Proteins at Interfaces III: Introductory Overview - American Chemical

Chapter 1

Proteins at Interfaces III: Introductory Overview Downloaded by UNIV OF SUSSEX on December 13, 2012 | http://pubs.acs.org Publication Date (Web): December 12, 2012 | doi: 10.1021/bk-2012-1120.ch001

Willem Norde,1,2 Thomas A. Horbett,3 and John L. Brash*,4 1Department

of Biomedical Engineering, University Medical Center Groningen, Groningen, The Netherlands 2Laboratory of Physical Chemistry and Colloid Science, Wageningen University, The Netherlands 3Departments of Bioengineering and Chemical Engineering, University of Washington, Seattle, Washington 98195 4School of Biomedical Engineering, Department of Chemical Engineering, McMaster University, Hamilton, Ontario, Canada L8S 4L8 *E-mail: [email protected]

In this chapter, we provide a review of current research on proteins at interfaces under the headings: physicochemical aspects, computer simulation of protein adsorption, biological function of adsorbed proteins, resistance to protein adsorption, and experimental techniques for the study of protein surface interactions. All of these areas are represented in the various chapters in the book. This chapter gives a broader context into which the individual, specialized chapters can be placed and we have attempted to point out the connections. We intend this chapter to be of help to the community at large, and in particular to beginning students and new investigators wishing to make a contribution to the field.

1. Physicochemical Aspects Proteins are very complex polymers. They are polyamino acids built from twenty two different amino acids, linked together via peptide bonds. They vary in size, polarity and charge. Depending on the distribution of the polar and apolar amino acids along the polyamino acid chain, the protein molecule is more or less amphiphilic. This is one of the more general or overriding reasons why © 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Downloaded by UNIV OF SUSSEX on December 13, 2012 | http://pubs.acs.org Publication Date (Web): December 12, 2012 | doi: 10.1021/bk-2012-1120.ch001

proteins are very surface active. Furthermore, some amino acids in the polymer chain contain a cationic group, some an anionic group, and others are uncharged. Proteins may therefore be classified as polyampholytes. Just as an almost infinite number of words can be written by using the twenty six letters of the alphabet, an endless number of polyamino acids may be formed using the twenty two amino acids, each one with its own amino acid composition and distribution. As with words, only a fraction (but still amounting to millions) of all possible sequences are “meaningful”, that is, are represented in nature as proteins, each with its own specific function. Understanding the behavior of proteins at interfaces may start from that of the simple, coiled polymers. First, like the simple polymers, proteins adsorb by attaching several segments to a surface (like a centipede on a fly trap) resulting in a poor ability to desorb (1–3). When the affinity for attachment of the various molecular segments is sufficiently reduced by environmental changes (e.g., temperature, pH, ionic strength, etcetera), the protein may leave the surface. Also, protein molecules may be displaced from the surface by adding components that have a higher affinity to adsorb. Second, because of their ionic groups proteins show adsorption patterns typical for polyampholytes, that is, strong pH-dependence, the more so the lower the ionic strength, with a maximum adsorbed amount at isoelectric conditions (4). In other aspects the adsorption behavior of most proteins deviates from that of the simple polymers. In solution the simple polymers adopt flexible high-entropy structures, but when adsorbed at an interface their entropy is lower. In proteins, in particular globular proteins in an aqueous medium, the polyamino acid chain is folded up to shield the apolar moieties from contact with water resulting in a more or less compact structure of which the exterior is relatively hydrophilic and the interior more hydrophobic. Obviously, the ionic groups reside primarily at the water-exposed surface of the protein molecule. Thus, unlike the simple polymers, proteins have limited conformational freedom or, in other words, are low-entropy structures (5). For reasons explained under “Protein adsorption affinity”, upon adsorption the protein may undergo structural rearrangements towards a higher conformational entropy. Against this background we will discuss some theoretical and phenomenological aspects of protein adsorption and its applications. 1.1. Protein Adsorption Affinity Adsorption data are often presented in the form of adsorption isotherms, where, for constant temperature, the adsorbed amount Γ per unit mass or, preferably, per unit surface area of the sorbent, is plotted against the protein concentration cp in solution, after adsorption. Protein adsorption isotherms tend to belong to the “high affinity” category, displaying a steep initial rise and a strong resistance to desorption by dilution. There is no reason to expect the isotherms to be of the Langmuirian type, because the premises of the Langmuir theory are usually not fulfilled: the adsorption is not at all or only partly reversible, lateral interactions cannot be excluded, and the attachment is usually not site-determined (6). The observation that adsorption isotherms for (globular) proteins show 2 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


well-developed plateau values, unlike the isotherms for coiled polymers that tend to increase with increasing concentration in solution, suggests that, even though structural changes may occur, the protein molecules do not unfold to attain a loopy structure at the sorbent surface. Indeed, different experimental approaches, such as ellipsometry, optical waveguide spectroscopy, quartz crystal microbalance, and AFM spectroscopy, point to relatively compact adsorbed protein layers (7–10). In some cases protein adsorption isotherms do follow a Langmuir pattern. This may be due to variation of the sorbent surface area occupied per protein molecule with varying protein concentration in solution. This phenomenon is discussed in more detail in section “Kinetics and dynamics of protein adsorption” as well as in reference (11). Obviously, analysis of the isotherm using the Langmuir theory (or modifications thereof) yields misleading conclusions, because the underlying conditions of that theory are not obeyed. Because of their complex nature, i.e., amphiphilicity, ambivalency, and structural features, adsorption of proteins is an intricate phenomenon involving different types of interactions. The main contributions to the adsorption process are from electrostatic, dispersion and hydrophobic forces, and, in many cases, from rearrangements in the structure of the protein molecules (6). The distance over which electrostatic interaction is effective, the so-called Debye length (12), is in the range of a few nm, depending on the ionic strength. More specifically, in a medium of 0.01 M ionic strength the Debye length is 3 nm and in 0.1 M ionic strength it is 1 nm. Dispersion forces between proteins and sorbents interacting across an aqueous medium are usually attractive but small, because of the small dimensions of protein molecules and the low value of the Hamaker constant pertaining to such systems (12, 13). When the protein and the sorbent are in close proximity (say, ≤ 0.5 nm) changes in the hydration of both components may strongly affect the adsorption. When the surfaces of the protein and the sorbent are both polar it is probable that some hydration water is retained in the contact zone between the two. However, if the surface of the protein and/or the sorbent is primarily apolar, dehydration strongly favors adsorption. Thus, when the protein and the sorbent repel each other electrostatically adsorption may occur because of overruling attractive forces. More quantitatively, the Gibbs energy of dehydration of one CH2 group is about 1 kBT, which corresponds to the Gibbs energy of adsorption of one monovalent ion at a surface having a potential of 25 mV. Still, because of the larger range of operation, electrostatic repulsion may give rise to an energy barrier that the protein has to surpass prior to deposition at the sorbent surface. Rearrangements in the protein structure may occur when a protein molecule encounters an interface where it can turn one side away from the aqueous solution. Then, upon adsorption the protein may be able to present part of its hydrophobic interior at the sorbent surface without exposing apolar residues to the water. As a consequence, intramolecular hydrophobic interactions become less important as a factor stabilizing the protein structure. Because hydrophobic interactions in the protein’s interior support the formation of ordered secondary structures (α-helices and β-sheets) (5), a reduction of these interactions destabilizes such structures. A decrease of α-helix and/or β-sheet content is therefore expected if the peptide units released from these structures can form hydrogen bonds with the 3 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


sorbent surface, as is the case for oxides like glass, silica, and metal oxides, or with remaining surface-bound water molecules. The decrease of ordered structure implies a higher conformational entropy and thus favors adsorption, possibly up to tens of kBT per protein molecule (6). If, however, in the non-aqueous contact zone no hydrogen bonding with the surface is possible, which is the case for apolar surfaces, adsorption may induce extra peptide-peptide hydrogen bonds promoting the formation of α-helices and β-sheets (14). Thus, whether or not adsorption at an apolar surface leads to an increased or decreased order in the protein structure depends on a subtle balance between energetically favorable intramolecular interactions (notably hydrogen bonding) and the ensuing changes in the conformational entropy of the protein. In this context the terms “hard” and “soft” have been introduced (15) to indicate the strength of the internal structural coherence in the protein molecule and, hence, its resistance against adsorption-induced conformational changes. The main conclusion is that interfaces cannot easily resist the adsorption of proteins. When the sorbent surface is hydrophobic, adsorption of any type of protein is very likely because dehydration of that surface easily outweighs electrostatic repulsion. When the sorbent is hydrophilic electrostatic interaction and/or protein structural changes may facilitate adsorption. Only when the surface is hydrophilic and the protein hard can electrostatic repulsion prevent adsorption from occurring. To achieve protein resistance, surfaces are modified, e.g., by applying a coating of hydrophilic strongly hydrated polymers or zwitterionic components. This matter is further discussed in Section 4, Resistance to protein adsorption.

1.2. Kinetics and Dynamics of Protein Adsorption As adsorption of proteins appears to be irreversible on practical time scales, the characteristics of the adsorbed molecules in their final state depend on their history, that is, on their preceding stages. Kinetics, in particular rates of adsorption relative to rates of structural changes, should be considered. During the last few decades various models for protein adsorption kinetics have been proposed. Because of the complexity of the protein and, possibly, the sorbent surfaces on the atomic level the models follow a mesoscopic approach, where the protein is considered as a particle and effective rate constants, particle-sorbent and particle-particle interactions are used. The models have in common that they account for the generally observed features of (partial) irreversibility of the protein adsorption process and deceleration of adsorption with increasing coverage of the sorbent surface. The models differ more or less with respect to the underlying assumptions. For instance, Bornzin and Miller (16) assume the sorbent surface heterogeneity to cause partial irreversibility, distinguishing regions where the protein molecules stick irreversibly and regions where they attach weakly and desorb upon dilution. Kurrat et al (17) interpret reversible and irreversible binding in terms of the number of bonds formed, without indicating whether this variation in the way of binding results from sorbent surface heterogeneity or from different orientations/conformations of the 4 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


adsorbed protein molecule. Obviously, when the sorbent surface is homogeneous the irreversibly adsorbed fraction will increase in time and ultimately the whole protein population at the surface will be irreversibly adsorbed. In the models proposed by Walton and Soderquist (18) and Beissinger and Leonard (19) time-dependent adaptation of the adsorbed protein structure to optimize interaction with the sorbent surface is accounted for: initially the protein adsorbs reversibly but during contact with the surface the desorption rate decreases gradually with time. Here too, the irreversible nature of the adsorbed layer increases with ongoing contact between the sorbent and the protein solution. The same assumptions, but, furthermore, an increasing molecular area (“footprint”) of the adsorbed protein molecule when it relaxes at the sorbent surface, is included in the model presented by Norde (20). According to this model, the growing fraction of irreversibly adsorbed, structurally relaxed, molecules at the expense of reversibly adsorbed unperturbed ones may result in an “overshoot” of protein at the surface during the course of the adsorption process (21–23). Perhaps the most successful, at least the most popular, description of protein adsorption is the random sequential adsorption (RSA) model, or modifications thereof (24–26). According to the RSA theory a single adsorbed molecule (or, for that matter, particle) that hits the sorbent surface sticks there and defines a zone around that particle that excludes the center of subsequently arriving molecules. Thus, for spheres of radius a each adsorbing particle blocks an area of π(2a)2. Therefore, at low surface coverage the area available for adsorption decreases four times faster than when the surface occupancy by the particles themselves is taken into account, with a corresponding decrease in adsorption rate. At higher surface coverage the area available for adsorption should be corrected for overlapping exclusion zones around the particles. For spheres the RSA model predicts adsorption saturation due to jamming at a surface coverage of 55%. For particle geometries deviating from spherical the jamming limit is lower, e.g., 40% for particles having an aspect ratio of 7.5. Experimental values for protein adsorption are usually higher than the jamming limits predicted by RSA. This may be due to the possibility of lateral diffusion of the adsorbed protein molecules, as has been reported by (27, 28) as well as by Sotres et al in Chapter 6. A fundamental problem in applying this or any model to actual data is that the area available to the protein molecule is never well known, because even very small, protein molecule sized deviations from flatness can accommodate protein molecules, yet are easily missed by surface area measuring methods. The RSA model may be modified to more accurately describe protein adsorption by including surface-induced changes in protein conformation and orientation (26). Such changes usually lead to a larger footprint and in the RSA model this is accounted for by an instantaneous and symmetric expansion of the particle to a given pre-set size. If no space is available for that expansion the particle permanently keeps its original dimensions. Further modification includes that, unlike the expanded particle, the non-expanded particles may leave the surface by desorption (26). The result could be that, under certain conditions, non-expanded particles are gradually displaced by expanded ones showing up as a maximum in the adsorbed amount (“overshoot”) during the sequential adsorption process. 5 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The RSA model has also been adapted to apply to mixtures of different proteins (26). Then, the population of each type of protein in the adsorbed layer is determined by its respective adsorption rate and molecular size. However, the outcome is not compatible with the experimentally observed “Vroman effect”, i.e., the transient change in composition of the adsorbed layer due to the displacement one kind of protein by a later arriving kind that has a higher adsorption affinity (29, 30). Indeed, the experimental data on sequential adsorption of antibodies and serum albumin, reported by Dupont-Gillain in Chapter 21, seem to be at odds with the RSA model. Each of these models is suspect in one way or another. The main problem is related to the complexity and heterogeneity of the protein molecule and the poor understanding of the mechanism underlying the time-dependent desorbability of the protein layer (31). Structural changes in the protein molecules are essential in adsorption kinetics and, because of the irreversibility of the adsorption process, for the final state and, as a consequence, for the biological activity of the adsorbed layer. Therefore, the dynamics of the relaxation of the proteins at the sorbent surface is considered in more detail. Relaxation, which usually implies a certain degree of spreading, leading to lower adsorption saturation Γsat, occurs with a certain characteristic time τr. The extent of spreading depends on the rate of relaxation compared to the time τf needed to fill the sorbent surface, in the absence of desorption. The value of τr depends on the protein’s resistance against deformation. For a given protein, the internal coherence usually decreases with increasing net charge. Indeed, the maximum value of Γsat (pH), often observed at the isoelectric point of the protein, may be ascribed to progressive conformational changes at pH values further away from the isoelectric point (6). Additionally, τr is influenced by properties of the sorbent-water interface, notably its interfacial tension. The higher the interfacial tension is, the stronger is the tendency to spread over it. Examples are given in references (32, 33). The value of τf is controlled by the supply rate (flux) of molecules that arrive at the sorbent surface and are able to deposit. Hence, τf scales inversely with the protein concentration in solution and linearly with the resistance to reach the surface (which is composed of the resistance to transport through the solution and the resistance associated with overcoming possible barriers for deposition). If τr/τf >>1, relaxation is completely inhibited because adjacent surface area is already occupied by newly depositing molecules before the previously adsorbed one has the time to spread. If, conversely, τr/τf

Proteins at Interfaces III: Introductory Overview - American Chemical

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