Proteins at Interfaces II - American Chemical Society

Chapter 21

Proteins at Surfaces Studied with the Surface Force Technique

Downloaded by CORNELL UNIV on October 1, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch021

Eva Blomberg and Per M . Claesson Laboratory for Chemical Surface Science, Department of Chemistry and Physical Chemistry, Royal Institute of Technology, S-100 44 Stockholm, Sweden, and Institute for Surface Chemistry, Box 5607, S-114 86 Stockholm, Sweden

Some results obtained by using the interferometric surface force technique for studying the interactions between adsorbed protein layers and between such layers and surfaces are presented. We have chosen to report results obtained for several types of proteins in order to emphasize differences and similarities in the behaviour. In this chapter we have also included sections describing the normal experimental procedure as well as some common difficulties which we have encountered during our studies of proteins with the surface force technique. It is hoped that these sections can be of use for readers that has no or a limited experience with this technique. The surface of proteins are generally very heterogeneous with positive and negative charges, groups with hydrogen bonding abilities, as well as non polar hydrophobic regions. This complexity of the protein surface means that each type of protein can interact with other molecules and surfaces in a great number of ways. There are possibilities for ionic interactions (both repulsive and attractive), hydrogen bonding, hydrophobic interaction, hydration forces, acid-base interactions, and, of course, the always present van der Waals force. The most important driving forces for protein adsorption are often regarded to be hydrophobic interaction and ionic interactions, combined with an entropy gain caused by conformational changes of the protein during the adsorption (7,2). Due to the assymetry of the protein surface, the importance of all of these interactions during an adsorption event will depend on the orientation of the molecule. Considering the large difference in overall size, shape and flexibility of different types of proteins (see e.g. Figure 1) it is of course not possible to treat proteins as a homogeneous group of molecules. Instead, a large flexible glycoprotein such as mucin will have completely different solution and interfacial properties than a small compact protein like lysozyme. For this reason one should be very cautious about drawing general conclusions about the behaviour of proteins at interfaces based on studies of only a few types of proteins. Nevertheless, the need of science (and scientists) to be able to predict the behaviour of as yet not studied systems is ever present. In this chapter we have grouped the proteins studied into four classes, compact globular, soft globular, amphiphilic and random coil like proteins. This distinction is of course not strict but we feel that different proteins which fall into the same category w i l l have at least some aspects 0097-6156/95/0602-0296$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Proteins at Surfaces

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Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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PROTEINS AT INTERFACES II

of their interfacial behaviour in common, even though they certainly also will behave differently in many ways. A number of review articles on somewhat similar subjects to what is presented here has recently been published. Among these Leckband and Israelachvili (3) focus mainly on molecular recognition, whereas Luckham and Hartley in a review on interactions between biosurfaces have included a section discussing protein interactions (4). We hope that this chapter will serve as a complement to their presentations. In the limited space of the chapter we have chosen to discuss mosdy data obtained from our own research. Hence, this chapter is not a review of the existing knowledge in the field of surface forces and proteins. However, in the near future we intend to publish a review on the subject that also discusses the many important contributions from other research groups.


Surface Force Technique With the interferometric surface force technique (5,6) the total force acting between two macroscopic (A « 1 cm ) molecularly smooth surfaces in a crossed cylinder configuration is measured as a function of surface separation. This choice of geometry is made due to its experimental convenience. There will be only one contact position, which easily can be changed by moving the surfaces, and there is no problem with aligning the surfaces. One of the surfaces are mounted on a piezo electric tube that is employed to change the surface separation. The other surface is mounted on the force measuring spring. Muscovite mica, a layered aluminosilicate mineral, is the preferred substrate in these measurements due to the ease with which large molecularly smooth thin sheets can be obtained. The surface chemical properties of these surfaces can be modified in a number of ways including adsorption from solution, Langmuir-Blodgett deposition, and plasma treatment (in some cases followed by reactions with chlorosilanes). The mica sheets are silvered on their back-side and glued onto half-cylindrical silica discs (silvered side down), normally using an epoxy glue. When white light is introduced perpendicular to the surfaces an optical cavity is formed between the silvered backsides of the mica surfaces. From the wavelengths of the standing waves produced the surface separation can be measured to within 0.1-0.2 nm. The deflection of the force measuring spring can also be determined interferometrically, and the force is calculated from Hooke's law. 2

Typical Experimental Procedure and Data Evaluation The procedure used when studying the interactions between protein layers adsorbed to solid surfaces is briefly described below. The experimental results are sensitive to contaminants, particularly particles adsorbed to the surfaces, and therefore it is advisable to always make some control experiments before the actual research is started. Once the two surfaces are mounted in the surface force apparatus they are brought into contact in dry air. When the surfaces are uncontaminated the long-range force is due to the attractive van der Waals force and the adhesion between the surfaces is high. In the next step, the measuring chamber is filled with pure water or a weak aqueous electrolyte solution. Under these conditions it is well established that the long-range force is dominated by a repulsive electrostatic double-layer force and the short-range interaction by a van der Waals attraction, and that the measured forces are consistent with theoretical predictions based on the D L V O theory. Hence, it is easy to establish that the measured forces at this stage are as expected in an uncontaminated system. The adhesive contact between the surfaces in water (i.e. the wavelengths of the standing waves with the surfaces in contact) defines the zero


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surface separation. When everything appears as normal the salt concentration and pH is changed to the values preferred under the experiment. The forces are measured a few times and the results are checked for reproducibility and compared with previous results (if available). It is only at this stage that the protein is introduced into the measuring chamber and the true research can be started. The force is not measured between individual molecules but rather all molecules over an area of tens to hundreds p m contribute to the force. Hence, the measured total force is an average force that depends on the orientation of the molecules. When the driving force for adsorption of one part of the molecule is very different to that of the other part, e.g. surfactants on a hydrophobic surface, one orientation of the molecules will dominate. In other cases, like for many proteins on surfaces one can expect that different orientations on the surface are possible and the force measured is then an average over these orientations. The force (F ) is measured between crossed cylinders with a geometric mean radius /?, which is related to the free energy of interaction between flat surfaces per unit area (W) (7,8): 2


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where W{ represents the different types of forces acting between the surfaces. This relation is valid provided that R (about 2 cm) is much larger than the range of the forces between the surfaces (typically 10" cm or less). A requirement fulfilled in this experimental set-up. The local radius can be measured from the shape of the standing wave pattern. Experimentally it is found that under the influence of strong forces, particularly when the forces vary rapidly with surface separation, the local radius will change as the surfaces are pushed firmly together (9,70). This is due to the flattening of the glue supporting the mica surfaces. (Note that the measured surface separation is not influenced by the deformation of the glue since the compressed glue is located outside the optical cavity). When the shape of the surfaces changes with the separation the Derjaguin approximation is no longer valid. An increased understanding of the results can often be obtained by interpreting the measured total force in terms of various force contributions. However, it should be stressed that this procedure in itself is an approximation since all forces are interrelated and not strictly independent. Nevertheless, it is often a useful approximation. The most common procedure is to calculate the electrostatic doublelayer force and the van der Waals force for the system under investigation. The discrepancy between measured and calculated forces is interpreted in terms of other force contributions. A complication in this respect is that in general the location of the protein layer - solution interface is not well defined. This means that the location of the plane of charge, from which the double-layer force acts, is not well defined. A further complication is that the dielectric properties of the adsorbed protein layer is not well known and therefore the van der Waals interaction is hard to calculate with a good precision. In practise we have in our analysis assumed the plane of charge to be at the distance where the short-range force due to the compression of the protein becomes dominant. This is a reasonable choice since we know that at smaller distances the Poisson-Boltzmann treatment of the double-layer certainly does not hold. When highly charged proteins are present in solution it has been shown that they make a significant contribution to the electrostatic decay-length in low ionic strength solutions (77). For the results presented here, this effect is not important. For the van der Waals force we have chosen the same plane of origin as for the doublelayer force. Two extreme cases for the van der Waals force were considered, using either the Hamaker constant A = 2.2 x 10" J, equal to that for mica interacting 5

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across water, or A = 0, corresponding to no van der Waals force between the hydrated protein layers. From this discussion it should be clear that the comparison between theoretical and experimental force curves is not straightforward and that only large discrepancies should be regarded as significant. Compact Globular Proteins Lysozyme is a small compact protein with dimensions approximately 4.5 x 3 x 3 nm. It is positively charged at pH 5.6, the pH used in this study. The positive charges on the molecule are located in such away that it is difficult to use electrostatic considerations to predict the orientation of lysozyme on negatively charged surfaces. The forces acting between mica surfaces across a 10" M NaCl solution containing various amounts of lysozyme are shown in Figure 2. Without any lysozyme in solution the forces are well described by D L V O theory with a repulsive double-layer force dominating at large separations (apparent surface potential -85 mV) and an attractive van der Waals interaction predominating at distances below 2 nm. When lysozyme is introduced into the surface force apparatus to a concentration of 0.002 mg/ml the forces changes dramatically (Figure 2). The repulsive double layer force is much reduced (magnitude of the apparent interfacial potential 16 mV) and a steric repulsion due to the adsorbed layer is present at distances below about 6 nm. This corresponds to contact between side-on oriented lysozyme on each surface. A further increase in lysozyme concentration to 0.02 mg/ml does hardly affect the interfacial potential. However, the thickness of the adsorbed layer has increased significantly to about 9-10 nm. This corresponds to contact between end-on oriented molecules. A further increase in lysozyme concentration to 0.2 mg/ml hardly affects the measured forces. We can draw three conclusions from these results. First, when the lysozyme concentration is increased some molecules adsorb in an end-on orientation and we most likely end up with a layer composed of proteins adsorbed end-on and side-on. Second, the additional adsorption of the charged proteins hardly affects the interfacial charge. Hence, due to the low dielectric constant of the adsorbed layer free charges are not easily incorporated, and upon adsorption acid-base equilibria are shifted towards the uncharged state and small counterions co-adsorb. This confirms the conclusion made by Norde based on electrophoretic mobility measurements and electron spin resonance (ESR) spectroscopy (72,13). Third, the layer thicknesses obtained are consistent with the size of the molecules in solution. Hence, no global changes in the protein structure occur upon adsorption or upon applying an external compressive force. In this respect lysozyme behaves like other small compact proteins like insulin, (77,14), cytochrome C (15-17), RNAse (18), and myelin basic protein (16,17). It should be noted, however, that small changes in the protein structure do take place upon adsorption of lysozyme onto silica. This can be seen clearly using e.g. CD-spectroscopy (79). Such small changes in the structure of the protein is not easily detected with the surface force technique. At the higher protein concentrations a weak non-electrostatic repulsion is observed at separations between 12 and 10 nm. The force in this distance regime is more repulsive on approach than on separation. These findings indicate that a second layer of lysozyme is weakly associated with the firmly bound one, most likely through association via the hydrophobic patch located opposite to the active cleft. It is the work needed to remove this layer from the contact zone that gives rise to the repulsion observed between 12 and 10 nm. Clearly, the layer does not reform during the time it takes to measure the force profile. However, the outer layer does reform when the surfaces are left apart. The formation of an outer layer is consistent with the fact that there is an attractive force between the adsorbed (mono) layers (Figure 2) and that lysozyme molecules form dimers in solution at high enough concentrations (20). It should be emphasized that the surface force technique readily detects the


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presence of any weakly adsorbed outer layer on weakly charged surfaces since the presence of this layer affects the weak long-range forces. A similar layer is not so easily detected by e.g. ellipsometry which primarily measures the adsorbed amount, a quantity which hardly is affected by a few molecules adsorbed in an outer layer. More information about the forces acting between lysozyme coated surfaces can be found in 27,22 Insulin is also a small compact protein. It readily associates in solution to form dimers, tetramers and hexamers (23,24). The overall shape of some of the different solution species is shown in Figure 1. The adsorption of insulin on mica (77) and hydrophobized mica (14) and the resulting interaction forces have been studied with the interferometric surface force technique. On hydrophobized mica it was possible to follow the build-up of the adsorbed layer. The forces measured across hydrophobized mica (i.e. mica coated with a Langmuir Blodgett layer of dimethyldioctadecyl ammonium bromide) across a solution containing 1.9 mg/ml insulin crystals at pH 7.3 are shown in Figure 3. Under this solution condition the fraction of monomers, dimers, tetramers and hexamers are 0.09,0.65,0.26, and 0.01, respectively. As the adsorption proceeds the layer thickness and the repulsive doublelayer force, dominating at D > 12 nm, increases. The results are consistent with an initial adsorption of monomers or dimers in side-on conformation. The layer thickness also increases with the adsorption time indicating formation of hexamers on the surface. Hence, a more dramatic build-up of the protein layer is observed for insulin than for lysozyme. It seems plausible to assume that whenever there is possibilities for attractive interactions between proteins, more than one layer of proteins may eventually adsorb. This has particularly clearly been demonstrated for adsorption of cytochrome C at the isoelectric point on mica (75). Soft Globular Proteins Human serum albumin (HSA) is a globular but rather flexible protein with an overall dimension of 14 x 4 x 4 nm. It consists of three globular units held together by short flexible regions. This structure was initially confirmed for fatty acid free H S A crystallized from polyethylene glycol ( M « 400 g/mol) by X-ray diffraction studies using multiple isomorphous replacement (MIR) (25). However, more recent more accurate MIR X-ray diffraction studies of the same protein instead indicate that the overall shape can be approximated with an equilateral triangle with sides of about 8 nm and average thickness of 3 nm (26). Considering, the rather flexible regions linking the globular parts of HSA it is not obvious which overall shape adsorbed H S A will adopt, which complicates the interpretation of the force data in terms of protein orientation and monolayer adsorption versus multilayer adsorption. The adsorption of fatty acid containing HSA at a low ionic strength (10 M NaCl) at pH 5.5, where the protein is weakly negatively charged, onto negatively charged mica and the resulting surface forces have been investigated (27,28). The forces measured depend strongly on the HSA concentration in bulk solution as illustrated in Figure 4. In all cases the long-range force is dominated by a repulsive double-layer force originating from the charges on the mica surface and on the HSA. When the HSA concentration is 0.001 mg/ml a hard wall repulsion is encountered at a separation of about 2 nm. This distance is less than the smallest cross-section of HSA. Hence, it is clear that when the number of molecules adsorbed on the surface is small the conformation of the protein can easily be changed by an external compressive force, indicating a rather limited structural stability. On separation, an attractive minimum is observed at a distance of about 4-5 nm. This corresponds to the expected thickness of one monolayer of H S A adsorbed side-on. At higher concentration the data do not provide any evidence for surface denaturation. This, w

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Distance (nm) Figure 2. The forces measured between mica surfaces across a 10-3 M NaCl solution at p H 5.6. The forces were measured as a function of lysozyme concentration, 0.002 mg/ml (filled circles), 0.02 mg/ml (open squares), and 0.2 mg/ml (filled squares). Open circles represent the force measured in a pure 10" M NaCl solution at pH 5.6. The solid curves represent the forces calculated from the D L V O theory (A = 2.2 x 10" J and K = 9.6 nm). 3

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however, does not prove that no changes in conformation occur upon adsorption. For instance at a concentration of 0.01 mg/ml the range of the non-DLVO force increases to about 7-8 nm, close to the expected thickness for one side-on monolayer adsorbed on each surface. These results do not show conclusively that no molecules are adsorbed end-on. Rather, the correct conclusion is that i f some end-on oriented molecules exist on the surface then the force needed to change the orientation from end-on to side-on is small compared to the electrostatic double layer force. When a high compressive force is applied the thickness decreases to about 5-6 nm. No, or a weak, adhesion force is observed between the layers. When the HSA concentration is even larger, 1 mg/ml, the range of the non-DLVO repulsion is 12-15 nm. This is considerably larger than expected for a side-on monolayer on each surface, demonstrating that some molecules are adsorbed at an angle to the surface. Under the action of a high compressive force the layer thickness is reduced to about 8-9 nm. No adhesion between the surfaces is observed under these conditions. We note that the protein do adsorb onto mica despite that it has the same net sign on its charge as the surface. This means that either some positively charged regions adsorb to the surface or that the driving force for adsorption is not due to chargecharge interactions. It has been concluded by Norde (29) that one important reason for protein adsorption is an increase in entropy due to structural changes. It seems likely that this is important for the case of HSA on mica since the adsorbed layer is considerably more compressible than that formed by e.g. lysozyme, and that the HSA layer thickness at low adsorption densities is not consistent with the dimension of the protein in solution. At high ionic strength, 0.15 M NaCl, it was found that a significant fraction of the proteins initially adsorb end-on. This is illustrated in Figure 5, that displays the forces measured during the first and a subsequent approach of the surfaces. On the first approach a repulsive force is experienced at distances below about 37 nm, corresponding roughly to the size of end-on molecules interacting with an electrostatic double-layer force. Upon further compression the increase in repulsion with decreasing separation is very limited until the surfaces reach a separation of slightly below 10 nm. The final position of the surfaces under a high compression is 4.4 nm. We interpret these findings as evidence for a pressure induced change in orientation of the initially end-on oriented fraction to side-on. When the surfaces are kept apart only some HSA molecules do change back to an end-on orientation as evidenced by the considerably less long ranged forces observed upon a subsequent approach (Figure 5). The forces operating between mica surfaces in the presence of bovine serum albumin (30) have been reported to be considerably more long-ranged than those acting in the presence of HSA. At present we do not have any explanation for this difference, but the subject requires further investigations. Amphiphilic Proteins Some proteins have an amphiphilic character with one large hydrophobic region separated from a large hydrophilic region. These proteins are often membrane bound, or, like casein, act as stabilisers for hydrophobic emulsions. In many cases this type of protein can be expected to have a strong preferential orientation on hydrophobic surfaces. This has been shown to be the case for proteoheparan sulfate, a glycoprotein with a rather hydrophobic peptide region with attached strongly negatively charged hydrophilic glycosaminoglycan side chains. For this protein the peptide chain adsorb strongly to hydrophobic surfaces whereas the polysaccharide chains do not adsorb (31). The forces measured between proteoheparan sulfate coated hydrophobic surfaces across a protein-free aqueous salt solution is shown in Figure 6. In a 0.1 m M



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Distance (nm) Figure 4. The forces as a function of separation between mica surfaces immersed in 10 M NaCl at pH 5.6. The solution also contained HSA at concentrations of 0.001 mg/ml (circles), 0.01 mg/ml (squares) and 1 mg/ml (triangles). Filled and unfilled symbols represent the force measured on compression and decompression, respectively. The solid curves represent the forces calculated from the D L V O theory (A = 2.2 x 10" J and i c = 8.5 nm). -3

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Distance (nm) Figure 5. The forces as a function of separation between mica surfaces immersed in a solution containing 0.01 mg/ml HSA and 0.15 M NaCl at p H 5.6. Forces measured on the first approach is represented by unfilled circles, and forces measured on the second approach by filled circles.





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NaCl solution a repulsive double-layer force dominates the long-range interaction. At a separation below 9 nm a steric repulsion due to compression of the glycoprotein is predominating. As the surfaces are separated, a small attractive minimum is noted at a separation of 13 nm. An addition of CaCl2 to a concentration of 1.25 m M results in a significant decrease in the long range repulsive force. However, after addition of CaCl2 the decay length of the force is no longer consistent with an electrostatic force. Instead the force is of steric origin and most likely due to interactions between the carbohydrate chains. We note that the compressed layer thickness also decreases somewhat upon addition of CaCl2. On separation a comparatively strong attraction is observed at a separation of 10 nm. The increase in adhesion observed upon addition of CaCl2 can be rationalized as resulting from a decreased repulsive interaction rather than invoking a calcium dependent attractive force. A further addition of CaCl2 to 2.5 m M results in a reduction of the long-range steric force but no further decrease in the compressed layer thickness. These observations can be rationalized in terms of the decreased electrostatic repulsion between charged segments within the carbohydrate chains resulting in an entropically driven chain contraction and thus a less long ranged steric repulsion. Note that the strong electrostatic repulsion observed before addition of CaCl2 precludes any determination of the range of the long range component of the steric force under this condition. For further information see 31. Random Coil-Like Proteins Mucin is a very large linear and flexible glycoprotein. About 80% of the weight is due to oligosaccharides that are clustered in regions flanked by stretches composed predominantly of amino acids. It is an important type of glycoprotein since it covers many internal surfaces in the body and thus will be one of the primary molecules that e.g. drugs will interact with. The forces between hydrophobic surfaces precoated with a layer of rat gastric mucin (RGM), a weakly charged mucin, have been investigated (32). In this case it was found that the forces were predominantly of steric origin as evidenced by the very weak salt dependence (Figure 7). It was also noted that for the more highly charged pig gastric mucin it was very difficult to measure (quasi)equilibrium forces due to a very slow relaxation of the adsorbed layer (32), indicating that viscous forces may be of importance for the protective function of mucins. The interaction forces between mucin coated surfaces have also been studied by Perez (33). Several theoretical models for the forces operating between polymer-coated surfaces have been developed for quasi-equilibrium (restricted equilibrium) situations (34-36). They predict that the same forces should be measured on approach and on separation. This is often the case when forces between surfaces coated with homopolymers are measured under poor solvency conditions (37). Quasi-equilibrium forces have also been observed for surfaces coated with heterogeneous polymers which adsorb strongly via specific anchor groups utilizing electrostatic forces (38) or hydrophobic interactions as for proteoheparan sulfate or ethylhydroxyethyl cellulose (39). The nonadsorbing segments may then experience good solvency conditions and for sufficiently high adsorbed amounts the polymercoated surfaces will repel each other (38). However, when the polymer-surface interaction is neither very strong nor very weak nonequilibrium forces are experienced as a rule rather than as an exception. This implies that the displacement of polymers from between the surfaces and/or slow conformational changes occur. Clearly, relaxation effects in adsorbed polymer layers, including mucin, and their consequences for the forces acting between polymer-coated surfaces are very important.


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Distance (nm) Figure 6. Comparison of the forces obtained between proteoheparan sulphate layers adsorbed at hydrophobized mica as a function of the distance of separation at different excess electrolyte concentrations. The forces were measured on approach and on separation in 0.1 m M NaCl (squares), 1.25 m M CaCl2 (circles) and 2.5 m M CaCl2 (triangles). Filled symbols represent forces measured on approach and unfilled symbols forces measured on separation.

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Protein-Surface Interactions The desorption of proteins, like for many other polymers, is very slow. This makes it possible to first replace the protein solution with a protein-free aqueous solution and then remove one of the protein coated surfaces and instead insert one bare mica surface. At this stage it is possible to measure the forces between one protein coated surface and one bare surface. Results from such measurements for lysozyme and for human serum albumin are shown in Figure 8. As expected, replacing one of the protein coated surfaces with bare mica results in a halving of the contact separation. More interesting is that the adhesion force between the mica surface and the protein coated surface is high, considerably higher than that between two protein coated surfaces. We also note that the adhesion force is higher between negatively charged mica and H S A than between mica and lysozyme despite that H S A is weakly negatively charged and lysozyme positively charged. Again, this points to a rather limited importance of the net charge of the protein in comparison with the protein flexibility for the protein-surface interaction. Despite the strongly attractive force measured it was possible to separate the surfaces and remeasure the same force on a subsequent approach. This indicates that the proteins are most strongly bound to the surface that they initially adsorb on, and that only a limited (if any) material transfer between the two surfaces takes place. This is not very surprising since the proteins are asymmetric and will orient in such a way that they interact most favourably with the surface they adsorb on. When a second surface is brought into contact with the protein coated surface it will interact less favourably with the opposite side of the protein. It is, however, interesting that even during several minutes in contact the proteins do not reorient to interact equally favourably with both surfaces. Pressure Induced Changes in Adsorbed Layers. A practical problem that has been observed when studying proteins on surfaces with the surface force technique is that irreversible changes in the adsorbed layer may take place when a strong compressive force is applied. This has to do with the fact that many proteins are neither very strongly or very weakly bound to the surface causing them to be pushed out from between the surfaces only at such high forces that the surfaces have started to flatten (due to the deformation of the supporting glue). Under such conditions molecules at the edge of the flat region can leave the contact zone whereas those in the middle of the flat region will be trapped and pushed together (40). (This phenomenon is related to elastohydrodynamic lubrication (41)). When the surfaces are separated again the "lump" of proteins that has formed in the middle of the contact region will often remain at the contact position for a very long time giving rise to long-range repulsive forces. A comparison between the forces observed before and after such pressure induced changes in the layer has occurred is seen in Figure 9. Hence, when studying proteins one should always determine how readily such changes in the adsorbed layer occur, and never apply such strong forces. As a comparison weakly bound molecules will be removed from between the surfaces already when a weak force is applied (e.g. the outer protein layer of lysozyme in Figure 2), whereas strongly bound molecules always will remain at the same position on the surface. Conclusions From this study it is shown that the surface force technique is suitable for the study of several aspects of proteins on surfaces, such as long-range forces, contact forces, molecular orientation and compressibility. It was found that small compact proteins



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Distance (nm) Figure 8. Forces between one protein coated surface and one bare mica surface across a 10" M NaCl solution. The forces measured in the case of H S A are represented by circles. Filled symbols represent forces measured on approach and unfilled symbols forces measured on separation. The insert show the forces in the case of lysozyme, represented by squares. 3

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Distance (nm) Figure 9. Forces as a function of separation between lysozyme coated surfaces (squares) before (filled symbols) and after (unfilled symbols) irreversible pressure induced changes have been introduced by applying a strong compressive force. The insert show the forces between HSA coated surfaces (circles) before (filled) and after (unfilled) pressure induced changes in the layer has taken place.




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like lysozyme and insulin did not show any large conformational changes upon adsorption. When the adsorption had reached equilibrium the adsorbed lysozyme nearly neutralised the surface charge of the mica substrate. When separating the lysozyme or insulin monolayers from contact, an adhesion force is present, demonstrating the existence of an inter-protein attraction that is involved in the adsorption of an outer layer. It was found that negatively charged human serum albumin adsorbs onto negatively charged mica. Small surface induced structural changes take place upon adsorption and further structural changes can be induced by applying an external compressive force. Hence, it seems plausible to believe that compact globular proteins do not change conformation to the same degree as more soft/flexible proteins do. The forces between proteoheparan sulfate and between mucin layers, that expose polymer chains towards the solution, contain an important steric component, similar to that acting between surfaces coated with nonbiological polymers. Literature Cited 1. Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267. 2. Wahlgren, M. Adsorption of Proteins and Interactions with Surfactants at the Solid/Liquid Interface, PhD-thesis, University of Lund, 1992. 3. Leckband, D.; Israelachvili, J. N. Enzyme Microb. Technol. 1993,15,450. 4. Luckham, P.F.;Hartley, P. G. Adv. Colloid Interface Sci. 1994, 49, 341. 5. Israelachvili, J. N.; Adams, G. E. J. Chem. Soc. Faraday Trans. I. 1978, 74, 975. 6. Parker J. L.; Christenson H. K.; Ninham B. W. Rev. Sci. Instrum. 1989, 60, 3135. 7. Derjaguin, B. W. Koll. Z. Z. 1934, 69, 155. 8. Israelachvili J. N. Intermolecular and Surface Forces - with applications to colloidal and biological systems, Academic Press, London, 1991, 2nd edition. 9. Pashley, R. M. J. Colloid Interface Sci. 1981, 80, 153. 10. Horn, R. G.; Israelachvili, J. N.; Pribac, F. J. Colloid Interface Sci. 1987, 115, 480. 11. Nylander, T.; Kékicheff, P.; Ninham, B.J.Colloid Interface Sci. 1994, 164, 136. 12. Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 285. 13. van Dulm, P.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1981, 82, 77. 14. Claesson, P. M.; Arnebrant, T.; Bergenståhl, B.; Nylander, T. J. Colloid Interface Sci. 1989, 130, 130. 15. Kékicheff, P.; Ducker, W. A.; Ninham, B. W.; Pileni, M. P. Langmuir 1990, 6, 1704. 16. Afshar-Rad, T.; Bailey, A. I.; Luckham, P. F.; MacNaughtan, W.; Chapman, D. Colloids Surf. 1988, 31, 125. 17. Luckham, P. F.; Ansarifar, M. A. British Polymer J. 1990, 22, 233. 18. Lee, C.-S.; Belfort, G. Proc. Natl. Acad. Sci. USA 1989, 86, 8392. 19. Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. 20. Deonier, R.C.;Williams, J. W. Biochemistry 1970, 9, 4260. 21. Tilton, R. D.; Blomberg, E.; Claesson, P. M. Langmuir 1993, 9, 2102. 22. Blomberg, E.; Claesson P. M.; Fröberg, J.C.;Tilton, R. D. Langmuir 1994, 10, 2325. 23. Pekar, H. K.; Frank, B. H. Biochemistry 1972,11,4013. 24. Holloday, L. A.; Ascoli, M.; Puett, D. Biochimi. Biophys. Acta 1977, 494, 245. 25. Carter, D.C.;He, X.-M.; Munson, S. B.; Twigg, P. D.; Gernert, K. M.; Broom, M. B.; Miller, T. Y., Science 1989, 244, 1195. 26. He, X.-M.; Carter, D.C.,Nature 1992, 358, 209.



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27. Blomberg, E.; Claesson, P. M.; Gölander, C.-G.J.Dispersion Sci. Technol. 1991, 12, 179. 28. Blomberg, E.; Claesson P. M.; Tilton, R. D. J. Colloid Interface Sci. in press. 29. Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. 30. Fitzpatrick, H.; Luckham, P. F.; Eriksen, S.; Hammond, K. Colloids Surf. 1992, 65,43. 31. Malmsten, M.; Claesson, P.M.; Siegel, G. Langmuir 1994, 10, 1274. 32. Malmsten, M.; Blomberg, E.; Claesson, P. M.; Carlstedt, I.; Ljusegren, I. J. Colloid Interface Sci. 1992,151,579. 33. Perez, E.; Proust, J. E. J. Colloid Interface Sci. 1987, 118, 182. 34. de Gennes, P. G. Adv. Colloid Polymer Sci. 1987, 27, 189. 35. Scheutjens, J. M. H. M.; Fleer, G. J. Macromolecules 1985, 18, 1882. 36. Klein, J.; Pincus, P. Macromoleules 1982,15,1129. 37. Patel, S. S.; Tirrell, M. Annu. Rev. Phys. Chem. 1989, 40, 597. 38. Hadziioannou, G., Patel, S., Granick, S., and Tirrell, M. J. Am. Chem. Soc. 1986, 108, 2869. 39. Malmsten, M.; Claesson, P.M.; Pezron, E.; Pezron, I. Langmuir 1990, 6, 1572. 40. Blomberg, E.; Claesson, P. M.; Christenson, H. K. J. Colloid Interface Sci. 1990, 138, 291. 41. Roberts, A. D.; Tabor, D. Proc. R. Soc. London 1971, A325, 323. RECEIVED March 1, 1995


Proteins at Interfaces II - American Chemical Society

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