Protein Adsorption at Solid-Liquid Interfaces: A Colloid-Chemical

Chapter 2

Protein Adsorption at Solid-Liquid Interfaces: A Colloid-Chemical Approach W. Norde, J. G. Ε. M . Fraaye, and J. Lyklema

Downloaded by CORNELL UNIV on September 2, 2016 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch002

Department of Physical and Colloid Chemistry, Agricultural University, De Dreijen 6, 6703 BC Wageningen, Netherlands

Protein adsorption on solid surfaces is discussed from a colloid chemical and thermodynamic point of view. Information is mainly obtained from adsorption isotherms, (proton)titrations, electrokinetics and calorimetry. The adsorption behavior of human plasma albumin and bovine pancreas ribonuclease at various surfaces is studied. The differences in behavior between the two proteins are related to differences in the structural properties. Furthermore, the essential role of the low molecular weight electrolytes in the overall protein adsorption process is stressed. Since the beginning of this century the adsorption of proteins at phase boundaries has been investigated for various reasons. A vast amount of l i t e r a t u r e , including several review a r t i c l e s (e.g., 1,2,3,4), i s available now. Most of the published work deals with adsorbed amounts and only during the last few decades have issues such as adsorption mechanisms and structure of the adsorbed molecules been discussed. Surveying the l i t e r a t u r e , i t appears that the i n t e r f a c i a l behavior of proteins i s a controversial subject. The main reason i s that many studies have been performed under i n s u f f i c i e n t l y defined conditions and/or that conclusions have been drawn on the basis of too scanty experimental evidence. Furthermore, the theoretical description of adsorbed layers of simple, f l e x i b l e polymers i s s t i l l in i t s infancy (5,6). As the structure of proteins i s much more complex than that of those simple polymers, theories of polymer adsorption need to be greatly extended to become applicable to proteins. Clearly, our current knowledge of protein adsorption mechanisms and of the structure of the adsorbed layer i s far from complete. In a number of ways the adsorption of polymers, including proteins, d i f f e r s from that of low molecular weight substances. A polymer molecule attaches to the sorbent surface v i a several segments. Even i f the adsorption free energy per segment i s low, 0097-6156/87/0343-0036$06.00/0 © 1987 American Chemical Society

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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NORDE ET AL.

Protein Adsorption at Solid-Liquid Interfaces

37

say, 1 kT, attachment of tens of segments adds up to a large adsorption free energy of some tens of kT for the whole molecule. As a result, polymer molecules do not readily desorb i n the pure solvent but they may be displaced by adding other (macro)molecules that adsorb with a larger (segmental) free energy. The three-dimensional structure of a protein molecule i s the net result of interactions inside the molecule and interactions between the protein and i t s environment. Adsorption involves the transfer of protein from solution to the sorbent surface and the concomitant displacement of solvent and, possibly, other components from that surface. The r e s u l t i n g environmental change may induce alterations i n the protein structure, which, i n turn, may affect the b i o l o g i c a l a c t i v i t y of the protein. Needless to say, s t r u c t u r a l variations upon adsorption are of great relevance to the various p r a c t i c a l applications of immobilized proteins. It i s evident that elucidation of the i n t e r f a c i a l behavior of proteins i s not a simple matter and requires contributions from several d i s c i p l i n e s . In recent years considerable progress has been made i n applying spectroscopic techniques to proteins i n the adsorbed state (e.g., 7,8,9). In such studies a (small) part of the molecule is analyzed i n d e t a i l . In our laboratory we study protein adsorption from a more c l a s s i c a l , colloid-chemical point of view. Arguments are derived from experimental data r e f e r r i n g to whole protein molecules or to layers of them. Information i s obtained from adsorption isotherms, proton t i t r a t i o n s and both e l e c t r o k i n e t i c and thermochemical measurements. Recently, t o p i c a l questions such as r e v e r s i b i l i t y of the adsorption process and changes i n the protein structure have been considered. This more h o l i s t i c approach has produced some insights that could not easily be obtained otherwise. Adsorption

Isotherms

The shape of the equilibrium isotherm (adsorbed amount Γ as a function of the concentration c i n solution) yields information about the free energy of adsorption. For most f l e x i b l e , highly solvated polymers h i g h - a f f i n i t y isotherms are obtained, i . e . isotherms i n which the i n i t i a l part coincides with the Γ-axis after which a l e v e l l i n g off takes place to a (pseudo-) plateau. However, isotherms for globular proteins often show a f i n i t e i n i t i a l slope. They develop well-defined plateaus at rather d i l u t e concentrations i n solution ( c < 1 g dm ). In interpreting adsorption isotherms, a d i s t i n c t i o n should be made between very low coverage ( i n i t i a l part of the isotherm), where the protein molecules interact with the sorbent surface only, and high surface coverage (adsorption plateau), where l a t e r a l interactions between the adsorbed molecules play a role as well. In the l i t e r a t u r e not much attention has been paid to the i n i t i a l part of the isotherm. Due to a n a l y t i c a l l i m i t a t i o n s , the trends i n this region often are rather uncertain. With human plasma albumin (ΗΡΑ) adsorbing on either polystyrene latex (10) or single crystals of polyoxymethylene (11) i t has been observed that the slope of the i n i t i a l part of the isotherm becomes less steep with increasing pH. Anticipating the discussion i n the following section,


38

PROTEINS AT INTERFACES

this could be caused by an increased number of carboxyl groups of the protein oriented towards the sorbent surface. Furthermore, on polystyrene i t was found that the adsorption of ΗΡΑ i n the low surface coverage region increased with increasing temperature, except at the i s o e l e c t r i c point (i.e.p.) of the protein where the adsorption appeared to be independent of the temperature. According to Clapeyron's law a positive value f o r (6r/ôT) implies an endothermic adsorption process under i s o s t e r i c conditions. Although with protein adsorption i s o s t e r i c conditions are d i f f i c u l t to establish, the q u a l i t a t i v e conclusion i s that at pH# i.e.p. the adsorption enthalpy i s p o s i t i v e . Hence, under those conditions, adsorption must be entropically driven. We w i l l return to this subject i n section 5. Usually, the plateau-value, Γ , of the isotherm corresponds roughly to a close-packed monolayer of native molecules i n a side-on or end-on orientation. It indicates that, at least at solution concentrations that are not excessively high, multilayers are not formed. In various systems i t i s observed that Γρ(ρΗ) i s at a maximum i n the i s o e l e c t r i c region of the protein molecule (e.g., 7,12,13,14). For example, Γ (ρΗ) curves for ΗΡΑ at several sorbents are shown i n Figure 1. The occurrence of a maximum at the i.e.p., independent of the nature of the sorbent, suggests that the charge of the protein molecule greatly influences Γ . In p a r t i c u l a r , f o r ΗΡΑ on polystyrene surfaces ample evidence has been collected to conclude that the reduction i n Γ on either side of the i.e.p. i s Ρ due to s t r u c t u r a l rearrangements i n the adsorbing molecules, rather than to increased l a t e r a l repulsion. Since the trends i n Γ (ρΗ) f o r the other surfaces are similar, they also may be caused by changes in the structure of the ΗΡΑ molecule. It i s , furthermore, remarkable that ΗΡΑ and many other proteins adsorb spontaneously on hydrophilic surfaces, even i f the surface has the same charge sign as the protein (e.g. ΗΡΑ on hematite, pH > 6.8). Under such conditions dehydration of the sorbent surface and o v e r a l l e l e c t r o s t a t i c interaction oppose the adsorption process. Therefore another contribution, originating from the protein molecule, drives the adsorption. Although the Γ (ρΗ) pattern, as shown i n Figure 1, i s quite common, i t i s not followed by a l l proteins. As an example, Γ_(ρΗ) for bovine pancreas ribonuclease (RNase) on d i f f e r e n t sorbents i s shown i n Figure 2. On polystyrene latex, Γ i s e s s e n t i a l l y independent of the pH of adsorption. The value of Γ i s comparable with that of a complete monolayer of native RNase molecules. I t suggests that only minor s t r u c t u r a l changes, i f any, occur and that they are not affected by the protein charge (the i.e.p. of RNase i s pH 9.3). The a f f i n i t y of RNase for the uncharged, less hydrophobic polyoxymethylene crystals i s so low that no s i g n i f i c a n t adsorption can be detected. With the hydrophilic hematite, RNase adsorption occurs only i n the pH range where the protein and the sorbent are oppositely charged. Thus, i n contrast to ΗΡΑ, RNase does not adsorb on hydrophilic surfaces, except i f i t i s aided e l e c t r o s t a t i c a l l y . I t i s concluded that the factor that dominates ΗΡΑ adsorption i s less strong, or absent, i n RNase. It i s probable that these differences i n the adsorption behavior between ΗΡΑ and RNase are related to differences i n s t r u c t u r a l properties between these two proteins. This w i l l be discussed further below.


c

ρ

ρ

ρ

ρ

ρ

ρ


2.

NORDE ET AL.

39



- mg m

pH

o^-tFigure 1. Plateau values for the adsorption of human plasma albumin on polystyrene latex (Δ), s i l v e r iodide (*), polyoxymethylene (•) and hematite (·). E l e c t r o l y t e : 0.01 M KNOo or 0.05 M KNO3 (for adsorption at polyoxymethylene). Τ = 22 C. e

1.5-

mg rrf

1.0-

0.5-

pH

Figure 2. Plateau values for the adsorption of bovine pancreas ribonuclease on polystyrene latex (o), polyoxymethylene ( • ) and hematite (x). Conditions as in Figure 1.


40



Adsorption

(Ir)reverstbllity

As indicated, protein adsorption isotherms are often not of the h i g h - a f f i n i t y type. However, desorption of the protein into the pure buffer does not occur s i g n i f i c a n t l y within hours or days. This observation lends support to the suggestion that, after adsorption, s t r u c t u r a l changes occur in the protein molecule in order to adapt i t s structure to the new environment. Such s t r u c t u r a l changes lower the free energy of the system, and, hence, the a f f i n i t y between protein and sorbent surface increases after adsorption. The condition of simultaneous detachment of several segments from the sorbent surface can be expected to slow the rate of desorption into the pure buffer, r e l a t i v e to the rate of adsorption. However, proteins may desorb readily on changing other conditions, e.g. pH or ionic strength of the medium or by adding a component that has a larger (segmental) free energy of adsorption (11). Moreover, r e l a t i v e l y fast exchange between adsorbed and dissolved protein molecules has been observed by various investigators (e.g., 15,16,17,18). For ΗΡΑ, removed from the sorbents hematite, s i l i c a and polyoxymethylene, the molecular structure has been compared with that of the native molecule, on the basis of their c i r c u l a r dichroisra spectra (11). It was found that after desorption the h e l i x content of ΗΡΑ is some twenty to t h i r t y percent lower. This reduction is v i r t u a l l y independent of the type of sorbent and the desorption method. It suggests that the change in the h e l i x content is related to properties of the protein molecule i t s e l f . It i s s t i l l not clear to which extent the adsorption and the desorption step affect the protein structure. It i s furthermore interesting that the h e l i x reduction i s larger for the samples with lower r e v a l u e s . This supports the e a r l i e r conclusion that a reduced Γ value r e f l e c t s further structural rearrangements in the protein molecule. It i s noted that the decrease in the helix content of desorbed ΗΡΑ found by us i s considerably less than that reported by others (19). RNase removed from hematite surfaces did not show s i g n i f i c a n t a l t e r a t i o n of i t s h e l i c a l content. This i s in agreement with our interpretation of the constant Γ (ρΗ) f o r this protein. ρ

ρ

Charge Effects In an aqueous medium, proteins and s o l i d surfaces are usually charged. In both systems the charge i s neutralized by counter- and co-ions, that are partly physically bound and partly d i f f u s e l y d i s t r i b u t e d . The charge d i s t r i b u t i o n in and around a protein molecule and at a sorbent surface i s schematically represented in Figure 3. When the protein approaches the surface the e l e c t r i c a l double layers overlap, giving rise to a r e d i s t r i b u t i o n of charge. This can have a s i g n i f i c a n t impact on protein adsorption. Tn some systems, e.g. RNase with a hydrophilic sorbent surface, o v e r a l l e l e c t r o s t a t i c repulsion between the protein and the sorbent prevents adsorption. With other proteins, e.g. ΗΡΑ, interactions between charged groups do not play a decisive r o l e . Interactions between charges on proteins and surfaces are


2.

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NORDE ET AL.

screened by low molecular weight ions in the system. On increasing the ionic strength, larger values of Γ are usually observed (10,20,21). If this i s found i n the case where the protein and the sorbent are oppositely charged, i t indicates that Γ i s primarily influenced by charge-charge interactions within, or between, protein molecules rather than between protein and sorbent. The influence of ionic strength cannot always easily be interpreted. It has been reported that the effect of ionic strength on ΗΡΑ adsorption depends on the type of sorbent used (12). Furthermore, adsorption may be sensitive to the type of ion present (22,23). The conclusion i s that the role of low molecular weight ions i s more complex than just e l e c t r o s t a t i c screening between interacting charges. Proton t i t r a t i o n data (11,24) point to the uptake of H ions by molecules of ΗΡΑ and RNase upon adsorption on polystyrene p a r t i c l e s . E s p e c i a l l y the pK of the carboxyl groups undergoes a considerable s h i f t to higher values. It i s inferred that a r e l a t i v e l y large f r a c t i o n of the carboxyl groups faces the negatively (!) charged polystyrene surface. The t i t r a t i o n data also reveal the different adsorption behavior between ΗΡΑ and RNase. For RNase the s h i f t i n the t i t r a t i o n curve i s e s s e n t i a l l y independent of the pH of adsorption, whereas for ΗΡΑ the s h i f t increases the further the pH of adsorption is away from the i.e.p. of the protein. These results confirm the conclusions i n the foregoing section: the structural perturbation, i f any, i n RNase i s not sensitive to the pH of adsorption and the structure of adsorbing ΗΡΑ i s progressively altered on moving the pH away from the i.e.p. Electrophoresis experiments (25,26,27) lead to the conclusion that ions, other than Η , are also transferred between the solution and the adsorbed protein layer. Figure 4 shows the charge a l t e r a t i o n due to the transfer of ions (including H ) f o r plateau-adsorption of ΗΡΑ and RNase on various surfaces. The trends are i n q u a l i t a t i v e agreement with expectations according to e l e c t r o s t a t i c interaction between the protein and the sorbent surface. The conclusion i s that charge-charge interactions are easily neutralized by adsorption of low molecular weight ions. Based on a model we published some ten years ago (28), the amount of co-adsorbed ions was estimated. Direct determination of ion uptake, using the radionuclides Na , Ba and the paramagnetic Mn^ ion has semi-quantitativily confirmed the model predictions (23). According to the model, the ion uptake has an e l e c t r o s t a t i c reason, i . e . i t happens to prevent accumulation of net charge i n the low d i e l e c t r i c contact region between the protein and the sorbent surface, which would otherwise result i n high values f o r the e l e c t r o s t a t i c potential. In addition to transfer of charge, the uptake of ions involves transfer of matter. Hence, the molar Gibbs energy, g, of transfer of an ion i between the solution s and the protein layer ρ contains both a chemical and an e l e c t r i c a l term: ρ

ρ


+

+

+

g

P t

"

g

8 4

-

(

μ

Ρ 1

-

μ

β 4

)

+ z F t

( Ψ

Ρ

Ψ

"

8

)

where i s the chemical potential of i i n the phase indicated by the superscript, ψ the e l e c t r o s t a t i c potential, z^ the valency of i and F the Faraday constant. As water usually i s a better solvent f o r ions than the proteinaceous layer, μ J* - μ^ > 0, i . e . , the chemical δ




©

Θ

layer sorbent

Figure 3. Schematic i l l u s t r a t i o n of the charge d i s t r i b u t i o n i n and around a dissolved protein molecule and at a sorbent surface.

-μ€ c m "

~7

Protein Adsorption at Solid-Liquid Interfaces: A Colloid-Chemical

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