Retention, Unfolding, and Deformation of Soluble Proteins on Solids

Chapter 2

Retention, Unfolding, and Deformation of Soluble Proteins on Solids 1

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S. C. Goheen , B. M. Gibbins , J. L. Hilsenbeck , and J. V. Edwards Downloaded by UNIV OF GUELPH LIBRARY on August 10, 2012 | http://pubs.acs.org Publication Date: August 10, 2001 | doi: 10.1021/bk-2001-0792.ch002

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Pacific Northwest National Laboratory, Batelle Boulevard, Richland,WA99352 Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, L A 70124 The behavior of proteins on solids has been studied over several decades. Recent developments have expanded the variety of methods that can be used to examine protein-surface interactions. This paper summarizes the adsorptive behavior of proteins on various surfaces including cotton and synthetic polymers. In general, it is postulated that surfaces act like catalysts for protein unfolding. We give specific examples of the unfolding of cytochrome c and show a lowering of the unfolding temperature from solution to an anionic surface. Additionally, we show how chromatography can be used to screen adsorbents, such as cotton for possible commercial applications. The ability of each surface chemistry to act as a catalyst for unfolding is discussed.

This chapter focuses on the adsorption of proteins and discusses the application of protein adsorption to cotton. Cotton is a special natural product that is nearly pure cellulose. Table I displays the composition of cotton. Cotton was not used because of its high purity, but rather for the potential application as a gauze to assist in wound healing. Whether the purity of cotton helps its success in wound healing is not clear. However, using a pure substrate for absorptive studies helps in the understanding of its adsorptive properties. Benefits to studying the adsorption of proteins to cotton include not only wound healing, but also fabric cleaning and staining. PROPERTIES O F C O T T O N A N D C E L L U L O S E The molecular structure of cellulose is well known. Cellulose is typically nonionic but has polar characteristics. Its hydrophilic properties help cotton resist excessive adsorption of water soluble proteins. This resistance is due to the matrix

© 2001 American Chemical Society

In Bioactive Fibers and Polymers; Edwards, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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21 having a greater attraction for water than protein. We modified cotton to enhance its binding properties for a specific protein, elastase. This allowed us to study the protein binding properties of various cotton derivatives (1).

Downloaded by UNIV OF GUELPH LIBRARY on August 10, 2012 | http://pubs.acs.org Publication Date: August 10, 2001 | doi: 10.1021/bk-2001-0792.ch002

Table I. Chemical Composition of Cotton Composition of Cotton Proportion ofdry Fiber weight (%) Cellulose Protein Pectin Wax Ash Other Substances

94.0 1.3 1.2 0.6 1.2 1.7

The information for this table was from "Encyclopedia of Chemical Technology, 4 Edition, Jacqueline I. Kroschwitz executive ed., Copyright 1993 by John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc. th

THE IMPORTANCE OF UNDERSTANDING PROTEIN ADSORPTION Protein adsorption is crucial to numerous industrial processes. Examples are biomaterial fouling and barnacle attachment. Barnacles absorb after receiving chemical cues from the surface (2). Bacterial adhesion has also been attributed to protein absorption and is known to take place in two stages. These have been described as adhesion of the extracellular membrane proteins in the primary and secondary minimum (2). The primary minimum involves molecular recognition. The secondary minimum is governed mainly by non-specific van der Waals forces. Similarly, protein adsorption to biomaterials involves two stages. The first is a reversible binding in which the native protein retains its shape. The second involves protein unfolding or spreading on the surface (3). This stage is irreversible, although severe conditions can cause these proteins to desorb (4). Protein adsorption from protein mixtures can be complex. For example, when plasma proteins from whole blood bind to biomaterials, albumin often binds initially and is later displaced by fibrinogen. Fibrinogen can then be displaced by other blood proteins (5). This is the Vroman Effect. Although the Vroman Effect was discovered for blood proteins, it may take place in other situations in which multiple components can bind. PROTEINS Proteins are polymeric amino acids and are the major component of dried living material. Some proteins are glycosylated (glycoproteins), still others are closely associated with lipids (membrane proteins, and lipoproteins). Each amino acid has a different chemical characteristic so that when they are assembled in a protein, they give the protein a unique series of chemical properties. Each protein varies by the


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sequence and/or number of amino acids. The amino acid sequence is partially responsible for the tertiary (3D) structure. The other contribution to protein structure occurs during the folding process (6). The final, native 3D structure is typically near or at the lowest energy state of the polypeptide under native conditions. Proteins can be classified in a number of different ways. For example there are enzymatic and non-enzymatic proteins. They can be classified by other means, such as those which are conjugated and non-conjugated, soluble and membrane-bound, or by their origin (viral, bacterial, plant, animal, blood, milk, etc.). There are believed to be about 40,000 different proteins in humans, with a large number of variations for each protein type. For example, it is believed that there are over 1 million different forms of the blood protein fibrinogen in each human (7). This large variety of proteins and protein forms suggest that when different types of proteins are present, the adsorption of one or more of them to any given surface is more likely than if they all had similar properties. Each surface may have an affinity to at least one of the thousands or millions of proteins available. W H E N D O PROTEINS A D S O R B ? Proteins are more likely to adsorb when surfaces have the least attraction for water. Water can prevent proteins from reaching a surface, minimizing protein adsorption (8). When protein adsorption does occur, the subsequent biological response depends on how the binding takes place. That is, the details of the adsorption process are important. A n example is the adsorption of blood proteins and blood coagulation. Surfaces with either high or low interfacial energy with water cause proteins to adsorb, but only surfaces with low interfacial energy with water accelerate blood coagulation (9). Some implants appear to resist coagulation more than others, probably due to the manner in which proteins bind to them. Another important consideration is how strongly water binds to the surface. A strongly bound water layer can prevent proteins from penetrating and binding. Yet, even strongly hydrophilic supports will promote protein binding and a biological response (6). This needs to be investigated further in order to improve the life expectancy of implant materials. Protein adsorption is a well-established field that melds the disciplines of surface science and biochemistry. In this field, significant knowledge can be gathered from some simple measurements. For instance, proteins can be loaded onto a surface from solution and the total amount of absorbed protein can be measured. This is the approach used for developing adsorption isotherms. Example isotherms are shown in Figure 1. Here, proteins were adsorbed either to a porous diethylaminoethyl (DEAE) derivatized agarose, or alumina. From these examples, one can observe that large amounts of protein can be bound to the D E A E support (Figure la) except when high concentrations or amounts of NaCl are present (IgG, Figure lb). However, albumin and fibrinogen remained bound after introducing 0.15 M NaCl, indicating they would not desorb under these near-physiological conditions. In contrast, IgG would not bind at that concentration, due to the ability of NaCl to occupy its ionic binding sites.


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ADSORPTION ISOTHERMS Adsorption isotherms are used not only to examine the amount of binding on a surface, or in a sorbent, but also to estimate the number of layers bound. When the protein structure or dimensions are known, and the surface is non-porous, plateaus in the isotherm (such as those observed in Figure lc) could help elucidate the molecular orientation (assuring close-packed monolayer, bilayers, and etc. are formed). Figure 1 compares the amount of protein bound on alumina and a porous D E A E support. Surface area is usually a known variable used to create an isotherm. For this study, the mass of the beads was known, but not the surface area. Therefore, mass was used in place of surface area. When the size of the adsorbed molecule is much smaller than the dimensions of the pores, the surfaces of the pores are available for adsorption. However, proteins can be larger than some pores, which restrict the available total area. The area, including that from the pores,

Initial Protein Concentration (mg/mL)

Figure la. Adsorption Isotherm for Albumin, Immunoglobulin G, and Fibrino Dilute (5mM) Tris Buffer (pH 7.4) on DEAE BioGelA.


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Initial Protein Concentration (mg/mL)

Figure lb. Adsorption Isotherm for Albumin, Immunoglobulin G, and Fibrino 0.15 MNaCl and (5 mM) Tris Buffer (pH 7.4) on DEAE BioGel A.


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T

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Protein Concentration in Solution (mg/mL)

Figure 1c. Comparison ofAdsorption Isotherms from Alumina and DEAEfor Fibrinogen and Albumin in Dilute (5 mM) Tris Buffer (pH 7.4).

must be included when the area is indicated in an isotherm. Pores complicate the determination of available surface area and the accurate interpretation of the isotherm. For alumina (Figure 1c), the pores were much smaller than the diameter of either of the blood plasma proteins shown. However, the pore size of agarose varies in commercially available gel permeation products (Figure 1-c) but in this case it was large enough (Bio-Gel A-0.5m, Bio Rad Laboratories, Hercules, C A , exclusion limit = 500,000 daltons) for most proteins to penetrate the beads. The significance of this difference is that the D E A E support could adsorb a greater protein mass due to its greater surface area per gram of material. However, alumina bound more protein than the porous D E A E , possibly due to the formation of multiple layers of protein on the alumina surface. O T H E R M E T H O D S F O R STUDYING PROTEIN ADSORPTION Adsorption isotherms are often used to define the amount of material adsorbed under equilibrium or steady state conditions. Other methods of studying surface properties include examining the surface tension. Surface tension influences the extent and rate of protein adsorption (10). The surface tension between water and a solid can be measured by placing a drop of water on the surface and measuring the contact angle. The contact angle is related to the affinity of the water for the surface. The water adhesion tension is negative for hydrophobic surfaces, resulting in a greater



26 contact angle. Polar surfaces such as silica have a much higher water adhesion tension, resulting in water spreading. It would be difficult to investigate the interfacial tension between a protein in solution and a surface in this manner. Proteins are generally in their native structure when they are in an aqueous buffer. When a protein binds to the surface in a multicomponent solution (protein, buffer, and water), the interfacial tension changes, as does the contact angle. In such a dynamic system, the contact angle would have to be followed with time and the data may be difficult to interpret. However, the surface tension between water and various solids has been measured, and correlated with various surface phenomena for which protein adsorption is believed to be responsible. One example is blood coagulation (9). Here, blood was believed to coagulate more rapidly when the water adhesion tension of a surface was increased. Protein binding does not necessarily correlate with biological response. There is another factor, which is probably related to the orientation and organizational structure of the bound protein. Liquid Chromatography Surface-protein interactions have also been studied by a variety of more recent technologies such as ellipsometry, N M R , fluorescence, Auger spectroscopy, etc. A method we have used to study protein adsorption is liquid chromatography (LC). L C is typically used to perform separations to help purify components of a mixture. The separation mechanism is by adsorption-desorption processes between the analytes and packing material surface. This affinity, or recognition process, is commonly used to purify proteins. L C allows us to examine three different properties of protein adsorption. One of these properties is the capacity of the material to adsorb protein. The same parameter is measured by adsorption isotherms (11). L C also allows the exploration of the relative retention of proteins on a surface chemistry (12). A third property examined by L C is the kinetics of the unfolding of proteins on surfaces (13, 14). Measuring the capacity of a sorbent for protein by L C involves monitoring the breakthrough during continuous loading (11). A family of data points can be generated in this manner in which either the temperature or eluent concentration is is varied. A large amount of information can be gathered this way, but care must be taken to quantify the more and less permanently bound protein, since losses for some proteins is a time-dependent phenomena (13, 16). Other phenomena for protein losses to surfaces may be concentration and temperature dependent (10). Relative retention of proteins is a typical measurement in L C , but its relevance to sorption is often under-emphasized. For many, the purpose of L C is in the separation and purification of components, such as proteins. The sorbent properties are not the focus of the study, and become a secondary consideration. However, for the surface scientist, elution order and relative retention help define the binding properties of the protein for the sorbent. One example of the significance of this approach is in the testing of biomaterials for protein adsorption. A n easy method for testing whether a protein will bind would be to pass it through a column containing particles of the biomaterial in the presence of physiological buffer. Those proteins which elute will not absorb. Another more definitive approach is to use a gradient that passes through


27 the physiological strength buffer. Using this approach, the relative binding strength can be determined (13). To illustrate this, Figure 2 shows a chromatographic separation of the three major proteins in blood plasma. These are albumin, immunoglobulin G (IgG), and fibrinogen. The sorbent is a weak ion exchange material, D E A E . Approximately one out of every 100 or 200 available sites on the

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Retention, Unfolding, and Deformation of Soluble Proteins on Solids

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