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Electrostatic and van der Waals Contributions to Protein Adsorption: Comparison of Theory and Experiment Charles M. Rotht and Abraham M. L e n h o P Center for Molecular and Engineering Thermodynamics, Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716 Received January 12, 1995. In Final Form: May 30, 1995@ Electrostatic and van der Waals interactions are important in many colloidal phenomena and as such have been studied and described extensively. In a previous paper (Langmuir 1993,9,962), we described a model for the adsorption equilibrium constant of proteins at charged surfaces based on these effects, calculated using molecular properties of the proteins. Here, we compare the predictions of this model to experimentallydetermined equilibrium constants obtained using total internal reflectance fluorescence (TIRF)spectroscopy. With this surface-sensitivetechnique, equilibrium constants were obtained over 4 orders of magnitude as the ionic strength was varied over a decade and a half. Lysozyme adsorbed preferentiallycompared to chymotrypsinogenA at low ionic strengths while the opposite trend was observed at high ionic strengths. The model, in its various degrees of complexity, is able to predict the trend observed in equilibrium constant with varying ionic strength, to predict the values approximately,and to discern differences between the behaviors of the proteins studied. The utility of this model in understanding the interplay between charge and size-dependentforces is demonstrated,as is its potential for predictive calculations.
Introduction The adsorption of proteins to interfaces is a phenomenon that has been observed extensively and exploited frequently,yet it is not understood mechanistically. In many instances, the adsorption ofproteins is an undesired effect, such as in adsorption to biomaterials, fouling of membranes in dialysis, and fouling of bioprocessingequipment. On the other hand, the adsorption of proteins has been used extensively as a basis for separation in chromatography. The many various forms of chromatographyincluding affinity, reversed-phase, hydrophobic interaction, and ion-exchange-testify to the variety of functionalities inherent in proteins, as exhibited by their delicate interactions with interfaces, as well as by their sensitivity to environmental conditions. A quantitative understanding of protein adsorption is not simple to develop,as many different mechanisms have been postulated for the adsorption ofproteins. Certainly, the intermolecular forces-including electrostatic, dispersion, and solvation-between a protein molecule and the surface can be invoked to explain adsorption but several other effects may be implicated as well. Solution thermodynamics seems to play a role, as the extent of adsorption often correlates with the solubility of proteins under the same condition^.^,^ Very specific interactions between proteins and surfaces are also important, as is their packing on a surface. Furthermore, conformational changes can occur to a protein as its environment changes or as it interacts with a surface,6-8
* Author to whom correspondence and reprint requests should be addressed. + Present affiliation: Shriners Burns Institute and Massachusetts General Hospital, Boston, MA 02114. Abstract published in Advance A C S Abstracts, September 1, @
1995. (1) Norde, W.Adu. Coll. Interf Sci. 1986,25, 267. (2) Lu,D. R.; Lee S. J . ; Park, K. J.Biomater. Sci. Polym. Edn. 1991, 3, 127. (3)Lu, D.R.; Park, K. J.Biomater. Sci. Polym. Edn. 1990,1, 243. (4)Melander, W.; Horvdth, C. Arch. Biochem. Biophys. 1977,183, 200. ( 5 ) Shibata, C.T.; Lenhoff, A. M. J.Colloid Interface Sci. 1992,148, 469. (6) Kondo,A.;Oku, S.;Higashitani, K. J.Colloid Interface Sci. 1991, 143,214.
possibly providing an additional driving force for adsorption. Complicating matters further has been the finding that protein molecules can interact with neighbors on a surface, moving along the surface: reorienting themselves with respect to the surface,1° or even forming arrays of long-range order. 1,12 Our goal is to develop quantitative mechanistic models of protein adsorption, and this precludes accounting simultaneously for all of the effects mentioned above. Instead, we focus on a well-defined system in which many of these effectsmay be assumed to be unimportant, leaving a smaller set that can be modeled more rigorously. In particular, we focus on systems that we believe are controlled by electrostatic and van der Waals interactions, which have formed the basis for much of colloid science. These interactions are direct manifestations of electromagnetic phenomena and can be described aptly by continuum field equations, which have been solved for a number of simple geometries. The interplay between these has proven to be important in the stability ofcolloidal suspensions in terms of their tendency for flocculation or agg~egati0n.l~ Also, these interactions have been utilized to explain surface tension effects and electrokinetic phenomena.13J4 Proteins lie at the small end of the colloidal size range, so colloidalforces are pertinent but relatively weak (often on the order of a few kT). Nonetheless, this magnitude of interaction is sufficientto influence profoundly a protein molecule, because of its small size and the fact that the energy of unfolding is itself on the order of 10 kT.15 Biophysicists have used molecular electrostatics compu(7) Norde, W.; Favier, J . P. Colloids Surf 1992,64,87. (8)Clark,S.R.;Billsten, P.; Elwing, H. Colloids Surf B: Biointerfaces 1994,2,457. (9)Tilton,R. D.; Robertson, C. R.; Gast, A. P. J.Colloid Interface Sci. 1990,137,192. (10)Lee, C . S.; Belfort, G. Proc. Natl. Acad. Sci. U.S.A. 1989,86, 8392. (11)Haggerty, L.; Watson, B. A.; Barteau, M. A.; Lenhoff, A. M. J. Vac. Sci. Technol. B 1991,9 , 1219. (12)Haggerty, L.; Lenhoff, A. M. Biophys. J. 1993,64,886. (13)Hunter, R. J.,FoundationsofColloid Science; Oxford University Press: London, 1986;Vol. 1. (14)Hough, D. B.; White, L. R. Adu. Colloid Interface Sci. 1980,14, 3.
0743-7463/95/2411-3500$09.00/00 1995 American Chemical Society
Protein Adsorption Theory and Experiment tations, in which the protein is treated as a colloidal particle with continuum dielectricproperties and arbitrary shape and charge distribution, to describe the energetics of quite specificmolecular interactions, such as enzymesubstrate binding, residue pK, shifts, ion binding, and cofactor electrochemistry.16-21 The interplay of electrostatic and van der Waals interactions has been used previously to describe protein adsorption, but in a more qualitative sense. Ruckenstein and =evez2 described a “potential-barrier chromatography”, in which the kinetics of a colloidal protein in overcoming electrostatic repulsion with the surface (to which it eventually adsorbs because of van der Waals attraction) would provide the basis for separation. Another gr0up23p24 has redefined ion-exchange chromatography as “electrostatic interaction chromatography”,in order to emphasize the primary mechanism behind their model. With the inclusion of van der Waals interactions, they were able to fit literature ion-exchangeretention data over a wide range (about a decade) of ionic strength. In a previous paper,25we have developed a model for protein adsorption, also based on these interactions, that emphasizes molecular parameters as a startingpoint in order to facilitate use as a predictive tool. For the case where a protein molecule can be approximated as a sphere, we showed that the interactions can be described as a function of the protein size and charge, the charge density on the surface, the Hamaker constant describing the mutual polarizability of the protein and surface in a particular solvent medium, and the ionic strength. Furthermore, we showed that over a decade in ionic strength, the equilibrium constant is likely to change by several orders of magnitude, and we demonstrated the effect of the other physical parameters on the relationship between equilibrium constant and ionic strength. In this work, we compare the predictions of this model, implementedin varying degrees of complexity,with direct measurements of the equilibrium constant (or, equivalently, the Henry‘s law constant) for adsorption of proteins onto flat quartz slides. This constant is determined as the slope of the adsorption isotherm at low concentrations; in this regime, adsorption should be dictated by proteinsurface interactions, without complications owing to protein-protein interactions either laterally on the surface or in the bulk phase. While analytical chromatography can give a measure of the equilibrium constant for adsorption, previous measurements of protein adsorption isotherms in well-defined geometries have focused primarily on the plateau region of the isotherms. The two main reasons for this are the high-affinity nature of many protein adsorption isotherms and the difficulty of measuring small amounts of adsorbed protein. In order to overcome the latter difficulty and accomplish the detection of low quantities of adsorbed solute (on the order of ng/cm2), we have used the technique of total (15) Creighton, T.E., Proteins. Structures and Molecular Principles; W. H. Freeman & Co.: New York, 1984. (16) Matthew, J. B.; Richards, F. M. Biochemistry 1982,21, 4989. (17)Klapper, I.; Hagstrom, R.; Fine, R.; Sharp, K. A.; Honig, B. Proteins 1986, 1, 47. (18) Bashford, D.; Karplus, M. Biochemistry 1990,29, 10219. (19) Bashford, D.; Karplus, M. J. Phys. Chem. 1991,95, 9556. (20) Gunner, M. R.; Honig, B. Proc. Natl. Acad. Sci. U.S.A. 1991,88, 9151. (21) Zhou, H.-X. Biophys. J. 1993, 65, 954. (22) Ruckenstein, E.; F’rieve, D. C. AZChE J. 1976,22, 276. (23) Stihlberg, J.; Jdnsson; B; Horvdth, C. Anal. Chem. 1991, 63, 1867. (24) Stihlberg, J.; Jonsson; B; HorvAth, C. Anal. Chem. 1992, 64, 3118. (25) Roth, C. M.; Lenhoff, A. M. Langmuir 1993, 9, 962.
Langmuir, Vol. 11, No. 9, 1995 3501 internal reflectance fluorescence (TIRF) spectroscopy,26 which provides an ideal system for the controlled study of protein adsorption. The three primary advantages of this technique are the ability to monitor adsorption in situ, the excellent sensitivity due to quantitation of adsorbed amount by direct measurement of a property proportional to concentration (i.e.,fluorescenceintensity), and the possibility of measuring adsorption directly without altering the molecular structure of the protein. Many other techniques exist for measuring protein adsorption isotherms, including solution d e p l e t i ~ nra,~~ diolabeling,28electron spectroscopy for chemical analysis (ESCA),29ellip~ometry,~~ refle~tometry,~~ streaming potential?l and direct observation using scanning probe microscopies.12 With the possible exception of the last, which is a very laborious method of measuring an adsorption isotherm, none of these techniques is able to match TIRF in its potential ability to provide precise determination of adsorbed concentrations for both kinetic and equilibrium studies. The proteins lysozyme and chymotrypsinogen A were used in the study. Both are relatively small, globular proteins that exhibit strong intrinsic fluorescenceand that are relatively stable structurally, as measured by their adiabatic compre~sibilities.~~ Each carries a net positive charge at pH 7, at which all experiments were performed, and they provide a contrast in both size and charge that puts the model capabilities to a demanding test. The adsorbent employed was an acid-washedquartz slide. We hypothesize that, for such a moderately charged, hydrophilic surface and protein not prone to conformational change, adsorption should be dominated by electrostatic and van der Waals interactions. In comparingthe results to model predictions made using molecular properties of the protein and surface, the interplay between the electrostatic effects, from the standpoint of the protein characterized primarily by its charge, and the van der Waals contributions, characterized primarily by its size, is borne out by the experimental results. This finding not only validates the model as describing the primary mechanismsof adsorption under the conditions employed but also suggests the possibility of rational manipulation of adsorption equilibria by varying the physical properties of the surface, using the model predictions as a guide.
Principles A. Experimental. (1) TIRF Spectroscopy. TIRF, along with its absorption analogs such as ATR-FTIR, is a surface-sensitive technique that allows for detection of fluorophores localized close to the adsorbing surface. The basic principle of TIRF is the excitation of fluorescence near the surface by means of an exponentially decaying evanescent wave formed at the surface-solution interface due to the total internal reflection of an incident light beam. The implementation in practice is typically within a flow cell, with the incident beam entering through a prism that is optically coupled to the adsorbing surface. The amount of fluorescence detected due to emission throughout the cell can be shown to be5J8 (26) Watkins, R. W.; Robertson, C. R. J.Biomed. Mater. Res. 1977, 11, 915.
(27) Haynes, C. A.; Sliwinsky, E.; Norde, W. J.Colloid Znterface Sci. 1994,164,394. (28) Hlady, V.; Reinecke, D. R.; Andrade, J. D. J. Colloid Znterface Sci. 1986, 111, 555. (29) Galander,C.-G.;Hlady, V.; Caldwell, K.; Andrade,J.D. Colloids Surf 1990,50, 113. (30) Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986, 79, 1. (31) Elgersma, A. V.; Zsom, R. L. J.;Lyklema, J.;Norde, W. Colloids Surf 1992, 65,17. (32) Gekko, K; Hasegawa, Y. Biochemistry 1986,25, 6563.
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3502 Langmuir, Vol. 11, No. 9, 1995
in which the fluorescence results from a sequence ofevents beginning with excitation at wavelength I,, followed by emission at wavelength A,, and ending with detection and conversion into a digital signal. AS a result, the observed fluorescence(F)is proportional to the probability of absorption (proportional to the chromophore extinction coefficient E ) , the fraction of excited light at lex emitted at Aem (4) (which is the product of the probability of emission, known as quantum yield, and the fraction of emission at A,,), and the fraction of radiation detected (fd),and it also depends on the concentration distribution of fluorophores in the cell [C(z)],the intensity of radiation at the exciting wavelength (I)(as a function of distance z into the cell),and the depth ofthe cell (L).The absorption and emission probabilities E and 4 are characteristic of the fluorophore and, to some extent, its solvent environment, whereas f d and the intensity at the interface (1,)are characteristic of the instrumental setup. In this work, the intrinsic fluorescence of the aromatic groups within the protein was utilized (i.e., excitation at 280 nm, emission maximum at ca. 340nm);the penetration depth (characteristic length of exponential deca of the The evanescent wave) for our system is ca. 1000 advantages here are the absence of a fluorescent tag alteringthe chemical structure and possibly the exhibited function of the protein, as well as a reduced penetration depth for the evanescent wave, relative to that obtained in the visible or infrared, since the penetration depth is proportional to excitation wavelength. The disadvantage is the fact that proteins without several tryptophan residues do not exhibit sufficient fluorescence for the sensitivity required in obtaining adsorption equilibrium constants, thus limitingpotential choices ofproteins. More information concerning the details of TIRF principles can be found (2) Calibration. A calibration procedure is required to relate the fluorescence intensity (usually in arbitrary units) to a surface concentration. The uncertainties in this procedure have constituted the biggest drawback to the use of TIRF as a quantitative te~hnique.~' Although the evanescentwave used to promote protein fluorescence is quite short-ranged, some fluorescence due to nonadsorbed protein is detected and must be accounted for in the calibration. Furthermore, light scattered at the point of total internal reflection has been found to contribute to the bulk fluorescence and must also be discounted a p p r ~ p r i a t e l y .Therefore, ~~~~ the total fluorescence can be written as
i.
First, to determine the relative intrinsic fluorescence yields of the various fluorophores used in this work, their fluorescence was measured in conventional fashion in a cuvette. For very low concentrations, i.e., ECI
