Reversibility and the Mechanism of Protein Adsorption - American

Chapter 2

Reversibility and the Mechanism of Protein Adsorption 1

2

Downloaded by PENNSYLVANIA STATE UNIV on March 11, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch002

Willem Norde and Charles A. Haynes 1

Department of Physical and Colloid Chemistry, Wageningen Agricultural University, P.O. Box 8038, 6700 Wageningen, Netherlands Biotechnology Laboratory, University of British Columbia, 6174 University Boulevard, 237 Wesbrook Building, Vancouver, British Columbia V6T 1Z3, Canada 2

Detailed adsorption isotherm data are combined with thermodynamic arguments in an effort to determine whether protein adsorption to solids is a reversible or irreversible process. We then examine the dominant driving forces for protein adsorption and present a thermodynamic framework for understanding the protein-adsorption process which is consistent with results from our study of process (ir)reversibility. Phenomenologically, a system is in equilibrium if no further changes take place at constant surroundings. At constant pressure P and temperature T, the equilibrium state of a system is characterized by a minimum value of the total Gibbs energy G. Any other state, away from this minimum, is nonequilibrium and there will be a spontaneous transition (i.e. a process) towards the equilibrium state provided the energy barriers along this transition are not prohibitively large. By definition, a process is reversible if, during the whole trajectory of the process, the departure from equilibrium is infinitesimally small, so that in the reverse process the variables characterizing the state of the system return through the same values but in the reverse order. Since a finite amount of time is required for the system to relax to its equilibrium state upon changing the conditions, investigations of the reversibility of a process must be designed such that the time of observation exceeds the time required for the system relaxation. In this paper, we address the question whether adsorption of proteins from aqueous solutions to solids is a reversible process with respect to variations in the bulk-solution protein concentration. The answer to this question, which is too often ignored in literature, determines what thermodynamic criteria apply to the protein adsorption process and also provides information about affinities between proteins and sorbent surfaces. We then examine the dominant driving forces for protein adsorption and present a thermodynamic framework for understanding the protein-adsorption process which is consistent with results from our study of process (ir)reversibility. A complementary set of adsorption-isotherm, isothermal-titration-microcalorimetry, potentiometric-titration, and differential-scanning-calorimetry data are used to argue that three effects, namely, structural rearrangements in the protein molecule, dehydration of the sorbent and protein surfaces, and redistribution of charged groups in the interfacial layer, usually make the primary contributions to the overall driving force for adsorption. 0097-6156/95/0602-0026$12.00/0 © 1995 American Chemical Society In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

2. NORDE & HAYNES

Reversibility and Mechanism of Protein Adsorption

Adsorption Isotherms and Reversibility Adsorption data are often presented as adsorption isotherms, where, at constant T, the amount adsorbed T is plotted against the concentration of sorbate c in solution (see Figure 1). For adsorption to be reversible with respect to variation of c , an increase in c from a value corresponding to point A in Fig. 1 to that corresponding to point C should result in a change in the adsorbed amount that is independent of the manner in which Cp has been changed. In this case, T should increase to its value at c = c (C) regardless of whether the change in concentration is made in one step [i.e., c = c (A) -> Cp = Cp(C)] or in multiple steps [e.g., via path A B ' B C ' C in Fig. 1]. Reversibility also requires that a decrease in c from its value at point C to its value at point A " result in a reduction in T to the value corresponding to point A , again independent of path. Therefore, in a reversible adsorption process, the ascending (increasing concentration in the bulk) and descending (decreasing concentration in the bulk) branches of the isotherm must overlap at all c . Only for such reversible processes can the adsorption isotherm be used to determine the (equilibrium) binding constant K, from which the thermodynamic functions of state (i.e., Gibbs energy of adsorption AadsG* enthalpy of adsorption A js//, and entropy of adsorption A^S) can be derived by applying reversible thermodynamics. p

p

p

p

p

p

p


p

p

a(

r

Cp Figure 1: Adsorption isotherm distinguishing reversible and irreversible pathways. Protein adsorption from aqueous solution often results in high-affinity isotherms where the initial slope of the ascending branch merges with the T-axis as depicted in Fig. 2a. Plateau values are reached at very low bulk protein concentrations and the region in which T depends on c is limited to values very near the T-axis. Verification of reversibility in such systems is difficult because precise measurement of the ascending and descending branches of the isotherm requires a method for determining bulk protein concentrations in very dilute solutions. Occasionally, lowaffinity isotherms are observed where the isotherm is distinguishable from the ordinate at low Cp (see Fig. 2b). In such systems, the form of the ascending branch is usually but not always (see refs. 1,2) independent of the number and size of c steps used in measuring the curve. However, when diluting such systems, V rarely if ever follows the same path backwards, thereby making the descending and ascending branches of p

p

In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

27

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PROTEINS AT INTERFACES II

the isotherm distinguishable. As a rule it is found that, when shear is excluded, dilution does not lead to detectable desorption of proteins from solid sorbents, particularly hydrophobic sorbents, even when the observation time is extended to several days and is therefore much longer than the relaxation time of the protein at the surface [2,3]. Such a deviation between the ascending and descending branches of the isotherm is defined as hysteresis. The occurrence of hysteresis indicates that at a given c the system has two equilibrium/meta-stable states: one on the ascending branch and the other on the descending branch. These two states are characterized by local minima in G which are separated by a Gibbs energy barrier that prevents the transition from the one state to the other and, hence, prevents the adsorption process from following a reversible path. The fact that the ascending and descending isotherms represent different equilibrium states implies that during the transition from adsorption to desorption a physical change has occurred in the system.


p

Irreversible Protein Adsorption In spite of the irreversible nature generally observed for protein adsorption, many authors erroneously interpret their experimental data using theories that are based on reversible thermodynamics. The most common example is the determination of A d G by fitting the ascending adsorption isotherm to the Langmuir or Scatchard equation [e.g., 4,5]. Another common approach involves calculation of the Gibbs energy of adhesion A dhG using the reversible thermodynamic result known as the Dupre equation a

s

a

AadhG = Ysp - Ysw - Ypw

(1)

where y is the interfacial tension of the interface indicated by the subscript and A jhG is the reversible work (at constant T and P) of forming a protein (p)/sorbent (s) interface at the expense of sorbent (s)/solution (w) and protein (p)/solution (w) interfaces [e.g., 6]. It is not clear how A ^ h G relates to A d G since the latter quantity reflects an irreversible process which, as shown below, often includes contributions from structural rearrangements in the protein molecule during adsorption; however, A ^ G and the quantity of interest AadsG are not equivalent. a(

a

s

Figure 2: Schematic representation of ascending and descending adsorption isotherms: (a) high-affinity isotherm, and (b) isotherms for which the descending branch shows a higher affinity between the protein and the sorbent surface.



2. NORDE & HAYNES


A persistent argument by those who continue to interpret protein-adsorption phenomena with reversible thermodynamics theories involves the assertion that the kinetics of protein adsorption are such that structural perturbations in the protein do not contribute to the driving force for adsorption because they occur after initial attachment of the protein to the interface [e.g., 7,8]. However, the Gibbs energy change A^G driving the protein-adsorption process refers to the global free energy required or released when taking a mole of protein in its native conformation in solution to its perturbed steady-state structure(s) on the sorbent surface. Thus, when they occur, protein structural rearrangements are an integral part of the adsorption process and cannot be ignored in any meaningful adsorption theory. The minimum error involved in treating protein adsorption as a reversible process can be estimated by calculating the entropy production due to the irreversibility of the process. In a closed system, the entropy change associated with any internal process can be written as AS = A^S + A[S

(2)

where A S is the reversible entropy exchange between the system and the surroundings and A{S is the internally created entropy in the system. For a reversible process A[S = 0 and for an irreversible process AiS > 0. According to Everett [9], Aads,iS can be calculated from the hysteresis loop (i.e., the closed-loop integral) between the ascending and descending branches of the adsorption isotherm e

(3) where T* is the adsorbed amount at the upper closure point of the hysteresis loop and R is the universal gas constant. Accurate solution of Eq. 3 requires detailed knowledge of r(c ) for both the ascending and descending isotherms, especially in the very-dilute c region. Regrettably, such data are rarely available. However, a minimum value for A ^ j S can be determined by letting the hysteresis loop close at the lowest experimentally detectable c . As an example, outlines of the ascending and descending isotherms for bovine serum albumin (BSA) on silica particles are shown in Fig. 3a [data can be found in ref. 10]. Replotting the data as shown in Fig. 3b and subsequent application of Eq. 3 above the lowest detectable c provides a minimum value for A ^ i S of 37 J K " mol" , indicating that irreversible entropy changes will lower the overall driving force for adsorption A ^G (at 298 K) by more than 11 kJ mol . It should be realized that if the ascending and descending branches of the ITT* versus In c plot do not coincide at concentrations below the detectable c limit (which in view of the shapes of the isotherms in Fig. 3b is almost certain), the true value of A ds,iS will be far greater than the minimum values calculated above. The hysteresis loop reflects a higher adsorption affinity for the descending branch of the isotherm as compared to the ascending branch. Assuming the sorbent surfaces before and after adsorption are identical, the following identity for the molar Gibbs energy of the protein g p

p

p

p

1

1

a

-1

p

p

a

p

g (adsorbed) - g (desorbed) < g (adsorbed) - g (native) p

p

p

p

(4)

must therefore hold. Thus, g (desorbed) > g (native) p

p


(5)

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PROTEINS AT INTERFACES II


indicating that the protein undergoes a physical change during the adsorption process. The nature of this physical change is illustrated by the transmission circular dichroism data of Norde et al. [11] for B S A adsorbed to and desorbed from silica. In these experiments, Norde et al. used morpholine to displace the adsorbed B S A after verifying that dilution did not lead to significant desorption. The average a-helix contents of B S A in its native, adsorbed, and desorbed (displaced) states are shown in Table 1. Adsorption to silica involves a severe reduction in the helical content of BSA, particularly when the surface coverage is low enough to allow for the increase in molecular volume which follows from a loss in ordered intra-atomic packing. Upon desorption from silica, B S A regains only a fraction of the helix content lost during adsorption and does not return to its native-state conformation (at least within the twoday observation time of the experiment). Table 1: a-helix content of bovine serum albumin in native, adsorbed to silica, and desorbed from silica states as measured by transmission circular dichroism (0 is the fractional surface coverage, i.e. 6 = T/TP , where TP is the plateau adsorption value) 1

pH

4.0 4.7 7.0

1

percentage a-helix content Adsorbed State q = 1.00 0 = 0.24 38 28

Native State 69 70 74

Desorbed State 50 51 55

Figure 3: Adsorption and desorption data for bovine serum albumin (BSA) on silica particles: (A) conventional representation of ascending and descending isotherms, and (B) replot of data according to Eq. 3, where the vertical dashed line indicates the lowest BSA detection level (c = 0.002 g dm-3; 0.05-M PBS; 25 °C). p

According to Eq. 5, re-incubation of desorbed BSA with fresh silica should show an adsorption affinity for silica which is stronger than that of native BSA. This is indeed the case. Time dependent reflectometry data [10] for adsorption of native and desorbed B S A to oxidized silicon wafers are shown in Figure 4a. Initial slopes of the reflectometry curves indicate that rates of adsorption are substantially higher for the previously (pre-)desorbed protein and, thus, that a higher fraction of the pre-desorbed


2. NORDE & HAYNES


protein molecules attach to the surface upon contact. As shown in Figure 4b, initial slopes of adsorption isotherms for native and pre-desorbed BSA also indicate that the latter conformation has a higher affinity for the silica surface.


1.5 - mg m~ ^

g dm''

Figure 4: Adsorption of bovine serum albumin (BSA) on silica: (A) adsorbed amounts from stagnation-point-flow reflectometry data (c = 0.01 g dm" ) where (x) is native BSA, (A) is BSA previously desorbed from silica by morpholine, and (A) and ( • ) are native B S A pre-exposed but not adsorbed (i.e., that remaining in the supernatant after equilibration) to silica and to morpholine, respectively; (B) adsorption isotherms for (O) native BSA and for (•) BSA previously desorbed from silica by morpholine (all experiments at 0.05-M PBS, pH 7,25 °C). 3

p

Dominant Driving Forces for Protein Adsorption The simplest realistic chronology of irreversible protein adsorption to a solid nonporous surface involves the three steps shown in Figure 5: (1) protein transport to the energetic boundary layer where the potential at the sorbent surface influences the rate of approach of the protein (including diffusion through the stagnant boundary layer), (2) interaction and attachment of the protein with the surface which may involve perturbations in protein structure, and (3) relaxation of the adsorbed protein to its steady-state conformation(s). As demonstrated by Northrup [12] and others [13], in the absence of convection, step (1) is a stochastic process which can be accurately described by the Langevin equation and Brownian-dynamics simulations. We will not concern ourselves further with this step of the adsorption process. Instead, we focus on steps (2) and (3), which are primarily controlled by direct forces (e.g., coulombic and hydration forces) between the protein, the sorbent surface, solvent (water) molecules, other adsorbed protein molecules in close proximity, and low-molecularweight ions in the interfacial region. The ability of water to hydrogen bond, the heterogeneous, usually charged surface chemistry of solid sorbents, and the amphipolar, amphoteric, compact nature of native proteins combined with their marginal structural stabilities suggest that no type of molecular interaction is unimportant in the adsorption process. However, our aim is to identify the nature and magnitudes of the dominant driving forces for globular protein adsorption, bearing in mind the irreversibility of the adsorption process.


31

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P R O T E I N S A T I N T E R F A C E S II

Native-State Conformation

Transport to Interfacial Region


©

Attachment to Surface

©

Structural © Further Rearrangements

Steady-State Perturbed Structure

Figure 5: Simplified chronology of irreversible protein adsorption to a charged solid. Table 2: Physico-chemical Properties of Model Proteins and Sorbent Surfaces Property

LSZ

ocLA

Molar Mass (D) 14,600 14,200 Dimensions (A ) 46x30x30 37x32x25 Isoelectric Point 4.2 11.1 Total Hydrophobicity (Jg ) - 7.6 - 5.8 % Apolar Surface Area 53 61 4.1 1.7 A N - D G (Jg" ) % a-Helix Content 42 26 Surface Charge Density (|iC n r ) Electrophoretic Mobility (10 m V " s" ) Electrokinetic Potential (mV) Hydrophobicity (contact angle of a sessile drop of 0.05-M PBS)

PS-

aFe203 (pH 9.5)

•23 -4.9 69 82°

- 2.9 -47

3

_1

1

2

-8

2

1

1

OP

Regardless of the mechanism and kinetics of the process, protein adsorption at constant T and p can only occur if the Gibbs energy of the system decreases: (6)

A ^ s G = AadsH - TAadsS

The irreversible nature of the protein-adsorption process eliminates the possibility of direct measurement of the overall driving force for adsorption, A dsG, as well as determination of A d S . This leaves A ds# and the heat capacity change upon adsorption, A dsCp> as the only directly measurable thermodynamic parameters a

a

S

a

a


2. NORDE & HAYNES


describing the irreversible adsorption process. Regrettably, such data are severely limited for protein adsorption systems [14-16]. Here we report A ds// and A dsC data for adsorption of two model proteins, hen egg-white lysozyme (LSZ) and bovine milk a-lactalbumin (aLA), to a negatively-charged polystyrene latex (PS-). As shown in Table 2, these proteins are of similar size, shape, and primary structure (40% sequence homology), but differ in native-state structural stabilities, hydrophobicities, and electrical properties. Electrokinetic, proton-titration, and chfferential-scanning-rmcrocalorimetry data are also reported for adsorption of these two model proteins to PS- and used to elucidate further those subprocesses involved in the adsorption process. a

a

p

Heats of Adsorption. A^ H represents the total enthalpy required or released when taking a mole of protein in its native conformation in solution to its perturbed steady-state structure(s) on the sorbent surface. The sign and magnitude of Aads// are therefore governed by a competition between the energetic subprocesses occurring within the protein molecule and between the protein and the sorbent surface. For instance, the total contribution from the electric field overlap, which includes the enthalpy change associated with net protein-protein and protein-sorbent coulombic interactions A ds//el> with low-molecular-weight ion (including proton) coadsorption in the interfacial layer A ds//ion-coad» and with formation of ion pairs between adjacent oppositely-charged residues on the protein and sorbent surfaces A ds//ion-pair, can be endothermic or exothermic depending on the solution pH, the ionic strength, and the nature and density of charges on the protein and sorbent.


S

a

a

a

400.0200.0-

E

-200.0-

r

-400.0-

Reversibility and the Mechanism of Protein Adsorption - American

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