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Langmuir 1994,10, 2325-2334
Interaction between Adsorbed Layers of Lysozyme Studied with the Surface Force Technique Eva Blomberg,* Per M. Claesson, Johan C. Froberg, and Robert D. Tilton? Laboratory for Chemical Surface Science, Department of Chemistry, Physical Chemistry, Royal Institute of Technology, S-100 44 Stockholm, Sweden, a n d Institute for Surface Chemistry, P.O. Box 5607, S-114 86 Stockholm, Sweden Received November 9, 1993. In Final Form: March 29, 1994@ The surface force technique was employed to investigate the adsorption of positively charged lysozyme onto negatively charged mica surfaces in M NaCl at pH 5.6 at lysozyme concentrations ranging from 0.002 to 0.2 mg/mL. At equilibrium the adsorbed lysozyme nearly neutralizes the surface charge of the mica at all bulk lysozyme concentrations investigated. Prior to charge neutralization the decay length of the long-range force is consistent with the electrostatic double-layerforce predicted by the DLVO theory. At low concentration, 0.002 mg/mL, a densely packed side-on oriented layer adsorbs on the mica surface. Above 0.02 mg of lysozymdml, a rather thick layer is adsorbed onto the surface. It consists of an inner, strongly bound layer of both side-on and end-on adsorbed proteins and an outer layer of weakly adsorbed proteins. An adhesion force is established upon contact ofthe adsorbed protein layers. The force measured between one lysozyme-coated surface and one bare mica surface is attractive at short separations. It was demonstrated that, at a concentration of 0.02 mg/mL, lysozyme adsorbs "irreversibly" with respect to dilution with M NaC1.
Introduction Proteins have an amphiphilic nature, and they therefore have a strong tendency to adsorb at different interfaces.' Considerable interest in the interaction between proteins and interfaces has developed in recent decades, due in large part to a number of technical applications where the interaction between proteins and a n interface is of great importance, such as biocompatibility, drug delivery,l and colloidal stabilitye2 The complex structure and charge distribution of proteins cause them to adsorb on a variety of surfaces in response to a combination of simultaneously acting forces. The most important driving force for protein adsorption has in some cases been identified as hydrophobic electrostatic i n t e r a c t i ~ n ,or ~ ~entropic ~ factors related to surface-induced conformational changes in the protein s t r u ~ t u r e Protein . ~ ~ ~ adsorption has in many cases been found to be mostly irreversible with respect to dilution of the bulk solution. However, desorption can be induced by changing the experimental conditions, eg., by changing the pH7,9or by adding surfactant^.'^-^^
* To whom correspondenceshould be addressed at the Institute for Surface Chemistry. Current address: Department of Chemical Engineering,Carnegie Mellon University, Pittsburgh, PA 15213-3890. Abstract published in Advance ACS Abstracts, May 15,1994. (1)Andrade, J. D. In Surface and Interfacial Aspects ofBiomedica1 Polymers; Andrade, J. D., Ed.; Vol. 2;Plenum Press: New York, 1985. (2)Dickinson, E. Food Hydrocolloids 1986,I, 3. (3)Norde. W. Adv. Colloid Interface Sei. 1986.25.267. (4)Elwing, H.; Welin, S.; Askedal, A,; Nilsson,'U.; Lundstrom, I. J. Colloid Interface Sci. 1987,119,203. (5)Young, B.R.; Pitt, W. G.; Cooper, S. L. J . Colloid Interface Sci. 1988,124,28. (6)Tilton, R. D.; Robertson, C. R.; Gast, A. P. Langmuir 1991,7 , @
971 n -. _".
(7)Norde, W.; Lyklema, J. J . Colloid Interface Sci. 1978,66, 257. (8) Arai, T.;Norde, W. Colloids Surf. 1990,51,1. (9)Hlady, V.; Ftiredi-Milhofer, H. J.Colloid Interface Sci. 1979,69, 460. (10)Wahlgren, M. C.; Amebrant, T. J . Colloid Interface Sci. 1991, 142, 503. (11)Elwing, H.; Golander, C.-G.Adu. Colloid Interface Sci. 1990,32, 317. (12)Bohnert, J.L.; Horbett, T. A. J.Colloid Interface Sci. 1986,111, 363.
The surface force technique can provide information about the interaction between adsorbed protein layers and the interaction between a n adsorbed protein layer and a solid surface. Comparing the dimensions of the protein molecule in solution with the adsorbed layer thickness and the range of the non-DLVO forces can provide information about the orientation and conformation of the adsorbed protein. The present study focuses on the adsorption of positively charged lysozyme onto negatively charged mica surfaces, and the interaction between adsorbed layers of lysozyme as well as between one lysozyme-coated surface and one bare mica surface. One aim of this study was to observe the buildup of the adsorbed layers with increasing protein concentration and with increasingadsorption time. The compressibility and the structural stability of the adsorbed lysozyme molecule were also addressed.
Experimental Section Materials. Chicken egg lysozyme (muramidase,mucopeptide N-acetylmuramoylhydrolase; EC 3.2.1.17; lot 89F-8275) was obtained from Sigma and sodium chloride (NaCl), suprapure grade, from Merck (used as received). Muscovite mica was received from M. Watanabe & Co., Tokyo, and Mica New York Corp., New York. The glue, an epoxy resin (Epon 1004),was received from Shell Chemicals. The water used in the experiments was treated by one of two alternative ways: (i) by decalcination,prefiltration,and reverse osmosis,followed by final purification in a modified Millipore purification unit which included, in order, two mixed bed ion-exchange cartridges, an activated carbon cartridge, a 0.2-pm in-line filter cartridge, an Organex cartridge, and a final 0.2-pm filter (all filters from Zetapore, all other cartridges from Millipore);(ii)by a Milli-RO lOPLUS pretreatment unit, including depth filtration, carbon adsorption, and decalcinationpreceding reverse osmosis, followed by a Milli-Q PLUS185 unit, which treats the feed water with UV light (185 nm 254 nm) before leading it into a Q-PAK unit-consisting of an active carbon unit followedby a mixed bed ion exchangerand finally an Organex cartridge-and a final 0.22pm Millipak 40 filter. Surfaceforce results were not affected by the choice of water purification method. Before use the water was degassed under vacuum.
+
(13)Rapoza, R. J.;Horbett, T. A. J . Colloid Interface Sci. 1990,136, 480.
0743-7463/94/2410-2325$04.50/00 1994 American Chemical Society
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BINDING ENERGY (eV)
Z = Io exp[-d/(A sin e)]
B)
300 299 298
297 296 295 294 293 292 291
potassium ions upon immersion in aqueous solutions are displaced by protons and other ions present in the solution (sodium ions and lysozyme molecules in this case). All proteins contain a high level of nitrogen, not present in the mica crystal, and hence, the ESCA signal resulting from the mica and the adsorbed protein can easily be distinguished. After the adsorption of lysozyme onto mica the N 1s photoelectron signal was measured and compared with the K2p signal, emanating from the mica surface. By using known sensitivity factors,16 and by taking into account photoelectron adsorption in the protein layer, the number of nitrogen atoms per unit area can be computed.16J7 From the number of nitrogen atoms per lysozyme molecule (193, derived from the known amino acid compositionla) and the molecular weight of lysozyme1* (14 500) the adsorbed amount was obtained. By measuring the intensity of the K 2p signal a t different incident angles, the reduced thickness d/A can be determined using the following equation?
290
BINDING ENERGY (eV)
Figure 1. ESCA peak signals for (A) N 1s and (B) K 2p for M NaCl solution, lysozyme adsorbed on bare mica from a at pH 5.6, containing 0.002 mg of lysozymelml.
Methods. Electron Spectroscopyfor Chemicalbalysis (ESCA). The adsorbed amount of lysozyme onto muscovite mica was measured by means of ESCA, using a Kratos (AXIS HS) ESCA spectrometer with a monochromatic Al Ka X-ray source (operating a t 15 kV and 20 mA), and a hemispherical analyzer. The integrated peak intensities for nitrogen 1s and potassium 2p signals were computed by using a desktop computer. Due to instrumental parameters, uncertainties in atomic cross sections, and the electron inelastic mean free path, the accuracy of the ESCA method for quantitative purposes is normally not better than 10%.14 In addition, a small random error results from difficulties in determining the correct peak intensities from the ESCA spectrum. This error was less than a few percent in our measurements. Examples of observed N 1s and K 2p spectral peaks are shown in Figure 1. Following the method of Herder et aZ.,15the adsorbed amount of lysozyme on mica was obtained from the intensities of the N 1s and K 2p photoelectron signals for a range of lysozyme bulk concentrations. Muscovite mica can be cleaved along the basal plane to create a molecularly smooth surface, virtually eliminating the problem of surface roughness. The surface composition of the basal plane of mica is well known. Exchangeable ions (mostly potassium, and to a lesser extent sodium) occur at a density of 2.1 x 1014cm+. These ions can be used as an internal standard for quantifylng the absolute number of adsorbed molecules or ions on the muscovite mica basal plane, as described p r e v i o u ~ l y .It~should ~ be emphasized that this method does not assume a stoichiometric displacement of the basal plane potassium by the adsorbed proteins. Instead, it is assumed that all (14)Briggs, D. Handbook of X-ray and Ultraviolet Photoelectron Spectroscopy; Heyden: London, 1977. (15) Herder, P. C.; Claesson, P. M.; Herder, C. E. J . CoZloidInterface Sci. 1987, 119, 155.
(1)
where d is the overlayer thickness, L is the inelastic mean free path of the photoelectrons in the sample, Z is the intensity of the K 2p photoelectron signal with an overlayer, ZOis the intensity without an overlayer, and B is the angle between the sample and the detector. The angle can be varied in a well-defined manner since the mica surface is molecularly smooth and flat. A plot of In Z versus -l/sin B should give a straight line with a slope equal to d l l . When the inelastic mean free path, L,ofthe photoelectrons in the sample is known, the thickness, d, of the overlayer can be quantified. Alternatively, when the thickness of the overlayer is known, the inelastic mean free path of the photoelectrons can be determined. The samples for the ESCA measurements were prepared in a laminar flow cabinet as follows. Mica sheets (freshly cleaved) were incubated in M NaCl solutions, pH 5.6, containing 0.001-0.2 mg of lysozymdml (in 12-mL test tubes made of Tefzel (poly(ethylenetetrafluoroethy1ene))). After incubation for 1718 h each test tube containing a mica sample was immersed into 1 L of pure water. The mica sample was then drawn out from the protein solution into the pure water (without exposure to air), gently shaken under water a few times, and then drawn through the aidwater interface. The reason for transferring the sample into water before taking it through the liquidlair interface is to avoid a Langmuir-Blodgett-type deposition of lysozyme. Finally, the sample was dried with a nitrogen jet. Surface Force Apparatus (SFA). Surface forces were studied with a Mark I1 surface force apparatus.lg The force was measured as a function of the separation between two molecularly smooth mica surfaces. The thin (1-3 pm) mica substrate was partially mirrored by silver evaporation on one side, glued (silver side down) onto optically polished silica disks, and mounted in the surface force apparatus in a crossed cylinder configuration. The distance between the two surfaces was determined interferometrically to within 2 A using fringes of equal chromatic order (FECO). The surface separation is controlled either by adjusting the voltage applied to a piezoelectric crystal rigidly attached to one of the surfaces or by a synchronous motor coupled by a cantilever spring to the other surface. Forces were determined from the deflection of this spring with a resolution of N. The force measured between crossed cylinders (FJ a distance D apart normalized by the local geometric mean radius (R)is related to the free energy of interaction per unit area between flat surfaces (Gf)according to the Dejaguin approximation:Z0 F,(D)IR = 2nGAD)
(2)
(16)Seah, M.P.In Practical Surface Analysis, 2nd ed.; Briggs, D., Seah, M. P., Eds.; John Wiley & Sons: Chichester, 1990;Vol. 1. (17)Blomberg, E.; Claesson, P. M.; Gdlander, C.-G. J . Dispersion
Sci. Technol. 1991, 12, 179. (18)Imoto,T.;Johnson,L.N.;North,A.C.T.;Phillips,D.C.;Rupley, J. A. In The Enzymes, 3rd ed.; Boyer, P., Ed.; Academic Press: New York, 1972;Vol. 7. (19)Israelachvili,J. N.; Adams, G. E. J . Chem. SOC.,Furuduy Trans. 1 1978, 74, 975. (20)Derjaguin, B. W.Kolloid-2. 1934, 69, 155.
Lysozyme Adsorption on Mica Studied with an SFA
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Table 1. Adsorbed Layer Characteristics at Adsorption Equilibriums [lysozyme] (mg/mL) 0 0.002 0.02
0.05 0.2
&on-DLVO
(A)
110-150 100-120 100-120 110-150
layer thickness
IqI
aredcharge (A21
(A)
0 60-70 90-100 80-90 90-100
(mv) 85 16 16 25 18
1700 13600 13600 8300 12000
96 96 96 96 96
(A)
K - ~
Dnon-DLVO is the distance where the measured force deviates from the force predicted by the DLVO theory. The layer thickness is the surface separation where a very strong repulsion is encountered. Here an increase in the compressive force hardly affects the surface separation. IYJ is the magnitude of the interfacial potential at large distance,the aredcharge is calculated from using the nonlinear Poission-Boltzmann approximation, and ~ - 1is the Debye length used for calculating the doublelayer force.
This approximation is valid when the radius (about 2 cm) is much larger than, and independent of, the surface separation. The radius used in eq 2 is that of the undeformed surfaces. However, under the action of strongly repulsive forces, particUlarly when the force varies rapidly with surface separation, the glue supporting the surfaces deforms and flattens.21 This increases the local radius, and invalidates eq 2 since R becomes a function of D. When an attractive force component is present, the gradient of the force with respect to the surface separation, W a D , may at some distance become larger than the spring constant, k. The mechanical system then enters an unstable region, causing the surfaces to jump to the next stable point.22 The adhesion force, F(O),normalized by the local mean radius of curvature, R, is determined by separating the surfaces from adhesive contact. The required force is calculated by the relation
(3) where F(Dj)is the force at the distance (Dj)to which the surfaces jumped on separation. Prior to the measurements in lysozyme solution, the surface forces were fist measured in 10-3 M NaCl at pH 5.6. ARer establishing that the measured forces were in agreement with the known interaction in pure electrolyte solutions, the experiment was continued. It should be noted that the measured forces at this stage are in agreement with the DLVO theory, showing no non-DLVO repulsion at short separations. In the next step the protein was injected directly into the electrolyte solution; separate experiments were carried out for each protein concentration. T w o different conditions during the adsorption process were used. In some experiments the adsorption was allowed to proceed with the surfaces only a few micrometers apart. In other experiments the adsorption was allowed to proceed with the two mica surfaces about 2-3 mm apart. All measurements were carried out at room temperature (20-25"). Each force curve plotted in the figures contains data from more than one measurement. TheoreticalAnalysis of the Data. The force profiles were analyzed within the DLVO theory for surface interaction, using additive contribution from the nonretarded van der Waals force and the electrostatic double-layer force calculatedin the nonlinear Poisson-Boltzmann (PB) approximation assuming interaction at constant surface charge. The calculations were performed according to the algorithm of Chan et ~ 1 . 2 3 In the presence of adsorbed proteins, the plane of charge and the origin of the van der Waals force were assumed to be located at the position of the hard wall repulsion (see column labeled layer thickness in Table 1). This corresponds to the proteidaolution interface when the layers are under a compressiveforce. In most cases two extreme cases for the van der Waals force were considered, using either the Hamaker constant A = 2.2 x 10-20 J for mica interacting (21)Parker, J. L.;Attard, P. J. Phys. Chem. 1992,96,10398. (22)Horn,R. G.;Israelachvili,J. N. J.Chen. Phys. 1981,76,1400. (23)Chan, D.Y.;Pashley, R. M.; White,L. R. J.Colloid InterfaceSci. 1980,77,283.
8.00
0.05
0.10
0.16
0.20
0.25
CONCENTRATION (mg/ml) Figure 2. Adsorbed amount of lysozyme as a fundion of the protein concentration, dissolved in lom3M NaCl at pH 6.6. The adsorption time was 18h. The adsorbed amount was measured by meam of ESCA. The dashed and dotted lines represent a hexagonal close packed side-on monolayer (2.07mg/m2) and end-on monolayer (3.10mg/m2), respectively. across water or A = 0, corresponding to no van der Waals force between the hydrated protein layers. The interfacial potentials obtained by fitting the DLVO theory to the measured force should be regarded as apparent values. This is due partly to difficulties associated with determining the plane of charge for the rough (on the molecular scale) lysozyme-coated surface~,1~ and partly due to the approximations in the PB theory, eg., the neglect of ion-ion correlation and ion size e f f e ~ t a . ~ 9 ~ ~ ReSUltS
ESCA Surface Analysis. The adsorption isotherm for lysozyme on muscovite mica after 18 h of adsorption in M NaCl solution a t pH 5.6, measured by means of ESCA, is displayed in Figure 2. The isotherm is of the high-afiinity type, and even a t lysozyme concentration as low as 0.001 mg/mL, the adsorbed amount is rather high, correspondingto nearly a hexagonal close packed end-on monolayer. A gradual increase in the adsorbed amount was observed when the concentration of lysozyme in the solution was increased, and a t a lysozyme concentration of 0.20 mg/mL the adsorbed amount corresponds to 4.0 mg/m2. The angle-resolved depth profiles for 0,001,0.02,and 0.2mg of 1ysozymdmLare shown in Figure 3. The values of d/A (1.17,1.24,and 1.57,respectively) were obtained from the slopes of the lines. The thickness of the uncompressed lysozymelayer was deduced from the range of the non-DLVO forces observed during the surface force measurements (see below). It was about 60 A on each surface for the surfaces prepared from 0.02and 0.2 mg of lysozymdml solutions. The inelastic mean free path, A, for the K 2 p photoelectrons calculated with these values is 48 and 38 A for 0.02 and 0.2 mg of lysozymdml, respectively. The inelastic mean free paths calculated this way are higher than the value of 35 A obtained by a theoretical approach for human serum albumin.26 The (24)yjellander, R.;Marcelja, S. J . Phys. Chem. 1996,90,1230. (25)Guldbrand,L.;Jhsson, B.; WennersMim,H.; Linse, P. J.Chem. Phys. 1984,80,2221. (26)Andrade,J.D.In Surface and Interfacial Aspects of Bwmedical Polymers;Andrade, J. D., Ed.; Plenum Press: New York, 1986;Vol. 1.
Blomberg et al.
2328 Langmuir, Vol. 10,No. 7, 1994 1O.OC'
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-1lsin 8 Figure 3. Angular dependence of the K 2p signal from mica M NaCl bearing an overlayer oflysozyme, adsorbed from a solution, pH 5.6,containing 0.001 mg of lysozymdd (filled squares), 0.02 mg of lysozymdd (open squares), and 0.2 mg of lysozyme/mL (filled circles). The results were fitted using linear regression to give the slope of the line, which represents dll.
theoretical inelastic mean free path is given by26
I = (M/~n)Ed(13.6ln(Ek)- 17.6 - 1400/Ek) (4) where M = the molecular weight of the molecule, n = the number of valence electrons in the molecule, Q = the density, and& = the kinetic energy (eV). It is reasonable to believe that Mln is roughly constant for different proteins since they are built-up of similar subunits. The density for lysozyme is slightly larger thanfor albumin,26-2s making the theoretical value of A slightly smaller. Since the experimentally determined inelastic path is a measure of the atom density of the layer, our results show that the average density of the adsorbed protein layers is less than that of the protein itself. This indicates the presence of voids between the adsorbed proteins, and the volume fraction of voids is smaller for layers obtained by adsorption from more concentrated solutions. This illustrates that the adsorbed protein layer becomes more compact as the bulk concentration of the lysozyme solution is increased. Surface Force Measurements. Figure 4 illustrates the force versus distance profilebetween two mica surfaces interacting across a M NaCl solution at pH 5.6 containing 0.002mg oflysozymelml. The force measured in pure (protein-free) M NaCl is also shown in Figure 4 (inset), and it is consistent with the force predicted from the DLVO theory. At large separations the interaction is dominated by a repulsive electrostatic double-layer force. When the surfaces are brought closer together, the attractive van der Waals force overcomes the repulsive force and dominates the interaction. This causes the surfaces to be pulled into strong adhesive contact from a separation of 20-25 A. The parameters used for calculating the double-layer force were a Debye length ( K - ~ )of 96 A and a surface potential (q) of -85 mV, cor(27) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry Part I: The conformation of biological macromolecules; W. H. Freeman and Co.: New York, 1980; Chapter 2. (28) Gekko, K.; Hasegawa, Y.Biochemistry 198625,6563.
DISTANCE (A) Figure 4. Normalized force as a function of surfaceseparation M NaCl solution at pH 5.6containing 0.002 mg across a of lysozyme/mL. The forces were measured on compression before (open circles)and after (filledcircles)the adsorptionhad come to equilibrium, after 4 and 45 h, respectively. The solid curves represent the forces calculated from the DLVO theory for % = -70 mV and K - ~= 96 A (4h) and % = f16 mV and K - ~= 96 A (45h). The upper line corresponds to A = 0, and J. The inset the lower line corresponds to A = 2.2 x shows the force normalized by the mean radius of the interacting M NaCl solution at pH 5.6. surfaces measured in a pure The solid curvesrepresent the forces calculated from the DLVO theory using the parameters given in Table 1. responding to a n apparent area per charge of 1700 A2. The van der Waals force was calculated by using the J for mica nonretarded Hamaker contantA = 2.2 x interacting across water. The forces between these same mica surfaces were measured again 4 and 45 h after addition of lysozyme a t a concentration of 0.002 mg/mL. During adsorption of the lysozyme the surfaces were kept far apart (2-3 mm). Figures 4 and 5 show that the interactions change during the adsorption. m e r 4 h of adsorption, before attaining equilibrium, the long-range interaction was still dominated by a double-layer force but with a slight decrease in the apparent interfacial potential (3 = -70 mV) compared to the situation in pure salt (K= -85 mV). When the surfaces approach a separation of about 60 A, a force wall is overcome and the surfaces are pulled into a hard wall repulsion located 30 A out from mica-mica contact. Upon decompression a strong adhesion force is observed, 420-30) x lo3 pNIm, acting between the lysozyme molecules and the mica surfaces (Figure 5) with a minimum located at a surface separation of 30 The forces measured after 24 and 45h of adsorption are similar to each other. Hence, the forces recorded correspond to adsorption equilibrium in a 0.002 mg of lysozymelml solution. These forces are shown in Figures 4 and 5. A double-layer force dominates the long-range interaction, but the magnitude of this electrostatic force is significantly lower than before the adsorption had come to equilibrium. The apparent interfacial potential is only f 1 6 mV, corresponding to a n apaprent area per charge of 13.6 x 103A2.When the surfaces approach one another, a n attractive force overcomes the weak double-layer force a t a surface separation of 120-150 A, and the surfaces are pulled to a hard wall repulsion located a t about 60 A. This distance corresponds to one single side-on oriented
A.
Lysozyme Adsorption on Mica Studied with an SFA
Langmuir, Vol. 10, No. 7, 1994 2329
I -1000
-2000
t-
AhA o
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-30000'. . 0
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DISTANCE (A) Figure 5. Force normalized by the local radius as a function of surface separation across a M NaCl solution at pH 5.6 containing0.002 mg of 1ysozymdm.L. The forceswere measured after different adsorption times, after 4 h ((0) compression and (0)decompression), after 24 h ((W) Compression and (0) decompression), and after 45 h ((A) compression and (+) decompression). layer of lysozyme molecules on each mica surface. The adhesion force observed on decompression (at D = 60 A) is about 10 times lower (-2000 pN/m) compared to the strength with only one side-on layer between the surfaces (Figure 5). In this case the attractive force arises from protein-protein interaction rather than from proteinmica interaction. The forces between mica surfaces across a 0.02 and a 0.05mg/mL lysozyme solution containing M NaCl at pH 5.6 are displayed in Figures 6 and 7, respectively. In the 0.02 mg/mL solution, the lysozyme was adsorbed onto the mica surfaces while keeping the surfaces far apart (2-3 mm). At this concentration, the forces measured after 1 h and after 16 h of adsorption are the same except for the adhesion values (see below). In 0.05 mg of lysozymdmL, the protein was adsorbed on mica with the surfaces close together (a few micrometers), and the forces were measured after the adsorption equilibrium (22 h) had been established. At large separations the interactions are dominated by a weak electrostatic double-layer force, with an apparent interfacial potential of f 1 6 (0.02 m@mL) and f 2 5 (0.05 mg/mL) mV, corresponding to a n apparent area per charge of 13.6 x lo3and 8.3 x lo3A2, respectively. A deviation from the force predicted by the DLVO theory (seeTable 1)occurs at a surface separation below 100-120 A. By increasing the compressive force, the surfaces can be pushed to about 90 A (45 &surface) h m mica-mica contact. When the surfaces are separated from contact, they initially follow the same profile as measured on compression. However, an attractive minimum is located at a surface separation of approximately 90-140 A. This is at a separation similar to that of the onset of the steric force measured on pushing the surfaces together. The magnitude of the adhesion force decreases
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DISTANCE (A) Figure 7. Force normalized by the local radius as a function of surface separation across a M NaCl solution at pH 5.6 containing0.05 mg of lysozymdd. The forces were measured &r the adsorptionhad come to equilibrium(22 h of adsorption). Filled and open symbols represent the forces measured on compression and decompression,respectively. and the separation where the minimum of the attractive force is located increases with the number of times the surfaces are compressed together. The forces measured across a 0.2 mg/mL lysozyme solution containing M NaCl at pH 5.6 are shown in Figure 8. The forces were measured before (less than 1 h) and after (more than 20 h) the adsorption had come to equilibrium. The force profile measured after a short adsorption time shows that no double-layer force is present. Hence, the micdysozyme system is uncharged. At a surface separation of approximately 130 A a n attractive force dominates the interaction and the surfaces are ulled into adhesive contact at a separation of 60 A (30&surface) from mica-mica contact. Afurther increase of the compressive force does not cause any significant decrease in surface separation. Hence, a layer of side-on oriented lysozyme molecules remains on each surface. On separation a n attractive minimum is located at a separation of 60 A. The lack of a repulsive double-layer force
Blomberg et al.
2330 Langmuir, Vol. 10,No. 7,1994
" 2000
3
1
0 I
0
*OO0r3000 --l
1000
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0
DISTANCE (A) Figure 8. Force normalized by the local radius as a function of surface separation across a M NaCl solution at pH 5.6 containing0.2mg of lysozymdml. The forces were measured compression and ( 0 )decompression) after 1h of adsorption((0) and after 24 h of adsorption ((W) compression and ( 0 )decompression). The solid curve represents the calculated van der Waals force using an effective Hamaker constant of 8 x J. and the well-defined layer thickness in this case make it possible to estimate the effective Hamaker constant from the jump position. The jump occurs when the gradient of the force (aFlaD) exceeds the spring constant (k). For a three-layer system, mica { 11, layer (2}, and solution {3}, the effective van der Waals force is approximately given byzg
R
where A 2 3 2 = the Hamaker constant for layer-solutionlayer, A 1 2 3 = the Hamaker constant for mica-layersolution, A 1 2 1 = the Hamaker constant for mica-layermica, A,ff = the effective Hamaker constant, D = the distance between mica surfaces, and T = the thickness of the adsorbed layer. The observed jump distance of 70 A gives A,a = 8 x J at D - 2T = 70 A, corresponding to a protein-water-protein Hamaker constant of 3.3 x J. This value is lower than the Hamaker constant J), infor hydrocarbon-water-hydrocarbon (5 x dicating that the adsorbed lysozyme layer under this condition contains a considerable amount of water. The force profile measured after the adsorption has reached equilibrium a t 0.2 mg/mL displays a very weak repulsive double-layer force dominating the long-range interaction. This force can be fitted with the DLVO theory, using a n apparent interfacial potential of f18 mV, corresponding to a n area per charge of 12 x lo3A2. When the surfaces are pressed closer together, a steeply rising repulsion is encountered at 110-150 A. By increasing the compressive force, the surfaces are pushed into a surface separation of 90-100 A (45-50 ksurface) from mica-mica contact. When the surfaces are separated from contact, the force initially follows the same profile as measured on approach. However, an attractive minimum is detected at a surface separation of 120-140 A.
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'
300
DISTANCE (A) Figure 9. Force normalized by the local radius as a function of surface separation. The forces were measured after 16 h of adsorption at a lysozyme concentration of 0.02 mg/mL in M NaCl solution at pH 5.6 (squares) and &r dilution with M NaC1, pH 5.6 (by a factor of 3000),for 3 h (triangles) and 16h (circles). Filled and open symbols represent the force measured on compression and decompression, respectively. In another series of experiments the protein was first allowed to adsorb on the mica surface from a 0.02 mg of lysozymelml solution, containing M NaCl at pH 5.6, for 16 h. The liquid was then removed from the measuring chamber, leaving a drop of protein solution between the mica surfaces. The protein concentration was then diluted by a factor of about 3000 by refilling the whole measuring chamber with protein-free 10-3MNaC1,pH 5.6. The forces measured before and after dilution (3 and 16 h) are illustrated in Figure 9. The magnitude ofthe electrostatic double-layer force and the compressed layer thickness were unchanged after 16 h of dilution, and the adhesion force observed upon decompression of the surfaces shows the same profile as before dilution. Thus, to a large extent the adsorption of positively charged lysozyme onto negatively charged mica is irreversible with respect to dilution with M NaCl. At this stage one of the surfaces was replaced with one bare mica surface, and the force between one lysozyme-coated surface and one bare mica surface was measured (Figure 10). As expected the compressed layer thickness decreased to half its previous value, i.e., 50 A, while the magnitude of the long-range electrostatic double-layer force and the adhesion force between one lysozyme-coated and one bare mica surface increased, compared to that between the two lysozyme-coated surfaces. In this case, calculation of the forcesusing DLVO theory for unlike charged surfaces interacting at constant charge was carried out using the elliptical integral formalism of Bell and P e t e r ~ e n .In ~ ~these calculations the potentials obtained for bare mica in M NaCl(-85 mV) on one surface and that obtained betwen lysozymecoated surfaces in 0.02 mg of 1ysozymdmL ( f 1 6 mV) for the other surface were used. Good agreement between the measured and calculated long-range forces was obtained when using the value -16 mV for the proteincoated surface. Hence, at least affer dilution the proteincoated surface is negatively charged. At distances below about 200 A,the measured forces were less repulsive than those calculated, indicating some charge regulation. (30) Bell, G. M.; Petersen, G. J. J . Colloid ZnteTface Sci. 1972, 41,
(29) Ninham, B. W.; Parsegian, V. A. J . Chem.Phys. 1970,52,4578.
542.
Langmuir, Vol. 10, No. 7,1994 2331
Lysozyme Adsorption on Mica Studied with a n SFA
so00
s.
L
YJ
~ l o O o ~
w
-1000
2
[
0 0
0 r
:
/' ,
DISTANCE (A) Figure 10. Force normalized by the local radius of the interacting surfaces as a function of surface separation. The forces were measured after 24 h of adsorption at a lysozyme M NaC1, pH 5.6,followed concentration of 0.02 mg/mL in M NaCl, pH 5.6 (by a factor of 3000). by dilution with Filled squares and circles represent the force measurd on compression and decompression, respectively, between two lysozyme-coated mica surfaces. Open squares and circles represent the force measured on compression and decompression, respectively, between one lysozyme-coated mica surface and one bare mica surface. The inset shows the force measured on compression on a logarithmic scale. The solid curves represent the forces calculated for DLVO theory for = -85 and f16 mV (one bare mica surface and one lysozyme-coated surface, respectively, and K - ~= 96 A). The parameters used for two lysozyme-coated surfaces are given in Table 1. The Hamaker constant for mica interacting across water (A = 2.2 x J) was used in both cases.
Discussion The System. Muscovite mica is a layered aluminosilicate mineral. Each layer has a large negative charge due to isomorphous substitution of aluminum for silicon. In the crystal this charge is compensated by ions (mainly potassium) located between the layers. When mica is immersed in aqueous solutions, the potasium ions on the surface are dissolved and exchanged for other cations, in this case protons and sodium ions. There are 2.1 x 1014 negative sites/cm2(48&/charged site)on the mica surface. However, due to ion adsorption the resulting surface charge density is much lower, and the apparent area per M NaCl at pH negative charge is about 1700 & in 5.5-6 (Figure 4, inset). Lysozyme is a rather compact protein with a n ellipsoidal sha e and approximate crystal dimensions of 45 x 30 x 30 The lysozyme molecule is cross-linked by four disulfide bonds, which contribute to its high conformational stability.'* Lysozyme carries a net charge of +9 per molecule at pH 5.6,31with most of the positive charge located around the active site cleR which in turn is situated on a side parallel to the molecule's long axis. On the side opposite the active cleR is a relatively large hydrophobic patch that may be involved in the dimerization oflysozyme, known to take place in concentrated solution^.^^ Double-LayerForce and Interfacial Charge. The magnitude of the repulsive double-layer force decreases
8.
(31)Tanford, C.;Wagner, M. L. J . Am. Chem. SOC.1984,76,3331. (32)Deonier, R. C.;Williams, J. W. Biochemistry 1970,9,4260.
when lysozyme is introduced into the solution and positively charged lysozyme associates with the negatively charged mica surfaces. The decay length of the doublelayer force in monovalent electrolyte solutions is accurately given by the Debye length (K-'), but it has been observed by Kbkicheff et al. that corrections are needed in order to take into account the effect of the highly charged protein.33 In our case, however, we found that the measured decay length was close to the theoretically expected value for a 10-3 M 1:l electrolyte solution, and this value was used in all calculations. Adsorption of a hexagonally close packed monolayer of side-on oriented lysozyme a t pH 5.6 would introduce a surface charge density of one positive charge per 130 &, more than an order of magnitude in excess of what is needed to neutralize the surface charge of mica in M NaCl at pH 5.6 (-1700 k). ESCA analysis of mica surfaces after immersion in a 0.2mg/mL lysozyme solution (see Figure 2) have shown that the adsorbed amount is considerably more (4.0 mg/m2)than that needed to neutralize the mica charge. Nevertheless, no recharging of the surface is observed. This can be explained by the following: (i)The adsorption of lysozyme is, as in the case ofcationic surfactant adsorption, followed by i o n - e ~ c h a n g e . ~ In~this * ~ ~case protons and sodium ions are displaced from the mica surface. (ii)Lysozyme adsorbs with most of the positive charges toward the mica surface. Hence, the ionizable groups are transferred to a low dielectric environment, leading to a n increase of the electrostatic self-energy. This causes a shift of all acidbase equilibria toward the uncharged state and results in a coadsorption of small ions into the adsorbed layer. This phenomenon was originally discussed by Norde and Lyklema. Short-Range Forces. The surface force technique measures the total force between the surfaces. We first analyzed this force using the DLVO theory. Even though it is not obvious where to locate the plane of charge or how to accurately take into account the van der Waals force between the adsorbed layers,17it is clear that additional forces besides van der Waals and electrostatic forces are present. Repulsive non-DLVO forces may be due to (i) removal of loosely bound molecules, (ii)compression and dehydration of molecules strongly bound to the surfaces, and (iii) deformation of the glue supporting the mica surfaces. The importance of the glue deformation has recently been considered theoretically.21 It was shown that this force contribution can be completely neglected when the force has the strength and range as observed for mica surfaces interacting across a M NaCl solution.21 For the present case glue deformation may affect the distance dependence of the short-range force when the surfaces are compressed firmly together. However, under these conditions, the force observed is close to that of a hard wall and we do not attempt to extract the force law. Hence, from now on, this force contribution will be neglected. At the lowest lysozyme concentration, 0.002 mg/mL, in the initial stage of adsorption (preequilibrium) a n attractive force starts to dominate the interaction a t distances below 60 A, and the surfaces are pulled into a strong adhesive contact (Figures 4 and 5). This attractive force is more long-ranged than the attractive van der Waals force predicted from the DLVO theory. We interpret this as follows: At this low concentration and short adsorption time the molecules adsorb on the mica surfaces as a loosely 7935
(33)Kbkicheff, P.;Ninham, B. W. Enrophys. Lett. 1BBO,12,471. (34)Rutland, M.W.; Waltermo, A.; Claesson, P. M. Langmuir 1992, 8,176. (35)van Dulm, P.;Norde, W.; Lyklema, J. J . Colloid Interface Sci. 1981,82,77.
2332 Langmuir, Vol. 10, No. 7, 1994 packed mainly side-on oriented layer. At a separation of 60 A,molecules on the opposing surfaces come into contact and some proteins intercalate to form a single lysozyme layer between the surfaces whereas others may leave the gap between the surfaces. When this occurs, the repulsive force decreases and the surfaces move inward. The final layer thickness of 30 A is consistent with one side-on oriented layer remaining between the surfaces. Once adsorption equilibrium has been established a more densely packed layer is adsorbed on each surface. This means that it is harder for the molecules to move along the surface and they can no longer intercalate or be pushed out. Instead an attractive force dominates the interaction from a distance of 120-150 A,where the surfaces are pulled into a hard wall repulsion located a t a separation of 60 A, consistent with one side-on oriented lysozyme layer on each mica surface. At the higher concentrations of lysozyme, 0.02-0.2 mgl mL, repulsive non-DLVO forces are present a t a surface separation below 110-150 A (most often a t 120 A). The outermost portion of the non-DLVOrepulsion has a rather poor reproducibility which suggests that this force is due to a n outer, weakly bound layer which is squeezed out from the contact zone when the surfaces are compressed together rather than a hydration force. That the possibilities for multilayer formation do exist is seen by the fact that there is a n attractive force between the adsorbed lysozyme layers (Figures 6-8). It is relevant that a t the bulk concentration used in these experiments lysozyme does not form dimers, but it does dimerize at higher concentration^.^^ In the small gap between the mica surfaces there is a higher local protein concentration than in bulk solution. It is likely that the molecular contacts that are involved in the solution-phase dimerization may also be responsible for the observed attraction between the adsorbed layers and the buildup of the outer layer observed a t the higher lysozyme concentrations. For insulin, a protein that also associates in solution, a similar but stronger adhesive force was observed and with time a multilayer was d e ~ e l o p e d . ~ ~ Buildup of the Adsorbed Layer. By comparing the dimensions of the lysozyme molecule with the range of the non-DLVOforces and the compressed layer thickness, some structural features of the adsorbed layer can be inferred. Much of this has already been discussed above and, for the highest lysozyme concentration, in a previous p ~ b l i c a t i o n Here . ~ ~ we instead focus on how the structure of the adsorbed layer developed with time and with increasing lysozyme concentration. I t should be stressed here that the long time scale of the adsorption process reported here is due to the experimental conditions where the surfaces are kept rather close together and the solution is unstirred. Hence, the adsorption process is slow compared to, for example, a n ellipsometric experiment with a single surface and a stirred solution.1° A further consequence is that the adsorption kinetics in the surface force experiment will depend on the injection rate and the absolute distance between the surfaces during equilibration. This is illustrated in Figure 11where the adsorption of 0.002 mg of 1ysozymeImL onto the mica surface was allowed to proceed under two different conditions. (i) In the first case the surfaces were kept only a few micrometers apart. Under these conditions the adsorption kinetics was so slow that after 20 h of equilibration only a very loosely packed layer of lysozyme was adsorbed onto the mica surface. By compressing the surfaces together, the (36)Claesson, p. M.; &nebrant, T.;BergensthH, B.;Nylander,T.J. Colloid Interface Sci. 1989,130, 130. (37) Tilton, R. D.; Blomberg, E.; Claesson, P. M. Langmdr 199S,9, 2102.
Blomberg et al. loooo
M
.
DISTANCE (A)
Figure 11. Force normalized by the local radius as a function of surface separation across a M NaCl solution at pH 5.6 containingO.002mg of lysozymdml. The forces were measured after two different adsorption conditions. The surfaces were kept close together, a few micrometers (open circles),and far apart, 2-3 mm (filledcircles),duringthe adsorption. The solid curves represent the forces calculated from the DLVO theory for 'I$ = -65 mV and K - ~= 110 A (0)and 'I$ = f 1 6 mV and K - ~= 96 A (0).The upper line corresponds to A = 0, and the J. lower line corresponds t o A = 2.2 x lysozyme molecules were forced to move laterally along the mica surface and intercalate to form one layer between the mica surfaces. Finally, they were expelled entirely from the gap by the application of a high enough compressive force. (ii)In the second case the adsorption was performed with the surfaces a few millimeters apart. Under these conditions, the adsorption kinetics was much faster and one densely packed lysozyme layer was formed on each surface. Hence, in our opinion the surface force apparatus used without a continuous stirring is not suitable for adsorption kinetics studies. Consequently the data obtained before equilibrium has been established do not provide any generally valid information of the adsorption kinetics. Nevertheless, these results are interesting since they provide some information about how the buildup of the adsorbed layer proceeds. When the lysozyme concentration is low (0.002 mg/mL), a single side-on oriented monolayer (thickness about 30 A) on each surface is obtained (Figures 4, 5, and 12a). When the lysozyme concentration is increased further to 0.02 mg/mL, the increased adsorption changes the situation. For instance, when adsorption equilibrium has been reached, a t a lysozyme concentration of 0.02 mg/mL or above, we have not observed forces that are consistent with molecules adsorbing exclusively in a side-on fashion. Instead, the range of the non-DLVO repulsion starts at a separation of 110-150 A,indicating the presence of molecules also adsorbed end-on and most likely a surface dimerization of molecules adsorbed side-on. By applying a higher compressive load, a final separation of 90-100 A (Figure 12b) can be reached, suggesting that some of the dimers are split up. This together with rearrangements leads to the observed decrease in the thickness of the layer between the surfaces. A similar trend has also been reported by Lee and Be1fo1-t~~ when studying ribonuclease A on mica. At short times incompressible side-on is
an
(38) Lee, C.-S.; Belfort, G. Proc. Natl. h a d . Sci. U S A . 1989, 86, 8392.
Lysozyme Adsorption on Mica Studied with an SFA
+
lmAI
+
Jump
No jump
Figure 12. A schematic illustration of the structure of the adsorbed lysozyme layers on mica at two different Concentrations: (A) 0.002 mg of lysozymdml showing two mica surfaces with side-on proteins at a surface separation of 120-150 where the surfaces jump to a separation of 60-70 A, corresponding to one layer of side-on oriented molecules on each surface; (B)0.2mg oflysozymdml showingthat initial contact occurs via pairs of side-on dimers at 120 A. Continued compressionsqueezes out one protein from each dimer contact, allowing a layer with a thickness of 90-100 to be developed, corresponding to contact of three side-on and/or two end-on protein molecules.
adsorbed on each surface,while at longer adsorption times the ribonucleaseA moleculesreorient to an (atleast partly) incompressible end-on monolayer on each surface. They could also correlate the change in surface orientation with changes in enzymatic activity. The adsorption of proteins in more than one layer, as suggested for lysozyme in this study, has also been observed with the surface force technique for other proteins, including insulin on hydrophobic surfaces,36side-on layers of bovine serum albumin on and cytochrome c at the isoelectric point on mica.40 In the cytochrome c case it was observed that the outer weakly adsorbed layers could be squeezed out by compressing the surfaces closer together. In this context it should be noted that the number of proteins located in the outer layer is likely much less than in the inner layer. This means that the presence of an outer layer would be hard to detect with techniques that basically determine the adsorbed amount, eg., ellipsometry.1° With the surface force technique the situation is the reverse; a few molecules adsorbed in an outer layer, or adsorbed end-on, may dominatethe interaction. Hence, the presence of these molecules is emphasized. A small increase in the adsorbed amount with increased lysozyme concentration is observed in the adsorption isotherm measured by means of ESCA (Figure 2). By comparing the experimentallydetermined adsorbed amount with the calculated value for a hexagonal close packed side-on monolayer and end-on monolayer, one observes that even at the lowest lysozyme concentration investigated some end-on oriented molecules are likely to be adsorbed on the mica surface. This appears to be inconsistent with the layer thickness obtained by the surface force technique at (39) Gallinet, J.-P.; Gauthier-Manuel, B. Colloids Surf 1992, 68, 189. (40) Kbkicheff, P.; Ducker, W. A.; Ninham, B. W.; Pileni, M. P. Langmuir 1990,6, 1704.
Langmuir, Vol. 10, No. 7, 1994 2333 0.002 mg of lysozyme/mL (corresponding to a densely packed side-on oriented monolayer). This may indicate that the agitation in the system (Le., how rapidly adsorption occurs) influences the total adsorbed amount. At a protein concentration above 0.1 mg/mL the measured adsorbed amount is larger than that for a hexagonal close packed end-on monolayer, indicating the presence of an outer layer of loosely packed molecules which is consistent with the surface force data. We note that the conditions in the ESCA measurements are different from those in the surface force technique in the sense that the ESCA measurements are carried out under high vacuum. This means that when preparing the sample for the ESCA experiments the surface has to be drawn through the air/ water interface. This could cause some of the lysozyme molecules in the outer layer to desorb during the transport through the aidwater interface. Adhesion Forces. The attractive force between surfaces separated by one side-on oriented lysozyme layer is very strong (Figure 5). In this orientation the positively charged residues in the protein molecules can interact with the negative sites on the mica surfaces, at the same time as neighboring molecules may interact via their hydrophobic regions, clearly a favorable structure. A considerablyless favorable packing is obtained when one layer of lysozyme oriented both end-on and side-on is present between the surfaces. This is the situation when one clean mica surface was brought into contact with an adsorbed layer formed at 0.02 mg/mL. In this case only a few of the molecules (the ones oriented end-on)are able to bridge between the two mica surfaces. This can explain the much lower adhesion in this case (compare Figures 5 and 10). In a previous publication the force acting between one human serum albumin (HSA)-coatedsurface and one bare mica surface was investigated. The adhesion between the HSA-coated surface and the mica surface was much larger than between one lysozyme-coated surface and one bare mica surface after the adsorption had come to eq~ilibrium.~'Belfort and Lee have been studying the force acting between one mica surface coated with preadsorbed ribonuclease A (RNase A) and one bare mica surface.42 In this case the magnitude of the adhesion was smaller than for lysozyme-mica. Clearly, the shortrange adhesion force between the protein and the mica surface depends on the detailed structure of the adsorbed layer includingthe presence of end-on oriented molecules, the arrangement of charged groups, and possibly surfaceinduced conformational changes. Hence, at present we see no way of predicting these forces from a knowledge of the surface structure and the structure of the protein in the crystal or in solution. There is also an attractive force present when more than one lysozyme layer is present between the surfaces (Figures 6-8). The van der Waals force between the protein layers contributes to this attraction, but it is likely that other forces such as interactions between hydrophobic patches also contribute. Compressibility/Sttural Stability* By comparing surface force results for layers composed of flexible proteins with those for layers of proteins with a more compact structure, information concerning the relationship between layer compressibility and stability of the protein structure can be obtained. A protein with a large flexible structure like mucin adsorbs onto bare mica43and hydrophobized mica4 in a (41) Blomberg, E.; Claeeeon, P. M.; Tilton, R. D.J . Cotbid Inte$zce Sci. in press. (42) Belfort, G.; Lee, C.-S. Pmc. Natl. &ad. Sci. USA. 1991,88, 9146. (43) Perez, E.; prouet,J. E.J. Colloid Interface Sci. 1987,118,182.
2334 Langmuir, Vol.10,No. 7,1994
comparatively flat configuration considering the high molecular weight, M,, and large radius of gyration, RG, in solution. Mucin consists of a linear polypeptide chain with linked carbohydrates, and has a random-coil conformation in solution. The mucins used by us in an earlier study had a molecular wei h t of (5-25)x lo6and a radius ofgyration of 1900-2100 . The range of the steric force, about 1000 A, was small by comparison.44 It was also observed that the thickness of the adsorbed layer decreases with increasing charge density of the mucin used. Human serum albumin (HSA) is a globular protein with a rather flexible structure. It consists of three compact units, linked together by short flexible regions. The molecular weight is 66 000, and the overall crystal dimensions are 40 x 40 x 140 A. When HSA adsorbs onto bare mica at low concentrations, a small conformational change is induced and further changes can be induced by applying a compressive force.G At higher HSA concentrations it is harder to determine if conformational changes are induced by adsorption, but the adsorbed layer is c ~ m p r e s s i b l e .Measurements ~~ of the force between mica surfaces coated with bovine serum albumin (BSA), at pH 5.5, show a highly compressible protein layer.46 Globular proteins with more compact structures like myelin ba~icprotein,4~@ i n . ~ u l i n , 3ribonuclease ~,~~ A,38and cytochrome c40,47,48show a smaller tendency to change conformation when adsorbing onto solid surfaces. For all these proteins it has been observed that the adsorbed layer thickness is consistent with the dimensions of the proteins, and hardly any change in the layer thickness occurs upon compression. In the case of insulin adsorbing onto bare micamand onto hydrophobized mica36a firmly bound layer with a thickness consistent with that expected for a layer of hexamers or end-on oriented dimers on each surface is developed. The present study shows that lysozyme behaves essentially as the other compact proteins in the sense that no large structural changes are evident. We cannot, however, rule out the possibility that small
1
(44)Malmsten, M.; Blomberg, E.; Claesson, P. M.; Carlstedt, I.; Ljusegren, I. J. Colloid Interface Sei. 1992,151, 579. (45) Blomberg, E.; Claesson, P. M.; Christenson, H. K.J. Colloid Interface Sei. 1990,138, 291. (46) Fitzpatrick, H.; Luckham, P. F.; Eriksen, S.; Hammond, K. Colloids Surf. 1992, 65, 43. (47)Afshar-Rad, T.; Bailey, A. 1.; Luckham, P. F.; MacNaughtan, W.; Chapman, D. Colloids Surf. ISM,31, 125. (48) Luckham, P. F.; Ansarifar, M. A. Br. Polym. J. 1990,22,233. (49) Nylander, T.; Kbkicheff,P.; Ninham, B. J. Colloid Interface Sei. 1994,164,136.
Blomberg et al. changes in the structure may occur but escape detection with this technique. A measure of the structural flexibility ofproteins in solution is the adiabatic compressibility, ,&. -All the proteins studied by Gekko et aLZ8had a positive pBvalue, indicating a large internal compressibility. The partial specificvolume, the hydrophobicity, and the helix elements are factors that increase the adiabatic compressibility. Serum albumin has a higher BI!, value than lysozyme, cytochrome c, insulin, and ribonuclease A, illustrating that serum albumin has a higher compressibility than the more compact globular proteins. The surface force data for proteins on solid surfaces show the same trend, indicating as expected that the internal compressibility in solution is important for the structural stability of proteins on solid surfaces.
Conclusions From the present study it can be concluded that when lysozyme is adsorbed onto mica surfaces from solution, a t low lysozyme concentrations, one dense side-on monolayer M adsorbs irreversibly with respect to dilution with NaC1. When the protein concentration in the solution is increased to 0.02 mg/mL and above, end-on adsorption also occurs and some adsorption in a n outer, more mobile layer is detected. Adsorption of proteins together with small ions neutralizes the surface charge, and consequently only a very weak double-layer force is present between the lysozyme-coated surfaces. When separating the protein layers from contact, a n adhesion force is present, demonstrating the existence of a n interprotein attraction that is involved in the multilayer formation of lysozyme. The short-range bridging attraction between lysozyme and mica is dependent on the orientation of the adsorbed protein molecules. Side-on oriented molecules give rise to a much stronger adhesion force compared to end-on oriented molecules. By comparing the compressed layer thicknesses with the dimension of the lysozyme molecule, it can be concluded that the adsorption of lysozyme on mica does not induce any large-scale conformational changes in the protein molecule. Acknowledgment. We would like to thank Professor J a n Christer Eriksson for valued comments on the paper and Gilbert Carlsson and Anders Svensk for operating the ESCA spectrometer. E.B., P.M.C., and J.C.F. acknowledge the Swedish Research Council for Engineering Sciences (TFR) for financial support.
