails in a manner reminiscent of micelle formation. The resultant accumulation of charge on the surface of the complexl6 increases ita solubility.l5 Charge accumulation magnifies electrostatic interactions between the complex and the surface,as well as between neighboring complexes, possibly destabilizing the adsorbed layer. In addition to changing the forces between the protein and surface, and the electrostatic forces between neighboring proteins, surfactant binding may alter the protein conf~rmation'~-~~ and thereby change steric and other interactions between neighboring proteins. We undertake this study to examine the effect of anionic surfactant binding on the electrostatic and structural properties of adsorbed protein layers in order to understand the molecular eventa underlying desorption of proteins by surfactants, one aspect of the larger issue of competitive adsorption. The surface examined in this (13) Dickinson, E.; Woskett, C. M. In Food Colloids; Bee, R. D., Richmond, P., Mingins, J., Eds.; Raoyal Society of Chemistry: London, 1989; p 74. (14) Dickinson, E.; Euston, S. R.; Woskett, C. M. Prog. Colloid Polym. Sci. 1990, 82, 65. (15) Goddard, E. D.; Pethica, B. A. J. Chem. SOC.1951,2669. (16) Jones, M. N.; Manley, P. J.Chem. SOC.,Faraday Tram. 1,1979, 75, 1736. (17) Shirahama, K.; Tsujii, K.; Takagi, T. J. Biochem. (Tokyo) 1974,
75, 309. (18) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970,246,5161. (19) Wright, A. K.; Thompson, M. R.; Miller, R. L.Biochemistry 1975, 14, 3224. (20) Mattice, W. L.; Riser, J. M.; Clark, D. S. Biochemistry 1976,15, 4264.
0743-7463/93/2409-2102$04.00/00 1993 American Chemical Society
Langmuir, Vol. 9, No.8, 1993 2103
Adsorbed Protein-Surfactant Interactions study, mica, is hydrophilic and negatively charged. Thus, the anionicsurfactant will not adsorb to the surface,11J281*22 and we study the effects of surfactant binding to the adsorbed protein molecules. Using the surface force apparatusF3we obtain a direct measure of the force profiie between adsorbed layers. From the force profile we determine the adsorbed layer thickness and the response of the layer to applied forces. Information about the charge and structure of the adsorbed layer is obtained from interpretation of the force profiles within standard theoretical guidelines.
10000
(21) Amebrant, T.;Bllckstram,K.;JBneson,B.;Nylander, T. J. Colloid Interface Sci. 1989,128,303. (22) Complete absence of sodium dodecyl sulfate adeorption to mica hae been demonstrated with the surface force apparatus: Blomberg, E., Fraberg, J. C., Claeseon, P. M. Manuscript in preparation. (23) Israelachvili, J. N.; Adame, G. E. J. Chem. SOC.,Faraday Trans.
.
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Experimental Section Materials. Lysozyme is a rather stable protein, and it has received appreciableattention regarding both surfactantbinding and competitive adsorption. Although much of the data in the surfactantbinding literature pertains to sodium dodecyl sulfate, the considerable susceptibility of this moleculeto hydrolysis and consequent alteration of surface activity over time%prompted us to consider a more stable surfactant, sodiumdodecanesulfonate (SDSo),for these surface force experiments that typically take several days to complete. Chicken egg lysozyme, obtained from Sigma,and (SDSo (>99.4% pure), from Aldrich, were used as received. Water was treated by decalcination, prefiltration, reverse osmosis, and passage through a modified Millipore purification system with two mixed bed ion exchange cartridges, an activated carbon cartridge, an in-line0.2-pm-pore-sizeZetapore filter, an Organex cartridge, and a final 0.2-pm Zetapore filter. Water was degassed under vacuum immediately before use. Muscovite mica substrates were prepared by cleaving along crystalline planes in a laminar flow hood. With oneside protected by adhesion to a freshly cleaved mica backing sheet, the thin mica substratewas partiallymirrored by silver evaporation. Prior to the s t a r t of an experiment, the mica pieces were glued with an epoxy glue (Epon 1004,Shell Chemical) onto half-cylindrical silica lenses (with the clean mica side exposed) and mounted in a crossed cylinder geometry in the surface force apparatus. Methods. Measurements were conducted with a Mark IV surface force apparatus,% a modification of the design of Israelachvili." The technique is described in detail in the indicated references. Briefly, the distance between the two mica surfaces is determined with a precision of approximately2 A by multiple beam interferometry using fringes of equal chromatic order. The surface separation is controlled either by adjusting the voltage applied to a piezoelectric mount rigidly attached to one of the surfacesor by a synchronousmotor coupled by a double cantilever spring to the other surface. Forces are determined from the deflection of this spring. Experiments commenced by filling the instrument chamber with water and examining the adhesion of the mica surfaces to verify the cleanliness of the system. This was followed by introduction of NaCl to a fiial concentration of 109M at pH 5.6, whereupon the forces between the mica surfaces were measured. Provided that the measured forces were in accordance with previous meaurementsin pure electrolyte solutions,the surfaces were regarded as free of contamination, and the experiment was continued. It should be noted that the forces observed at this stage were entirely consistent with DLVO theory. After these preliminary checks, lysozyme solution was injected to a concentration of 0.2 mg/mL, mixed, and allowed to stand for at least 12 h prior to the fiist measurements of surface forces between adsorbed lysozyme layers. The lysozyme solution was then diluted by a factor of approximately3000by drainingthe chamber (being careful to leave a drop of solution between the surfaces
mo
0
100
200 300 DISTANCE (A,
400
500
Figure 1. Force normalized by the mean radius of curvature of the interacting surfacesmeasured in pH 5.6,0.2 mg/mL lysozyme solution containing 109 M NaCl (filled symbols). Unfiied symbols represent the force measured after 12 h of adsorption followed by a 3000-fold dilution. The arrow marks the position where the lysozyme-covered surfaces jump inward on approach. The solid curves representthe forces predicted by DLVO theory with 90= 18 mV and k = 96 A. The upper curve corresponds to A = 0 and the lower to A = 2.2 X 10-" J. Inset: Filled and unfilled circles represent the force measured on approach and separation, respectively. in order to avoid exposing the adsorbed layer to a three-phase contact line) and refilliig with 109 M NaCl solution. Surface forces were again measured over a period of 12 h to investigate the reversibility of adsorption. SDSosolutionswere then injected into the chamber, mixed, and allowed to equilibrate for at least 1 h before continuing surface force measurements. SDSo concentrations were increased by successive additions to the chamber while maintaining a constant NaCl concentration. AU measurements were conducted at 20 "C, and the pH was unaffected by the addition of SDSo.
Theoretical Calculations We analyzed the force profiies within the DLVO theory for surface interaction via additive contributions from the nonretarded van der Waals force and the electrostatic double-layer force (in the nonlinear Poisson-Boltzmann approximation invoking the assumption of interaction at constant charge). The calculations were performed according to the algorithm of Chan et aL26 The decay length of the double-layer force in monovalent electrolyte solutions is accurately provided by the Debye length (I+), and the theoretically expected values were used in all calculations, except where noted. The plane of charge and the origin of the van der Waals force were assumed to lie at the position of the force wall where the lysozyme layers are at their maximum compression (at a mica-mica separation distance D = 90 A). We considered two extreme cases for the van der Waals force, using either the Hamaker J for mica surfaces interacting constant A = 2.2 X across water or A = 0, corresponding to no van der Waals force between the hydrated layers. These assumptions, particularly regarding the location of the plane of charge, have been discussed previ~usly.~' Results Force profiles are shown in Figures 1-7. Each figure contains data from at least two separate measurements. To check for consistency, measurements have been made on multiple spots on the surfaces in each preparation, and the same behavior has been observed in two independent preparations.
1 1978. 74. 975.
(24) Czichocki, G.; Vollhardt, D.; Much, H. J. Colloid Interface Sci. 1983,96, 275. (25) Parker,J.L.;Christenson,H.K.;Ninham,B.W. Rev. Sci.Instrum. 1989,601, 3135.
(26) Chan,D. Y.;Paehley,R.M.; White, L. R. J. Colloidlnterface Sci. 1980. _.__ , 77.283. - -, (27) Blomberg, E.; Claesson, P. M.; Mlander, C.-G.J. Dispersion Sci. Technol. 1991,12, 179.
Tilton et al.
2104 Langmuir, Vol. 9,No. 8,1993 O0O0
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Figure 2. Normalized force between adsorbed lysozyme layers in 10-9M NaCl after addition of lo-' M SDSo. Filled symbols represent the force measured on approach when the system was left undisturbed for 30 min, and the unfilled symbols represent the force measured on approach immediately after the first measurement. The solid curves represent the DLVO predictions for 90= 30 mV and K-1 = 96 A, for A = 0 (upper curve) and A = 2.2 X 10-20 J (lower curve).
Figure 5. Normalized force between adsorbed lysozyme layers in 10-9M NaCl measured on approach 1h after addition of 8.3 X lo-' M SDSo (unfilled symbols) and 20 h after surfactant addition (filled symbols). The solid curves represent DLVO predictions for \ko = 53 mV and r1= 71 A, for A = 0 (upper curve) and A = 2.2 X 10-20 J (lower curve).
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Figure 3. Normalized force between adsorbed lysozyme layers in 10-9 M NaCl measured on approach 4 h after addition of 4 x lo-' M SDSo and 99 h after addition of surfactant (unfilled and filled symbols, respectively). The solid curves represent DLVO predictions for 9 0 = 43 mV and K-1 = 81 A, for A = 0 (upper curve) and A = 2.2 X lo-%J (lower curve).
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Figure 6. Normalized force between adsorbed lysozyme layers in 10-9 M NaCl and 8.3 X lo-' M SDSo measured on approach and on separation (filled and unfilled symbols, respectively).
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8 0 .
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Figure 4. Normalized force between adsorbed lysozyme layers in 10-8 M NaCl and 4 X lW M SDSo measured on approach and on separation (filled and unfilled symbols, respectively). The forces between mica surfaces across a 0.2 mg/mL lysozyme solution containing 10-3 M NaCl at pH 5.6 are shown in Figure 1. Under these conditions, lysozyme adsorbs to the surfaces and nearly neutralizes the charge. At large separations ( D > 200 A), a very weak repulsive double-layer force dominates the interaction. The decay length of this weak force cannot be accurately measured or calculated theoretically (given the uncertainty about the effect of large, multivalent proteins on the Debye length). Nevertheless, it is evident that the apparent charge per area is less than one per lo4 A2. At distances below 200 A there is an attraction resulting in a minimum
Figure 7. Normalized force between adsorbed lysozyme layers in 10-9 M NaCl after addition of 1.4 X 10-9 M SDSo measured on approach and on separation (filled and d i e d symbols, respectively). The solid curves represent DLVO predictions for 90= 58 mV and = 62 A, for A = 0 (upper curve) and A = 2.2 X J (lower curve). in the force profile (Figure 1 inset). When the surfaces are brought even closer together, a steeply rising repulsion is encountered. The onset of this short-range repulsion varies somewhat between different force runs and positions on the surface. It is always between 110 and 150 A, and in most cases is approximately 120 A. The part of the short-range force below 1lOA is more reproducible. When the compressive force is increased to approximately 105 pN/m, the surfaces come into a separation of 90-100 A from mica-mica contact. A further increase of the force does not cause any significant decrease in the surface separation. Hence, the final adsorbed layer thickness is 45-50 A on each surface.
Langmuir, Vol. 9,No. 8, 1993 2105
Adsorbed Protein-Surfactant Interactions When the adsorbed layers are separated from contact (Figure 1 inset), they initially follow the same force profile as that measured on approach of the surfaces. The force profile on separation deviates from that on approach at separations greater than 100 A. The attractive minimum is located at a distance of approximate 120-140 A. The adhesion force, denoted F(O), normalized by the mean radius of curvature R, is approximately-400 to -600 pN/m (Figure 1 inset). This is related to the interfacial tension, y s ~ and , the tension in the contact zone, yss, as
IF(O)/RI = 4
7 9 ,
-~ s s )
(1)
where the coefficient a is between 27r (small forces and rigid surfaces) and 3 4 2 (largeforces and softsurfaces).ms The coefficient 27r is probably most appropriate in this case. If the contact were ideal, the interfacial tension between the protein layer and the aqueous solution would be approximately 30-50 pN/m. This will be discussed later. After 12 h of lysozyme adsorption, we diluted the protein solution by a factor of 3000 with an aqueous 1 0 s M NaCl solution and measured the forces again. As illustrated in Figure 1, no significant change in the force profile occurs upon dilution, and the proteins are regarded as irreversibly adsorbed with respect to dilution. After dilution, the theoretical Debye length is 96 A. Using this value for rl, we estimate that the apparent interfacial potential is less than 18 mV, corresponding to charges occurrin at an apparent density of less than one per 1.2 X lo4 2. The forces between the protein-coated surfaces after addition of 10-4 M SDSo are displayed in Figure 2. The most important difference between these forces and those present without surfactant is that the surface charge has increased, whereas the final layer thickness remains unchanged. Reproducible measurements of the forces acting on approach (Le., independent of adsorption time over a period of 24 h) were obtained when the system was left undisturbed for at least 30 min between successive force runs. When the forces were measured a second time immediately after completion of the first approach/ separation cycle, the repulsive double-layer force was weaker, and a clear reduction in the slope of the force was observed in the distance range 120-100 A (Figure 2). The reduction in the double-layer force on a second approach suggests that surfactanh are eliminated from the adsorbed layer in the contact region and do not completely reenter the contact region prior to the start of an immediately successive force run. In other words, the forces measured on such second approaches do not reflect the equilibrium situation. No such dramatic behavior was observed at higher surfactant concentrations. The forces measured on separation in 10-4 M SDSo are always considerably less repulsive than those observed on approach for the same reasons. A weak adhesion (compared to that for surfactant-free layers) is observed on separation for short surfactant adsorption times but disappears at longer times. Figure 3 displays the forces measured on approach after increasing the surfactant concentration to 4 X 10-4 M. Clearly, the repulsive double-layer force is larger than that measured at lower surfactant concentrations. More surprisingly, the force increases over several days. This indicates that the amount of bound surfactant increases slowly with time. A possible explanation for this will be given later. At this surfactant concentration, the double-
1
(28) Johnson, K. L.; Kendall, K.;Roberts, A. D. Proc. R. SOC.London, A 1971,324,301. (29) Muller, V. M.; Yuahchenko, V. S.; Derjaguin, B. V. J. Colloid Interface Sci. 1983, 92,92.
layer force dominates the interaction down to a surface separation of 110-100 A. An additional repulsion appears to exist at smaller separations. Hence, the range of the extra repulsion appears to be less than at the lower surfactant concentrations. The layer thickness obtained under a high compressive force remains unchanged. The forces measured on separation (Figure 4) are consistently lower than those measured on approach. Hence, some surfactants are displaced from the contact zone when the surfaces are brought into contact, but the mass transport back into the contact zone is more rapid at this concentration than at 10-4 M, so that forces measured on subsequent approaches are equal. This mass transport effect is likely due to the increased lateral repulsions between the adsorbed SDSo molecules at the higher concentration. Further increases of the surfactant concentration to 8.3 X 10-4 M and again to 1.4 X 1 0 s M increase the charge at the interface. Just as at the lower surfactant concentration, the interfacial charge increases rapidly at first and continues to increase slowly when the system is left overnight (Figure 5). The repulsion observed on separation is somewhat smaller than that on approach in 8.3 X 10-4 M SDSo solutions, but we observe hardly any hysteresis in 1.4 X 1 0 s M SDSo (Figures 6 and 7). It is also evident that the non-DLVO repulsion is less pronounced at these higher surfactant concentrations. The f i i layer thickness of 90-100 A is independent of surfactant concentration. We note that similar trends in charging behavior due to SDSo binding have been observed in a third preparation (not presented here) using a higher ionic strength of 1:l electrolyte.
Discussion Structure of the Adsorbed Layer: Non-DLVO Force. The final contact between adsorbed lysozyme layers under a high compressive force is in all cases approximately 90 A out from mica-mica contact. A repulsion stronger than expected from DLVO theory extends about 20-30 A further out. No such non-DLVO repulsion was observed in 1 0 s M NaCl prior to addition of lysozyme. At least three factors may contribute to this non-DLVO repulsion observed between lysozyme layers (including those in dilute SDSo solutions). These are (i) removal of an outer, loosely bound protein layer (and surfactant when present), (ii) compression and dehydration of proteins firmly bound to the surfaces, and (iii) compression of the glue supporting the mica surfaces. Regarding the third possibility, deformation of curved surfaces can be caused by the unequal strength of surface forces acting at different points along the circumference, thereby causing the local radius of curvature to change. For this reason, care must be taken when discussing the distance dependence of short-range forces. It has been shown theoretically that such deformation can be completely neglected for mica surfaces interacting across a 1 0 s M NaCl solutionqmDeformation effects are typically observed easily for values of FIR exceeding approximately 104pN/m (based on the undeformed radius of curvature), avalue never reached in this work. Calculations also show that surface deformationeffects may occur at lower values for steep force profiles.30 The force profile for adsorbed lysozyme in the absence of SDSo is steep, but FIR is sufficiently low that deformationeffects remain negligible at the onset of the non-DLVOrepulsion. Finally, reported surface separations are the distances of closest approach (30) Parker, J. L.; Attard, P. J. Phys. Chem. 1992,96,10398.
2106 Langmuir, Vol. 9,No. 8,1993
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Figure 8. A schematic illustration of the structure of the adsorbed lysozyme layers on mica, showing that initial contact occurs via pairs of horizontal dimers at 120 A. Continued compression squeezes out one protein from each dimer contact, allowing the layer to relax through a series of possible protein contacts until a final layer thickness of 90 8, corresponding to contact of vertical proteins or three horizontal proteins. This structure assumes lysozyme behaves as a rigid 45 X 30 X 30 A ellipsoid (drawn to scale).
of the mica surfaces, determined from light reflections at the silvered backing layer on the mica substrates. This distance measurement is not altered by surface deformations. Hence, the position of the onset of non-DLVO forces provides useful information about the structure of the adsorbed layer in this investigation, as discussed below. A discussion of the structure of the adsorbed layer is possible by comparison of the dimensions of the lysozyme molecule, approximately a 45 X 30 X 30 A ellipsoid,31 the range of the non-DLVO forces (D= 120 A),and the final layer thickness of 90-100 A (45-50 A/surface) under high compression. The interpretation is not unequivocal, but two situations are possible. In principal, the final layer thickness data are consistent with either one monolayer of vertically oriented proteins remaining on each surface or with three layers of horizontal proteins between the surfaces (or a combination of these two configurations). The proposed layer structure is illustrated schematically in Figure 8. The rather poor reproducibility of the outermost portion of the non-DLVO repulsion observed between 110 and 150 (most often 120 A) suggests that this force is due to an outer, weakly bound layer (or layers) that is squeezed out from between the surfaces rather than a hydration repulsion. The location of the inward jump observed in the absence of SDSo could be predicted by DLVO theory only if the plane of origin of the van der Waals and double-layer forces were shifted outward to at least 150 A, even in the upper limit of the mica-watermica Hamaker constant. This is not realistic, particularly considering that we would have to allow the Hamaker constant to be drastically lowered by surfactant binding in order to explain the absence of an attractive force in SDSo solutions. A probable structure of the layer may be inferred by considering the structure of the lysozyme molecule in slightly more detail. Lysozyme carries a net charge of +9 per molecule at pH 5.6,32with most of the positive charges located on either side of the active site cleft. This suggests that an electrostatically favorable adsorption can be achieved with either a horizontal or vertical orientation (31) Blake, C. C. F.; Mair, G. A.; North, A. C. T.;Phillips,D. C.; Sarma,
V. R. R o c . R. SOC.London, B 1967,167,365.
(32)Tanford, C.; Wagner, M. L. J. Am. Chem. SOC.1954, 76, 3331.
Tilton et al.
of the protein on the negatively charged mica surface. It is likely that lysozyme adsorbs initially in a random distribution of monomers by strong electrostatic binding to the surface. The low coverage at early adsorption times allows the molecules to relax to a horizontal orientation, but as the surface coverageincreases,additional adsorption becomes more difficult due to packing constraints. Thus, the latter stages of adsorption may be dominated by proteins orienting vertically on the surface to present the smallest projected area. Consistent with the data, this situation would give rise to a force barrier at 90 A (see Figure 8). Adsorption of proteins in the weakly bound second layer, effectivelyrepresenting a surface dimerization, may introduce horizontally oriented proteins in contact with horizontal proteins in the first layer. Contact between two such dimers on opposite surfaces would produce the non-DLVO repulsion observed near 120 A. Considering that the proteins are hydrated and probably not perfectly oriented, this model is consistent with the data. Note that there is experimental precedent for the orientation of adsorbed proteins to change over time.33 Given the dynamic nature of binding in adsorbed protein layers, the proteins will likely have appreciable lateral freedom. Thus, surface dimers, or at least the outer halves of the dimers, may be expected to be easily expelled from the contact zone to relieve the applied stress under compression. It is important that the non-DLVO forces near 120A are similar on successiveapproaches at a given position, even when the lysozyme solution has been diluted. This reversibility suggests that the proteins are not expelled out of the adsorbed layer but along it, and that their lateral mobility allows them to diffuse back along the imposed concentration gradient into the original contact zone after the compressivestress has been removed. As noted in a prior p ~ b l i c a t i o nthere , ~ ~ is no inconsistency between our observations of irreversible adsorption and lateral mobility of adsorbed proteins. Further investigation of lysozyme adsorption on mica in the absence of surfactants will be presented elsewhere.35 We note that, in the absence of a specific attraction or a long-range attraction that extends beyond an adsorbed protein monolayer, multilayer adsorption is not expected for such a dilute protein solution. Two facts show that attractive forces do exist between lysozyme molecules. First, while lysozyme does not form dimers in bulk solution at the concentration of these experiments, it does dimerize at high so the occurrence of surface dimers is not surprising at the locally high protein densities at the interface. The molecular contacts involved in solution-phasedimerizationmay be involved in the surface dimerization. Second, the existence of an adhesive force when separating the protein-coated surfaces from contact shows that the required interprotein attraction for some sort of multilayer formation is satisfied. A similar but stronger adhesion force was observed between adsorbed insulin monolayers.37 Insulin also associates in solution (formingdimers and hexamers), and with time, it adsorbs as a multilayer. Effect of SDSo on the Structure of the Adsorbed Layer. We have addressed the effect of long-term exposure of a negativelycharged surface,carrying adsorbed (33) Lee,C.-S.;Belfort,G.Proc.NatZ.Acad. Sci. U.S.A. 1989,86,8392. (34) Tilton, R. D.; Robertson, C. R.; Gast, A. P. J. Colloid Interface Sei. 1990, 137, 192. (35) Blomberg, E.; Claesson, P. M.; Friiberg, J. C.; Tilton, R. D. Manuscript in preparation. (36) Deonier, R. C.; Williams, J. W. Biochemistry 1970,9,4260. (37) Claesson, P. M.; Amebrant, T.; Bergenstihl, B.; Nylander, T. J. Colloid Interface Sei. 1989, 130, 457.
Langmuir, Vol. 9,No.8, 1993 2107
Adsorbed Protein-Surfactant Interactions positively charged proteins, to nonmicellar solutions of an anionic surfactant. First of all,the results show that under these conditions the surfactant does not induce large-scale conformational change or desorption of the proteins adsorbed directly to the surface even for very long exposure times (several days). On the other hand, the non-DLVO repulsion observed at distances near 120 A becomes less pronounced and perhaps disappears at the higher surfactant concentrations investigated. Hence, the outer protein layer may be desorbed due to the surfactant. It should be noted that when plotted individually, several of the force profiles exhibit rather pronounced, steplike nonDLVO repulsions, but when multiple force profiles are superimposed, the form of this extra repulsion becomes almost featureless, due to the irreproducibility of the exact location of its onset. (Although a superposition of several measurements, Figure 3 illustrates the steplike form fairly well.) The conclusion that the non-DLVO forces become less pronounced at higher concentrations is not due to this graphical data superposition problem. Surfactants bind to proteins electrostatically a t low concentrations and via hydrophobic interactions,with the headgroupspointing out from the protein surface, a t higher concentrations.16 As the surfactant concentration increases the protein-surfactant complex becomes initially more hydrophobic than the protein itself and then decreases in hydrophobicity.ls In light of eq 1,this implies that the adhesion between adsorbed protein-surfactant layers should become stronger at low surfactant concentrations (more hydrophobic adsorbed layer) and decrease at the higher concentrations. Instead, we observed that the adhesion force disappears at even the lowest surfactant concentration. This suggests that the adhesion force is not simply related to the interfacial tension y s ~ The , lack of adhesion in the presence of surfactants may involve a degree of surfactant and/or protein expulsion from the contact zone and possibly disruption of specific interactions. Surfactant binding may disrupt the geometry required for the specific attration between lysozyme molecules. Thus, the value of yss is influenced by the surfactant. It is noteworthy that no adhesion exists between contacting full monolayers of human serum albumin adsorbed on mica,27where one might expect to observe interfacial tensions similar to those in the current system. At high surfactant concentrations (14 X lo4 M),we observed that the double-layer charge increases somewhat when the system is left undisturbed overnight. This demonstrates that surfactant binding to the adsorbed proteins continues slowly over long times. This is surprising, considering the rapid transport and adsorption kinetics of surfactants in general. It is likely that this increased adsorption is related to slow, small conformational changes induced by surfactants in the adsorbed layer. This process may be hindered to some extent by protein packing constraints. It is worth noting that no observable changes occur when lysozyme layers are exposed to surfactant-free NaCl solutions. This has been tested for a 1-day exposure of adsorbed lysozyme to protein-free and surfactant-free, lo3 M NaCl solution, and for several days exposure to lo3 M NaCl solutions containing lysozyme. Of course, our technique cannot detect conformational changes on length scales below a few angstroms. Lysozyme Adsorption on Mica and the Interfacial Charge. Our proposed model for the adsorbed layer (Figure 8) can explain its structural features and its response to applied force, but the issue of surface neu-
Table I. Adsorbed Layer Characteristics [SDSo] (M)9, (mV) aredcharge (A9 charge/protein r1(A) 0 18 11800 0.09 96 (96) lo-' 30 6840 0.17 96 (92) 4 X W 8.3 X lo-' 1.4 X 109
43 53 58
3790 2540 1960
0.31 0.46 0.60
81 (81) 71 (71) 62 (62)
Theoreticallycalculated Debye length is given in parentheses.
tralization upon lysozyme adsorption must also be addressed. Due to isomorphous substitution of aluminum for silicon, ionizable groups occur on the mica surface at a density of one per 48 A2,but counterion binding (protons and sodium ions in this case) results in a surface charge density of only one negative charge per 2000 A2 in 10-9 M NaCl at pH 5.5-6.0.38 Adsorption of a hexagonal closepacked monolayer of horizontal lysozyme molecules a t this pH would introduce positive charges at a density of one per a projected area of 130 A2. This is more than sufficient to neutralize the mica surface charge in 10-9M NaC1, yet no significant recharging is observed. There are two reasons for this. First, lysozyme adsorption is accompanied by ion exchange. In other words, protons and Na+ are displaced from the mica surface. Second, when lysozyme adsorbs with its positive charges toward the surface, the ionizable groups are transferred to a low dielectric environment, and the electrostatic self-energy increases. This shifts all acid-base equilibria toward the unchargedstate and carries co-ions into the adsorbed layer. The importance of coadsorptionof small ions with proteins was originally discussed by Norde and Lyklema.6*39 Surfactant Adsorption and Interfacial Charge. We estimated the charge of each lysozyme-SDSo layer from the magnitude of the double-layer force, fitting the measured force to the double-layer force calculated as described earlier. The difficulties associated with this procedure for adsorbed proteins have been discussed previou~ly.~'Because of the approximations in the nonlinear Poisson-Boltzmann model, particularly the neglect of ion-ion correlation effects, all fitted charges and potentials are underestimations of the real ones, and fitted values should be regarded as apparent. Nevertheless, for monovalentions and such small potentials as those observed in this investigation, the fitted values are quite accurate, and even for the highest charged case, the underestimation should not be more than 20 % A greater concern is the diffuse distribution of charge within the adsorbed layer and its implications for the true location of the origin of the double layer. Despite these difficulties, it is clear that SDSo adsorbs to the lysozyme-coatedsurface and increases its negative charge. The apparent area per charge and the number of adsorbed surfactants per lysozyme (charge/protein),calculated assuming an occupied area of 1165A2/lysozymein a hexagonal close-packed horizontal monolayer, are presented in Table I. (Data in the literature suggest that the surface should be saturated with lysozyme at the concentration used here.41) Even at the highest concentration, less than one SDSo molecule adsorbs per protein molecule. Of course, due to ion exchange processes there may not be a one-to-one correspondence between the number of surfactants bound and the interfacial charge. Nevertheless, it appears that only ~~
(38) Pashley, R. M. J. Colloid Interface Sci. 1981,80, 153. (39)van Dulm, P.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1981, 82, 77. (40) Attmd, P.; Mitchell, J.; Ninham, B. W .J. Chem. Phys. 1988,89, 4358. (41) GBlander, C.-G.; Hlady, V.; Caldwell,K.; Andrade,J. D. Colloids Surf. 1990, 50, 113.
2108 Langmuir, Vol. 9, No. 8,1993 small amounts of SDSo have adsorbed to the lysozyme layer, and not surprisingly,these amounts are insufficient to cause significant lysozyme desorption. Significant desorption may not be possible until the SDSo concentration approaches the cmc. Above the cmc, positively charged lysozyme can be removed from negatively charged silica surfaces by sodium dodecyl sulfate." However, the Krafft point of SDSo is approximately 35 O C t 2 so complete desorption by SDSo may never occur at the temperature of these experiments.
Conclusions When adsorbed from 0.2 mg/mL solutions, lysozyme and coadsorbing ions nearly neutralize the surface charge of mica to form a heterogeneous, partly mobile adsorbed layer. The adsorbed layer deforms reversibly in response to compressive force and consists of an inner, tightly bound layer and at least one outer, weakly bound layer. An adhesion force exists between compressed layers in contact, and this force may be related to specific interactions between amino acid residues involved in the lysozyme dimerization process that occurs in concentrated solutions. (42) Tartar, H.V.;Wright, K.A. J. Am. Chem. SOC.1939,61,539.
Tilton et al. The effects of SDSo on preadsorbed lysozyme layers may be summarized as follows. SDSo does not induce large-scaledesorption of lysozyme, although at the highest concentrations investigated, it appears to induce desorption of the outermost proteins. At all concentrations investigated, SDSobinding increasesthe interfacialcharge. SDSo is at least partially expelled from the contact region between two layers under compression. The interfacial charge accumulation that we observe at low surfactant concentrations probably represents the precursor to destabilization of adsorbed lysozyme layers on negatively charged surfaces in the presence of micellar anionic surfactant solutions. Since this is the first study of the effect of surfactant binding on the interactions between adsorbed protein layers, it is too early to generalizeon the basis of our results. While the amount of surfactant required to desorb proteins will vary for different systems, it is likely that the phenomenon of adsorbed layer chargingplays an important role in the process.
Acknowledgment. R.D.T. gratefully acknowledgesthe financial support of the National Science Foundation of the United States of America under Grant INT-9003988.
