Formation of Adsorbed Protein Layers

JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.

207, 186 –199 (1998)

CS985763

FEATURE ARTICLE Formation of Adsorbed Protein Layers Martin Malmsten Institute for Surface Chemistry, P.O. Box 5607, S-114 86 Stockholm, Sweden Received May 19, 1998; accepted July 14, 1998

1. INTRODUCTION

2. DRIVING FORCES FOR ADSORPTION

The adsorption of macromolecules has attracted considerable attention in the last few decades. Particularly for synthetic homopolymers, but also for copolymers, polyelectrolytes, and flexible polymers of natural origin, there has been considerable progress in the understanding of the mechanism of the adsorption, the adsorbed layer structure and formation, and the interaction between such layers (1). This development has involved the appearance of new methodologies and the application of others in a new context, as well as formulation of theoretical models for describing these systems. Also, the interfacial behavior of biopolymers, particularly that of proteins, has attracted much attention during the same period (2–5). Again, the progress has been considerable during the last few decades, but compared to the field of polymer adsorption more remains unclear in the understanding of the interfacial behavior of proteins. This is largely due to the higher complexity of protein adsorption, which poses experimental difficulties and has so far precluded a detailed theoretical understanding of the often intricate processes involved. However, it is the opinion of the author that much can be learned about protein adsorption from consideration of the adsorption of polymers, polyelectrolytes, and simpler colloids. This is not to say that proteins are simple polymers or colloids, but clearly they are also polymers and colloids and should therefore be subject to the same thermodynamics. In the present report, a brief overview of the formation of adsorbed protein layers will be given, with particular emphasis on the driving force for adsorption and the structure of the adsorbed layer during and after the adsorption process. When applicable, protein adsorption behavior will be compared to that of simpler macromolecules and colloids; similarities and differences will be emphasized. Note, however, that the specific examples chosen will be biased by the author’s own interests and experiences and that a complete overview of the field is not attempted. For this, the reader could consult, e.g., Ref. (2).

An important aspect of protein adsorption is its driving force, since this will affect, e.g., the adsorbed amount, the adsorbed layer structure, and the adsorption kinetics. Compared to simpler (uncharged) homopolymers, where the adsorption is largely driven by enthalpic interactions and opposed by conformational and translational entropy (1), the driving force for protein adsorption is generally more complex. Enthalpic contributions to the adsorption driving force may include, e.g., van der Waals interactions, hydrophobic interactions, and electrostatic interactions between oppositely charged surfaces and proteins or protein domains, whereas entropically based mechanisms may involve release of counterions and/or solvation water as well as reduction of the amount of ordered structure (e.g., a-helix or b-sheet) due to adsorption-induced conformational changes (2–6). These contributions will be discussed briefly below.

0021-9797/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

2.1. Effects of Protein–Surface Interactions The effects of protein–surface interaction on protein adsorption are rather complex due to the large number of contributions to the total interaction. In particular, they will depend on the protein net charge and charge distribution, the decay length of the electrostatic interactions, the occurrence of hydrophobic domains in the protein surface, and the size of the protein, to mention a few. Therefore, there are few rules without exceptions as to the effects of surface charge, hydrophobicity, etc., on protein adsorption. However, although there are numerous exceptions, most proteins tend to adsorb more extensively at hydrophobic than at hydrophilic surfaces (2– 4), which is illustrated particularly nicely by the behavior of hydrophobicity gradient surfaces (7). Furthermore, at least in some systems, proteins tend to adsorb more strongly at hydrophobic surfaces, as suggested, e.g., by results from protein–protein exchange experiments indicating a more limited exchange at hydrophobic than at hydrophilic surfaces (2, 8). Most likely, these effects are related to differences in the conformation of the adsorbed protein at hydrophobic and hydrophilic surfaces, as previously demonstrated, e.g., for fibrinogen, fibronectin, bovine serum albumin (BSA),

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FIG. 1. (a) Amount of ZZTn (circles), ZTn (diamonds), Tn (triangles), and oligo-Trp (squares) versus the number of Trp in the protein/peptide adsorbed from 0.5 M potassium phosphate buffer, pH 7.0, at methylated silica (open symbols) and silica and poly(ethylene oxide)-modified silica (all filled symbols). (b) Excess adsorption [Gex 5 Si(Fi 2 Fb), where Fi and Fb are the protein volume fractions in layer i and bulk solution, respectively] obtained from lattice mean-field model calculations for ZZTn (circles), ZTn (diamonds), and Tn (triangles) at methylated silica (data from Ref. (14)). (c) Adsorbed amount of dodecane-modified poly(acrylate) at methylated silica from 0.1 M NaCl versus the degree of hydrophobe modification (data from Ref. (16)).

immunoglobulin G (IgG), a-chymotrypsin, and cutinase (2, 9 –13). Also, the effects of the electrostatic protein–surface interactions are rather complex in protein systems. Somewhat simplistically, proteins may be divided into two classes according to their electrostatic behavior. For proteins displaying large conformational changes on adsorption (often referred to as “soft” proteins), nonelectrostatic driving forces contribute significantly to the adsorption, and therefore the adsorption does not generally behave according to electrostatic dictates. On the other hand, for proteins undergoing limited or no interfacial conformational changes (so-called “hard” proteins), adsorption tends to follow expectations from electrostatic considerations to a larger extent (2– 4). These systems therefore bear some resemblance to simpler colloidal systems in this respect. An interesting way to probe the effects of the protein–surface interaction on the adsorption is to make controlled variations in protein structure (e.g., by inserting hydrophobic or charged groups). Note, however, that when this approach is used, care must be taken to ensure that the changes do not significantly affect protein structure or structural stability or protein–protein and protein–solvent interactions. Otherwise, it will not be possible to differentiate the effects of protein–surface interaction from other effects. In an attempt to use this approach, Malmsten and Veide investigated the effects of hydrophobic peptide stretches on the

adsorption of the protein ZZ, corresponding to the IgG binding domain of staphylococcal protein A (14). By performing insertion of oligopeptides of the type (AlaTrpTrpPro)n and (AlaIleIlePro)n close to the C terminus in a region of less importance for the structure of this otherwise structurally rather stable protein, hydrophobic “tags” of various length could be introduced in the otherwise rather hydrophilic protein without significantly changing the protein structure or structural stability. [Later, these authors also investigated the effects on the adsorption of ZZ of simultaneously hydrophobic and charged peptide stretches of the type (AlaTrpTrpLysPro)n and (AlaTrpTrpAspPro)n, i.e., identical to one of the hydrophobic “tags” above, save for the additional negative and positive charge due to Asp and Lys, respectively (15).] While the (non)adsorption of the protein (and of the inserted oligopeptides) at hydrophilic silica and silica modified with poly(ethylene oxide) was not affected by the length of the oligopeptide, the adsorption at hydrophobic methylated silica increased with an increasing number of hydrophobic Trp (or Ile) groups (Fig. 1a). This was ascribed to an increasingly attractive interaction between the hydrophobic “tags” and the hydrophobic surface, since the peptide insertions had little effect on the protein structure and structural stability and since no adsorption was found at silica, the latter arguing against any substantial effects of the peptide insertions on the protein– protein and protein–solvent interactions. By use of a lattice

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FIG. 2. (a) Adsorption at methylated silica of b-lactoglobulin A (circles) and B (squares) from 0.01 M phosphate buffer (I 5 0.017). Shown also is the fraction of dimers in solution as calculated for the dissociation constants 63 and 8 mM for b-lactoglobulin A and B, respectively (data from Ref. (19)). (b) Relative amount (GT /G20°C) of EO99PO65EO99 adsorbed at silica (G20°C 5 0.35 mg/m2; open circles) and methylated silica (G20°C 5 2.1 mg/m2; filled circles) from water versus temperature. Shown also is the fraction of polymer molecules in micellar form (diamonds). The solid line represents the critical micellization temperature (data from Ref. (27)).

mean-field model for block copolymers, the authors were able to qualitatively model this behavior (Fig. 1b) (14). It is interesting to compare the results above to those displayed by simpler copolymer systems. Thus, for block copolymers consisting of both strongly adsorbing block(s) and weakly adsorbing or nonadsorbing block(s), the adsorption increases with an increasing length of the adsorbing block(s), at least up to a limit. As an example of this, Fig. 1c shows results obtained by Poncet et al. on the adsorption at methylated silica of poly(acrylate) modified with grafted dodecyl chains (16). As can be seen, the adsorption initially increases strongly with the degree of hydrophobic modification, whereas at high substitution degrees the adsorbed amount decreases again, which is a consequence of the formation of thinner adsorbed layers. This behavior is found also for simpler copolymer systems (1). Considering these examples it seems clear that ZZ with inserted Trp-containing oligopeptide stretches behaves analogously to simpler copolymer systems regarding the effects of hydrophobic modifications on the adsorbed amount. 2.2. Effects of Protein–Protein Interactions An important finding regarding the adsorption of homo- and copolymers is that the adsorbed amount increases with increasing molecular weight. This is also the case for polyelectrolytes, although the molecular weight dependence is generally weaker and also depending on electrolyte concentration (1). If proteins were to behave as copolymers and polyelectrolytes, one would therefore expect an increasing adsorption with an increasing molecular weight. However, the study of these effects is not entirely straightforward for proteins since the molecular weight may not be easily changed in the same manner as for synthetic polymer systems, and because a direct comparison between proteins with different molecular weights is typically precluded by simultaneously occurring differences, e.g., in interactions and structural stability.

An alternative approach for investigating molecular weight effects is to study how protein self-assembly affects the adsorption. Although some changes in interaction may be the result of the self-assembly, reasonably similar species differing strongly in molecular weight may be investigated. Studies of this type have been performed, e.g., for human serum albumin (HSA), BSA, b-lactoglobulin, and insulin (2, 17–26). For example, the adsorption of BSA monomers and dimers was investigated by Okubo et al., who found that the dimer adsorbs preferentially over the monomer at a number of surfaces and a range of protein concentrations (17). Similar findings were also obtained for HSA by Lensen et al. (18). The preferential adsorption of BSA dimers has also been discussed extensively by Belfort and Zydney (2). In a series of investigations, Elofsson et al. studied the adsorption of the two b-lactoglobulin variants A and B, differing primarily in their self-association behavior (19 –22). It was found that the adsorption of the two b-lactoglobulin variants displayed a shift in concentration-dependent adsorption corresponding to the difference in dissociation constant for the two proteins, suggesting that the dimer adsorbs preferentially over the monomer. This behavior is illustrated in Fig. 2a. However, analogous to self-associating polymers (see below), the effects of self-assembly on the adsorption of proteins can be rather complex, depending, e.g., on the surface properties. For example, Nylander, Arnebrant, and others have studied the adsorption of insulin in relation to its self-association (2, 23–26). It was found that self-assembly induced by nonspecific electrostatic screening or specific cations (Zn21) generally favors adsorption, although the quantitative effects of self-assembly vary with the surface properties. Also, investigations have been made with a mutated insulin not undergoing self-assembly, and it was found that at methylated silica, where the adsorption is believed to occur mainly through monomers, the mutated and the wild-type insulin adsorb to a similar extent in the absence of Zn21. On the other hand, at silica, where

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FIG. 3. (a) Solvency dependent adsorption of EO6PO37EO6 from water at methylated silica. The dashed line represents the phase separation limit, whereas the insert shows results obtained from lattice mean-field modeling of the adsorption (data from Ref. (30)). (b) Correlation between multilayer adsorption rate (Rm) and the second virial coefficient (B22) for BSA at quartz (data from Ref. (34)).

insulin adsorbs mainly in its hexameric form, no adsorption was found for the monomeric mutant. It is interesting to compare these results with findings on the effects of self-assembly on adsorption in simpler polymer systems. For example, Malmsten et al. investigated the adsorption of EO99PO65EO99 (EO and PO being poly(ethylene oxide) and poly(propylene oxide), respectively) at silica and methylated silica in relation to the self-assembly of this polymer (27). As can be seen from Fig. 2b, EO99PO65EO99 displays a temperature-dependent micellization, with a critical micellization temperature (cmt) and an increasing fraction of polymer molecules in micellar form with increasing temperature (i.e., the critical micellization concentration decreases with increasing temperature). At silica, where the EO segments are expected to adsorb preferentially, a dramatic increase in the adsorbed amount occurs just prior to solution micellization (Fig. 2b). This is most likely a result of interfacial aggregate formation, in line with the behavior of EO-containing low molecular weight surfactants at silica (28). On methylated silica, on the other hand, the PO segments adsorb preferentially, which means that both the driving force for, and the possibilities of, interfacial aggregate formation are largely eliminated, and therefore there is little effect of the solution micellization on the adsorption at this surface. Once more, this is analogous to the behavior displayed by EO-containing low molecular weight surfactants (29). Hence, also for simple copolymers, self-assembly affects adsorption in a rather complex manner, which may preclude interpretation in terms of simple molecular weight effects. However, just as for the protein systems discussed above, self-assembly tends to favor increased adsorption when the aggregate is the adsorbing entity. 2.3. Effects of Protein–Solvent Interactions When a macromolecule becomes less soluble, alternatives to a molecular solution will become relatively more favourable, and the system may therefore change its constitution by induced self-assembly (e.g., micellization in block copolymer

systems, oligomerization or aggregation in protein systems), phase separation, or increased surface activity. Naturally, the factors determining this behavior are the relative magnitudes of the macromolecule–macromolecule, macromolecule–solvent, and macromolecule–surface interactions, and therefore these effects are not strictly separable. Nevertheless, it is by now well known that on worsening the solvency conditions of polymers, their adsorption tendency increases (1). Therefore, the adsorbed amount generally increases on worsening the solvency conditions at a finite equilibrium concentration. For example, Fig. 3a shows the solvency-dependent adsorption of EO6PO37EO6. Analogous to a range of EO-containing polymers and surfactants, this copolymer displays a decreased solvency and eventually phase separation with increasing temperature. As can be seen from Fig. 3a, decreased solvency is accompanied by increased adsorption, and close to the phase separation limit, the adsorption diverges (30). Accompanying such changes in the adsorbed amount there is generally a change in the adsorbed layer structure, and for homopolymers and copolymers the adsorbed layer becomes more dense with decreasing solvency as a result of the system trying to reduce the number of polymer–solvent contacts (1, 31–33). For proteins, the situation is more complex than that for simpler homopolymers and copolymers since the structure of the proteins in itself is determined by a delicate balance of numerous interactions, most of which are altered by changing the solvency conditions (2– 4). Therefore, also the protein structure and structural stability may change drastically on changing the solvency conditions, an important example of this being the denaturation of proteins at high concentrations of, e.g., urea or guanidinium hydrochloride. Naturally, these solvency-dependent changes occur also for simpler polymer systems, but they tend to be of smaller impact than those displayed by proteins. Despite these complications, however, one would expect proteins to behave analogously to simpler polymers regarding

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solvency-dependent adsorption at least to some extent. Indeed, this is also what is found experimentally. For example, Asanov et al. investigated the adsorption of BSA from various solvents and found that on worsening the solvency by addition of ammonium sulfate, the tendency for aggregative adsorption increased (34). More precisely, an inverse correlation was found between the rate of multilayer adsorption, on one hand, and the second virial coefficient, on the other (Fig. 3b). Another case which could be at least partly related to solvency effects is the pH-dependent adsorption of a range of proteins and the maximum adsorption typically found close to the isoelectric point (IEP) (2– 4). As the protein net charge decreases, the protein–protein interaction becomes less unfavorable and the protein–solvent interaction becomes relatively less favorable. This, in turn, favors increased adsorption. For moderately stable proteins, however, also the protein structure and/or structural stability is markedly affected by the protein net charge, both in bulk solution (see, e.g., Ref. (35)) and for adsorbed proteins (2). Another explanation for the maximum adsorption close to the IEP could therefore be that the adsorbed protein molecules undergo larger structural alterations well above or well below the IEP, which should result in a smaller adsorbed amount than close to the IEP (see below). Indeed, the latter has been suggested to be the case, e.g., by Norde and Lyklema (36 –38), and by Buijs et al. (13). Another case related to the protein solvency is the increased adsorption frequently found on thermal denaturation of proteins. Examples include studies of fibrinogen by Schaaf et al. (39), and by Elofsson et al. (22) and Arnebrant et al. (40) on b-lactoglobulin. Related to denaturation are the findings by Chang et al., that acid-pretreated antibodies at hydrophobic surfaces display a significantly increased antigen-binding capacity compared to that of native antibodies (2, 41). Most likely, this is a consequence of an increased degree of orientation of the antibodies at the hydrophobic surfaces, since acid treatment is known to cause denaturation of the Fc part of the antibodies, thereby exposing hydrophobic groups and favouring the adsorption of the Fc part at the hydrophobic surface. This also illustrates the complexity of the solvency-dependent adsorption, since the increased adsorption tendency of both the intact antibodies and the Fc domains on denaturation of the latter is due to an increasingly attractive protein–surface interaction, an increasingly repulsive protein–solvent interaction (through the exposure of hydrophobic groups), and an increasingly attractive protein–protein interaction (as evidenced by the increased effective molecular weight of the proteins after acid treatment) (41). 2.4. Effects of Protein Conformational Stability As will be discussed further below, conformational stability is important for determining the state of adsorption of a protein, and notably the adsorbed layer structure and adsorption strength. However, the structural stability of a protein may also

affect the extent of adsorption. The origin of this is that in the native state the protein conformation is quite restricted, and hence the conformational entropy is low. On adsorption, numerous proteins tend to undergo conformational changes and frequently lose a fraction of their ordered structure (e.g., the content of a-helix or b-sheet decreases). Therefore, the adsorption process may be associated with a conformational entropy gain, which in principle can act as an adsorption driving force (2– 4). However, the issue of entropy changes associated with the adsorption process is somewhat complex, since also other mechanisms for an entropy change on adsorption can be anticipated, including, e.g., the release or binding of counterions and/or hydration water for both the protein and the surface. Furthermore, it has been found in numerous cases that for a given system, the degree of conformational change on adsorption decreases with increasing adsorbed amount (see below). Therefore, the relationship between the adsorption and interfacial conformational loss as a driving force for adsorption is not always straightforward. The issue of loss of ordered conformation as a driving force for adsorption of proteins has been discussed in depth previously and is outside the immediate scope of the present treatise (2– 4). 3. ADSORBED LAYER STRUCTURE

The structure of adsorbed protein layers is a complex issue, and different systems display widely different behavior. Somewhat simplistically, one could say that regarding structure and structural (shape) stability, overall protein structures are situated somewhere in the range between flexible “polymer-like” coil structures, stiff rods, and rigid colloidal particles. To a large extent depending on this structure and structural stability, different adsorbed layers will be formed. 3.1. “Flexible” Polymer-Like Proteins For flexible, polymer-like proteins, structural variations occur continuously, and on perturbing the system, e.g., by addition of cosolutes or by altering the electrostatic interactions through pH or the electrolyte concentration, rather large structural changes are typically the result. From this point of view, the interfacial behavior of flexible proteins could be expected to be rather similar to that of simpler macromolecules, such as synthetic homopolymers, copolymers, or polyelectrolytes. As examples of this type of protein, one could mention mucus glycoproteins and proteoglycans (42– 49). The former are large (Mr ' (5–25) 3 106) macromolecules forming the mucus protecting the “internal” surfaces of the body. These macromolecules are long, linear, and flexible structures consisting of subunits (Mr 5 (2–3) 3 106) joined end-to-end. Mucus glycoproteins, frequently referred to also as mucins, contain about 80% carbohydrate which occurs as oligosaccharides mainly present in “clusters” flanked by “naked” regions of the protein core. Mucus glycoproteins are thus segmented

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FIG. 4. (a) Schematic structure of mucus glycoproteins. Thick and thin sections represent oligosaccharide clusters and “naked” stretches, respectively. (b) Normalized force between rat gastric mucin adsorbed at hydrophobized mica. The NaCl concentration was 0.1 mM (circles), 10 mM (triangles), and 150 mM (diamonds). The insert shows the forces on a logarithmic scale (from Ref. (46)).

structures, where carbohydrate-rich regions alternate with protein-like structures (Fig. 4a). Malmsten et al. previously investigated the adsorption of mucus glycoproteins at a number of surfaces (46, 47). In particular, the adsorption and surface forces were investigated for two different mucins at hydrophobized mica surfaces. It was found that despite the very high molecular weight, the adsorbed layers formed were not excessively extended. More precisely, the adsorbed layers were significantly thinner ('50 – 80 nm) than twice the radius of gyration (Rg ' 200 nm) (Fig. 4b). It is therefore clear that a significant distortion from the solution structure occurs on adsorption for these proteins. Since it is known that mucins form (somewhat stiff) random coil structures (48), this indicates an adsorption behavior analogous to simpler systems, such as copolymers or copolyelectrolytes. This notion is further supported by the rather weak distance dependence of the force acting between adsorbed mucin coatings, the nonelectrostatic nature of this gradual repulsion, and the presence of dynamic effects for some mucin systems. Similar conclusions were reached also by Perez et al., albeit for a slightly different system (49). Other examples of polymer-like proteins are members of the proteoglycan family, such as proteoheparan sulfate, proteodermatan sulfate, and proteochondroitin sulfate (42– 45). The proteoglycans consist of highly carboxylated and sulfated glycosaminoglycan chains covalently attached to a protein core. They are therefore strong polyelectrolytes with a high linear charge density (typically about –100 mV in pure water). Malmsten et al. previously investigated the adsorption pattern and surface forces exerted by these proteins (42, 44). By analogy with the mucin glycoproteins discussed above, proteoheparan sulfate

was found to adsorb at hydrophobized mica in a layer much thinner than expected from the protein molecular weight. Furthermore, it was inferred from the nonadsorption of the polysaccharide glycosaminoglycan chains at methylated silica and from the interaction forces displayed that the proteoheparan sulfate adsorbs at hydrophobized surfaces with the hydrophobic backbone toward the surface and the hydrophilic and strongly negatively charged side chains protruding toward the bulk solution. On addition of Ca21, the adsorbed proteoheparan sulfate layer was found to contract, presumably as a result of both electrostatic screening and reduction of the linear charge density of the polysaccharide side chains of the glycoprotein due to specific Ca21 binding (42– 45). By comparison, Kurihara et al. found that decreasing the linear charge density of a poly(methacrylic acid) amphiphile adsorbed at hydrophobized mica through its lipid moiety by decreasing pH resulted in contraction of the polyelectrolyte chains (50). [Somewhat less straightforward results were obtained on increasing the excess electrolyte concentration.] Analogous results were obtained also for chitosan by Claesson and Ninham (51). Furthermore, Poncet et al. found for hydrophobe-modified poly(acrylate) at methylated silica that increasing the concentration of NaCl resulted in a thinner adsorbed layer despite a higher adsorbed amount at the higher electrolyte concentration (16). Considering these examples, it seems clear that the adsorbed layer structure in the case of flexible, coil-like, proteins is similar to that of simpler polymer systems. Having concluded this, however, it is important to note that the former systems may display a significantly richer behavior than the latter, as

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shown by the dependence of both the adsorbed amount and the adsorbed layer structure on the nature (as opposed to the valency and concentration) of the counterion in the case of proteoglycans (43, 44). 3.2. “Particle-Like” Proteins At the other extreme of the spectrum of protein shapes are the rigid particle-like structures. In principle, one would expect such proteins to behave in a manner similar to colloidal particles. In various investigations, inorganic particles (52), polymer latices (53–58), and oil-in-water emulsions (59) have been found to deposit at macroscopic surfaces mainly according to expectations from electrostatic considerations, e.g., displaying an increasing deposition (rate) with a decreasingly repulsive or increasingly attractive colloid–surface interaction. Although much can be learned from considerations of electrostatic and van der Waals interactions, as shown by Roth and Lenhoff (60, 61) and Johnson et al. (62), the situation is often more complex for proteins since hydrophobic interactions are frequently of importance. A typical example of proteins displaying this type of adsorption behavior is RNase (others are cytochrome c, subtilisin, and lysozyme). As has been discussed previously (3), this protein (with an IEP ' 9.5) displays an adsorption pattern which is largely determined by electrostatic and hydrophobic interactions. Thus, at hydrophobic and negatively charged latex particles, a significant adsorption is found for this protein over a wide pH range due to electrostatic and hydrophobic interactions. For uncharged and hydrophilic poly(oxymethylene), the hydrophobic interactions are less significant and the electrostatic interactions are repulsive due to image charge effects (6), which results in nonadsorption. For hydrophilic a-Fe2O3, adsorption is determined primarily by electrostatic interactions, resulting in significant adsorption at high charge contrast but no adsorption well below the IEP of the surface, i.e., in the absence of attractive electrostatic interactions. On adsorption of a “hard” protein at an interface, few or no structural alterations are expected. Depending on the packing densities and the shape of the proteins, different types of interfacial orientations can be anticipated. For reasonably spherical/symmetric proteins such as lysozyme (approximate dimension (3.0 3 3.0 3 4.5 nm)) the adsorbed layer is expected to consist essentially of an array of these proteins. This is indeed what has also been found experimentally. For example, Haggerty and Lenhoff investigated the adsorption of lysozyme at graphite surfaces with scanning tunneling microscopy (STM) and found ordered arrays of proteins (63). From the “lattice spacings,” these authors were able to extract information about the adsorbed amount as a function of protein and excess electrolyte concentration which was in good agreement with that obtained with total internal reflection fluorescence spectroscopy (TIRF). Furthermore, Malmsten investigated the adsorption of lysozyme at silica with ellipsometry (64) and

found an adsorbed layer thickness of 3 6 0.5 nm, which implies an adsorbed layer at this surface consisting of a monolayer of side-on adsorbed lysozyme molecules. Somewhat analogous conclusions were also reached by Blomberg et al. for mica surfaces using surface force measurements (65), by Su et al. using neutron reflectivity (66), and by Radmacher et al. using atomic force microscopy (AFM) (67). Note, however, that in this seemingly simple system there are numerous complications, since this protein has been reported to display a side-on to end-on transition (65), conformational changes on adsorption (68), pH-dependent interfacial aggregation (67), and concentration-dependent multilayer adsorption (64 – 66). In relation to the latter it is interesting to note that there seem to be large differences in the state of adsorption for early and late adsorbing proteins, since the former are poorly exchangable, whereas the latter are more extensively exchangable (69, 70). Interestingly, this is coupled to a large loss in enzymatic activity for the initially adsorbing proteins, whereas the enzymatic activity of the proteins adsorbing later in the adsorption process display an effectively higher activity (69). It is interesting to note that the readily exchangable fraction increases and the irreversibly adsorbed fraction decreases with an increasing bulk protein concentration (70). A possible explanation for this is that an increasing concentration results in an increasing degree of multilayer formation. Since multilayers can be expected to be more loosely adsorbed than the layer in direct contact with the surface (65), the formation of these should result in an effective increase in the degree of exchangeability and in the fraction of exchangeable proteins. However, considering the simultaneous occurrence of conformational changes and reorientation effects (65– 68, 71), the mechanism behind this effect is not trivial. Clearly, also for “hard” proteins the adsorption may be significantly more complex than that of simpler colloids. 3.3. “Soft Shape” Proteins For proteins falling between the extremes of random coil, rigid colloid, and stiff rod structures, the degree of structural rearrangements on adsorption is a more subtle issue. Depending on a range of parameters, including the protein stability, temperature, interfacial crowding, and protein–surface interactions, different degrees of interfacial conformational changes could be expected. As has been mentioned above, a repeating problem when trying to investigate adsorption mechanisms in protein systems is the fact that it is frequently quite difficult to separate the effects related to various types of interactions from those due to structural stability. It is therefore beneficial to these studies to use protein mutants, where selective changes have been made to the protein in order to modulate its interactions or structural stability (2). A particularly illustrative example of the use of protein engineering for investigating adsorption-induced conformational changes is that of T4 lysozyme (72–75), although also other protein mutants, e.g.,

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FIG. 5. (a) Content of a-helix in nonadsorbed (open diamonds) and adsorbed (open circles) T4 lysozyme mutants of different structural stability calculated from CD spectra. Also shown (filled diamonds) is the loss in a-helix content on adsorption from 10 mM phosphate buffer, pH 7.0 (data from Ref. (74)). (b) Normalized force between wild-type lysozyme (circles) and the Ile3 3 Trp T4 lysozyme mutant (diamonds) adsorbed at mica from water. Only forces measured on approach are shown (data from Ref. (73)).

human carbonic anhydrase (76) and tryptophane synthase a-subunits (77), have been used in this respect. McGuire et al. studied the adsorption at silica and methylated silica of a number of T4 lysozyme stability mutants obtained by point mutation in the Ile3 position. By comparing the structural stability with the degree of “elutability” of the adsorbed protein by a cationic surfactant (dodecyltrimethylammonium bromide, DTAB) these authors inferred that the less stable proteins undergo larger interfacial conformational changes (72). This conclusion was reached also by Billsten et al. based on circular dichroism (CD) investigations of T4 lysozyme adsorption at silica particles (74). Thus, Fig. 5a shows the helical content of T4 lysozyme mutants before and after adsorption at silica nanoparticles as well as the loss in helical content on adsorption. As can be seen, the loss of helical content on adsorption increases strongly as the structural stability of the protein decreases, despite the helical content before adsorption being essentially independent of the protein structural stability. That these conformational changes can be of considerable magnitude was recently shown by Fro¨berg et al. (Fig. 5b) (73). Using surface force measurements, these authors found when comparing the adsorbed layer structure at mica of wild-type lysozyme with that of an Ile3 3 Trp mutant (the latter 2.8 kcal/mol less stable than the former) that the adsorbed layer thickness for the less stable protein is much smaller (15–17 Å) than that of the wild-type (45–50 Å). Not unexpectedly, this also results in a significantly stronger adsorption of the less stable protein mutant. A major factor determining the degree of orientation, deformation, and conformational change on adsorption is the crowding in the adsorbed layer. For example, Morrissey and Fenstermaker showed that for g-globulin, the fraction of protein segments in direct contact with the surface decreases with increasing adsorbed amount (78). Thus, with increasing adsorption the g-globulin molecules either adapt an orientation more normal to the surface or undergo a smaller conformational change on adsorption. These effects have been illustrated

for similar systems by Chang et al. (41) and Buijs et al. (13) (see also discussion below). Furthermore, crowding-dependent conformational changes have been illustrated previously, e.g., by Kondo and Mihara for hemoglobin and myoglobin adsorbing at various colloidal particles (79) (Fig. 6), by Kondo et al. for catalase at silica (80), and by Maste et al. for savinase at Teflon and silica (81). These authors all found that the degree of structural loss on adsorption decreased with an increasing adsorbed amount. Thus, as the interfacial crowding increases the proteins are able to “spread” at the surface to a smaller extent. This is analogous also to the behavior of simpler polymers (1). Furthermore, Kondo et al. investigated the relationship between interfacial conformational changes and the activity for catalase at silica (80). Whereas the a-helix content of enzymes was the same for the adsorbed state at “high” adsorbed amounts and in bulk solution, helix content reduction was observed for lower adsorbed amounts. Parallel studies on the enzymatic activity indicated a reduction in the activity at the low adsorbed amounts. Furthermore, both the a-helix content reduction and the activity loss at low adsorbed amounts were

FIG. 6. Relative a-helix content of human hemoglobin adsorbed at silica particles as a function of the adsorbed amount at pH 8.0. The isoelectric point was 6.8 –7.0 (data from Ref. (79)).

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found to be more pronounced below the isoelectric point of the protein, where the protein and the particle had opposite electrostatic potential. These results indicate that at low coverage, where the protein–surface interaction dominates and the degree of crowding is low, the electrostatic attraction between the oppositely charged protein and particles at these conditions causes a conformational change of the protein, which in turn results in an activity loss. Although the observed low adsorbed amounts give rise to some questions about the importance of crowding in this system, the approach taken is interesting. Regarding the effects of the protein–surface interaction on interfacial conformational changes it is also interesting to note that Kondo and Mihara found a correlation between the extent of desorption on dilution, on one hand, and the degree of structural changes on adsorption, on the other (79). Based on this and the adsorbed amounts for the different materials investigated (silica, titania, and zirconia) it was inferred that a higher protein–surface attraction causes a larger degree of conformational change on adsorption, in line with the findings discussed above. Apart from interfacial conformational changes asymmetric proteins, whether “soft” or “hard,” can undergo reorientation processes during the adsorbed layer formation. Representative examples in the case of “hard” proteins include lysozyme and RNase (discussed above and below, respectively). In the presence of interfacial conformational changes, however, orientational aspects of protein adsorption are not always straightforward. For example, Malmsten investigated the adsorption of IgG at silica and methylated silica and found that for both of these surfaces, the adsorbed layer thickness was about 17–18 nm (64). Analogous results ('18 nm) were obtained by Heinrich et al. for silica using scanning angle reflectometry (82) and by Morrissey and Han for g-globulin at poly(styrene) colloidal particles (although at higher surface concentrations than those in the former investigations) (83). Furthermore, You et al. investigated the adsorption of IgG at mica modified with 3-(aminopropyl)triethoxysilane (APTES) with AFM and found an adsorbed layer thickness of 18 nm (84) under conditions similar to those used by both Heinrich et al. and Malmsten. Considering the molecular dimensions and structure of IgG, these results seem to indicate that IgG preferentially orients in an essentially end-on configuration at these surfaces. At the same time, however, numerous investigations have suggested that conformational changes do occur for these systems (2, 13, 78, 83), and therefore the issue of interfacial orientation is rather complex. This is emphasised also by AFM studies, showing the presence of both “strand-like” structures (84) and “dendrite-like” aggregates (85). Completely different behavior is displayed by fibrinogen. Thus, in a number of studies it has been found that the thickness of adsorbed fibrinogen layers ('15–30 nm, depending on conditions) is significantly smaller than the long axis of the protein ('45 nm) (64, 86 – 88). This may be interpreted as

indicating that fibrinogen adsorbs in an essentially “random” orientation at the surface. At the same time, however, fibrinogen has been shown to undergo a surface-dependent conformational change on adsorption (9), and therefore it is difficult to infer the interfacial orientation of fibrinogen from the adsorbed layer thickness alone. [In fact, microscopic studies indicate that the lateral structure of adsorbed fibrinogen layers may be quite complex (89 –91).] Nevertheless, from the adsorbed layer thickness and refractive index it can be inferred that adsorbed layers formed by this protein are characterized by a quite “loose” packing and (as for polymers) that the adsorbed layer thickness in the case of fibrinogen depends strongly on the degree of interfacial crowding (see below). An approach other than those based on comparison between the adsorbed layer thickness and the molecular dimensions was attempted by Kull et al. for investigating the effects of the protein–surface interaction on the adsorbed layer organization (92). In order to probe the adsorbed layer structures formed by b-casein at silica and methylated silica, these authors investigated how addition of endopeptidase Asp-N, which cleaves the N-terminal of the protein between residues (42– 43) or (46 – 47), affects the adsorbed amount and adsorbed layer thickness of b-casein at these surfaces. On the basis of the ellipsometric data obtained, the authors were able to suggest adsorbed layer structures for b-casein at silica and methylated silica. Note, however, that although the “enzymatic sensor” approach seems to have worked well in this case, care should be taken when using multicomponent protein systems since these frequently display complex adsorption patterns, including competitive adsorption, associative adsorption, and multilayer adsorption (2– 4, 93, 94). If any of these processes should occur in the type of experiment described above, interpretation of the experimental results in terms of structural parameters is precluded. 4. ADSORBED LAYER FORMATION

The formation of adsorbed protein layers is a complex process dictated by a number of factors. The kinetics of adsorption could be envisioned to depend on a number of steps, including mass transport to the surface (involving diffusion through a partially formed adsorbed layer), attachment at the surface, adsorbed layer rearrangements, reorientation of the adsorbing protein, and conformational changes. A number of these factors have recently been described (2). In the present treatise, only a few examples will be discussed as illustrations of effects relating to interfacial crowding, orientation, and conformational change. In the absence of orientational effects and interfacial conformational changes, one could perhaps expect adsorbed layer formation in protein systems to resemble that of simpler colloids, such as polymer latices or emulsion droplets. As has been observed, e.g., by Johnson and Lenhoff (53), Adamczyk et al. (54, 55), and Meinders et al. (57, 58), adsorbed layer formation by such entities involves the attachment of intact

FORMATION OF ADSORBED PROTEIN LAYERS

particles with an interfacial interparticle radial distribution determined by the interparticle interactions. As the latter become more repulsive, the particles within the adsorbed layer become increasingly separated. Furthermore, with an increasingly attractive or decreasingly repulsive particle–surface interaction, the adsorption rate increases, which is a result of an increasing probability of particle–surface separations smaller than that corresponding to the primary maximum in the particle–surface interaction profile (typically determined by electrostatic and van der Waals interactions) (6). Within the framework of the “random sequential adsorption” (RSA) model, adsorbed layer formation occurs through irreversible attachment of nonoverlapping particles, whereafter no structural alterations occur (54 –56). A notable feature emerging from the RSA model is that the maximal crowding within the adsorbed layer (frequently referred to as the jamming limit) is quite low, about 50% depending on the object shape. Thus, due to the lack of rearrangements quite dilute adsorbed layers are formed. In order to account for adsorbed layer rearrangements the RSA model has been modified in its so-called ballistic version to allow sterically excluded particles to roll over blocking particles in order to reach unoccupied surface (2). Naturally, this results in an increased adsorbed layer packing density, characterized by a jamming limit of about 61%. Furthermore, the RSA model has been extended to include interparticle interactions. Particularly for long-range repulsive electrostatic interactions, the picture changes somewhat, and the jamming limit decreases (2). Although the RSA model has proven quite valuable for describing the adsorption of colloidal particles, its application to protein adsorption is less straightforward due to multiple modes of adsorbed layer rearrangement (e.g., reorientation and conformational change), partial reversibility of adsorption, and the presence of multiple contributions to the total protein– protein interaction (cf. the discussion on lysozyme above). Nevertheless, at least some protein systems seem to adsorb largely according to expectations from (modified) RSA considerations (2, 95–97). For example, Ramsden investigated the adsorption of human apoferritin at Si0.62Ti0.38O2 under convective– diffusive conditions (95) and found that the adsorption kinetics is well described by the model by Schaaf and Talbot (98, 99). On the other hand, neither Langmuir kinetics (where the fraction of surface available for binding (f) decreases with the fraction of the surface covered by the adsorbate (u) as f 5 1 2 u) nor the low coverage approximation of the excluded area (f 5 1 2 4u) gives a satisfactory description of the complete adsorption process (Fig. 7). [Naturally, adsorption kinetics is strongly affected by a number of factors, such as long-range repulsive interactions, clustering, and multilayer formation. This is discussed in detail in Ref. (2). Unfortunately, most proteins typically display richer adsorbed layer formation than do simple colloids, including both adsorbed layer reorganizations, reorientations, and interfacial conformational changes. As two examples of structural aspects

195

FIG. 7. Adsorption kinetics of apotransferrin from 10 mM HEPES buffer, pH 7.4, at Si0.62Ti0.38O2 versus the adsorbed amount. Shown also are model fits based on the model by Schaaf and Talbot (solid line), f 5 1 2 4u (dashed line), and f 5 1 2 u (dotted line) (data from Ref. (2)).

of the adsorbed layer formation, let us consider IgG and fibrinogen (Fig. 8). As discussed above, the adsorbed layer thickness of the former may be interpreted to indicate an essentially end-on adsorption of the IgG molecules. This is supported also by adsorbed layer formation, showing a linear increase in the adsorbed layer refractive index (related to the adsorbed layer protein concentration) with increasing adsorbed amount, while the adsorbed layer thickness is essentially constant. This behavior, which is found by both ellipsometry (64, 86) and scanning angle reflectometry (82), indicates that little large-scale reorganizations occur within the adsorbed layer during the adsorption process. Note, however, that this seems not to be the complete picture. For example, Chang et al. found that acid pretreatment of antibodies may increase the degree of accessability of the Fab fragments to antigen binding, which is likely to be coupled to the interfacial orientation of antibodies (41, 100). Furthermore, Buijs et al. found that IgG’s undergo conformational changes on adsorption, depending on both the adsorbed amount and the surface properties (13). Moreover, Morrissey and Fenstermaker found that the fraction of segments in direct contact with the surface decreases with an increasing adsorbed amount for g-globulin at silica (78), again indicating that some crowding-dependent structural rearrangements and/or reorientations are occuring for these systems. Thus, it seems clear that processes other than the large-scale ones detectable with ellipsometry and reflectometry occur during the adsorption of these proteins. Completely different adsorption behavior is displayed by fibrinogen (Fig. 8b) (64, 86 – 88, 101). While initially adsorbing fibrinogen molecules seem to lie rather flat at the surface, an increasing adsorption results in an increased adsorbed layer thickness. Initially, the adsorbed amount increase is accomplished both by an increased adsorbed layer thickness and an increased protein concentration in the adsorbed layer. This suggests that an increased packing density causes the adsorbed

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FIG. 8. Adsorbed layer thickness (del, circles) and mean adsorbed layer refractive index (nf, diamonds) versus the adsorbed amount for (a) IgG and (b) fibrinogen adsorbing at methylated silica from 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.4 (data from Ref. (101)).

layer to grow normal to the interface. [Similar results have been obtained also for apolipoprotein B (102), fibronectin (103), C3, and C1q (93).] Interestingly, there seems to be an adsorbed layer protein concentration, beyond which further fibrinogen adsorption is facilitated largely by a compensation for the increased amount of material by an adsorbed layer growth normal to the surface, thus keeping the adsorbed layer protein concentration essentially constant during the latter part of the adsorption process. The fibrinogen adsorption process is a clear example of how interfacial crowding affects the adsorbed layer structure. In this respect, the adsorption of fibrinogen does not differ dramatically from that of simpler polymers. For example, for uncharged homopolymers or polyelectrolytes at high excess electrolyte concentrations, the interfacial layers at low coverage are characterized by polymers lying rather flat at the surface, thus forming thin adsorbed layers. With an increasing adsorbed amount, however, the lateral crowding increases, resulting in an increased adsorbed layer thickness (1). The issue of reorientation of protein molecules during the adsorbed layer formation was addressed in a nice study by Lee and Belfort for the structurally quite rigid RNase A at mica (104). From surface force measurements it was found that initially the adsorption occurred side-on, which by electropotential considerations was suggested to be due to electrostatic interactions between a positively charged domain of the protein and the negatively charged mica surface. Since the active site resides in the positive potential region, it is not surprising that the enzymatic activity displayed under these conditions was quite low (Table 1). With increasing adsorption time, however, the adsorbed amount increases, causing a reorganization of the adsorbed layer from side-on to end-on adsorption. Since this exposes the active site this reorganization results in increased enzymatic activity. [Although an alternative explanation of these findings could be a crowding-dependent conformational change on adsorption and concomitant activity loss, this seems less likely considering the structural stability of RNase A.]

Although the effects of factors such as surface properties, interfacial crowding, and protein structural stability on the degree of conformational changes as a result of adsorption have been investigated for a number of systems, including fibrinogen (9), IgG (13), hemoglobin and myoglobin (79), a-chymotrypsin and cutinase (12), and lysozyme and a-lactalbumin (105), much is still unknown particularly about the kinetics of adsorption-induced conformational changes of proteins. As an example of recent investigations on this it could be noted that Billsten et al. and Tian et al. monitored the timedependent adsorption-induced conformational loss of T4 lysozyme at silica (74, 75). As discussed above, mutants of this proteins of less structural stability tend to keep less of their ordered structure on adsorption. As shown in Fig. 9, the kinetics of the adsorption-induced conformational changes depend on the protein structural stability. More precisely, the larger the interfacial conformational changes, the faster they are. Although this behavior could perhaps be expected from the point of view of the energetic driving force for the structural alteration, this is a rather interesting result. By comparison with the surface force results by Fro¨berg et al. (73), one would expect these structural changes to be rather major, also involving

TABLE 1 Adsorption and Interfacial Activity of RNase A (Data from Ref. (104)) Adsorption time (h)

G (mg/m2)

Activity (Kunitz units/mg)

Thickness (Å)

1 4 14 24 36 48

1.7 2.1 2.3 2.5 2.5 2.5

20 6 10 37 6 8 78 6 5 91 6 5 96 6 5 93 6 5

28 34 38 42

Free solution

—

124 6 12

—

FORMATION OF ADSORBED PROTEIN LAYERS

FIG. 9. Molecular ellipticity at 222 nm as a function of time for the wild-type (circles), Ile3 3 Trp (diamonds), and Ile3 3 Cys (squares) bacteriophage T4 lysozyme adsorbing at silica particles from 10 mM phosphate buffer, pH 7.0 (data from Ref. (74)).

nearby protein molecules. Therefore, the conformational changes should be coupled to structural alterations within the adsorbed layer and possibly also to desorption of a fraction of the adsorbed molecules (see below), both of which could be expected to be slow processes, at least at high coverage. Apparently, this is not the case. Some more work in this direction was recently performed by Kondo and Fukuda, who investigated the rate of conformational changes on adsorption of human hemoglobin at a range of conditions (106). It was found that the rate of conformational change on adsorption was affected by the temperature, the adsorbed amount, pH, and the ionic strength. More precisely, faster conformational changes were favored by a high temperature, a low adsorbed amount, a pH close to the isoelectric point of the protein, and a low ionic strength. This behavior could be expected, since a higher temperature generally favors conformational changes, since crowding-dependent conformational changes are frequently observed (see above) and since an electrostatic repulsion between the similarly charged protein

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and particle at pH . IEP could be expected to oppose conformational changes to some extent due to an effectively weaker protein–surface attraction. A completely different approach for investigating these effects was taken by van Eijk and Cohen Stuart, who studied the adsorption kinetics of savinase as a function of the interfacial “spreading” time (107). It was found that the limiting adsorbed amount increases with the flux to the surface (Fig. 10). This is expected from the discussion above, since a higher flux provides less time for initially adsorbing molecules to spread at the surface due to crowding. Thus, at high flux, the area occupied by each adsorbed molecule is smaller and the adsorbed amount is higher. By a detailed investigation of the effects of the protein concentration and hydrodynamic conditions on the adsorption and the adsorption kinetics, and modeling the data with a “growing disk” model, the authors were able to estimate the spreading rate and extent of spreading of savinase at silica. Naturally, the “growing disk” concept is also compatible with frequently observed adsorbed amount maxima in time-resolved adsorption experiments, as discussed previously by Cohen Stuart and Ramsden (2), and analysis in terms of interfacial spreading has been performed directly based on time-resolved adsorption data (see Refs. (2) and (108)). 5. CONCLUDING REMARKS

There are considerable parallels between the interfacial behavior of proteins and that of simpler systems, such as polymers, polyelectrolytes, and colloids. This is the case for a number of features relating to the adsorption driving force, structural aspects of the interfacial layer, and the adsorbed layer formation. At the same time, however, proteins display a significantly richer behavior in relation to adsorbed layer formation, and much is still unknown about this process. In particular, future studies of effects of controlled variations regarding structural stability, protein–surface interactions, protein solvency, and protein self-assembly, with well defined

FIG. 10. (a) Effects of the polymer concentration on the adsorption of savinase at silica at pH 8 and I 5 10 mM. Shown are results obtained for 2.5 (circles), 7.5 (squares), 12.5 (diamonds), and 20 g/m3 (triangles). (b) Maximum adsorbed amount as a function of the flux obtained experimentally (circles) and from the “growing disk” model calculations (solid line) (data from Ref. (107)).

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proteins are required, and recent developments in protein engineering therefore have great potential. Furthermore, the development of analytical methods for investigating protein layers is essential, and the applications of techniques such as CD, AFM, and surface force measurements, neutron scattering and reflectivity, and optical methods in protein adsorption studies are promising. ACKNOWLEDGMENT This work was financed by the Foundation of Surface Chemistry, Sweden, the Swedish Research Council for Engineering Sciences (TFR), and the Institute for Research and Competence Holding AB, Sweden (IRECO).

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