Langmuir 1991, 7 , 2710-2718
2710
Manipulation of Hydrophobic Interactions in Protein Adsorption Robert D. Tilton,+ Channing R. Robertson, and Alice P. Gast' Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025 Received April 15,1991. In Final Form: August 1 , 1991 The generally positive correlationbetween the extent of protein adsorption and the hydrophobicity of the adsorbent surface suggests that the hydrophobic interaction is a major driving force for adsorption. The customaryapproach to evaluatingthe role of this force,comparison of protein adsorptionto a spectrum of solid surfaces spanning a range of hydrophobicities, is limited by incomplete controlover surface chemical variables other than the overall hydrophobicity. We evaluatethe importance of the hydrophobic interaction in protein adsorption, while maintaining constant surface properties, by addition of alcohols known to modulate the strength of the hydrophobic interaction in aqueous solutions. The responses of adsorption isotherms,dynamics, and lateralmobility of adsorbed proteinsto changesin solvent composition demonstrate the primary role of the hydrophobic interaction in the adsorption of ribonuclease A to polystyrene surfaces. Manipulating the magnitude of the hydrophobic interaction alters the adsorption mechanism through both fluid-phase transport and protein binding at the interface.
Introduction Numerous biotechnological applications depend on the function of proteins in the vicinity of or confined to interfaces. Examples include biocompatible materials, protein chromatography, solid-phase immunoassays, and biosensors. This has led to an extensive compilation of data regarding the adsorption of numerous proteins from solution to a spectrum of solid materials. One of the most frequently invoked hypotheses formulated from the trends in the literature is that the dominant driving force for protein adsorption is dehydration of nonpolar surface or protein functional groups. This is predicated on the generally positive correlation between the protein binding affinity of a particular material and its hydrophobicity.l+ Experimental evidence suggests that the hydrophobic interaction is the major determinant of protein adsorption, while protein structural stability and electrostatic effects, including ion coadsorption, are also influential.718 The notion that protein adsorption is governed solely by the hydrophobic interaction would be incorrect, however, as exceptions to this trend have been and will most likely continue to be dis~overed.~+l~ For example, fibrinogen often displays adsorption tendencies that do not correlate with surface hydr~phobicity.~ Indeed, a balance of polar and dispersion components of the surface energy may be of equal or greater importance than the overall hydro-
* To whom correspondence may be addressed. Current address: Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213-3890. (1)Young, B. R.;Pitt, W. G.; Copper, S. L. J. Colloid Interface Sci. 1988, 124, 28. (2)JBnsson, U.;Ivarsson, B.; LundstrBm, I.; Berghem, L. J. Colloid Interface Sci. 1982,90,148. (3)Young, B. R.;Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988.,~~ 125. 246. (4)Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstrbm, I. J. Colloid Interface Sci. 1987, 119, 203. (5)Darst, S.A.; Robertaon, C. R.; Berzofsky, J. A. Biophys. J. 1988, t
I - - -
.
53. 599. ~.
(6)Darst, S.A. Ph.D. Dissertation, Stanford University, 1986. (7)Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (8)Van Dulm, P.; Norde, W.; Lyklema, J. J. Colloid Interface Sci.
--. . ..
1981.82. 77 ..__ ,
(9)Cheng, Y. L.;Darst, S. A,; Robertson, C. R. J.Colloid Interface Sci. 1987, 118, 212. (10)Absolom, D. R.; Zingg, W.; Neumann, A. W. J. Biomed. Mater. Res. 1987,21, 161. (11)Baszkin, A.; Lyman, D. J. J. Biomed. Mater. Res. 1980, 14, 393. (12)Norde, W.;MacRitchie, F.; Nowicka, G.; Lyklema, J. J. Colloid Interface Scc. 1986, 112, 447.
phobicity." Interpretation of adsorption data on a variety of materials having different hydrophobicitiesis obstructed by the lack of adequate control over all aspects of surface chemistry, including the identity of exposed functional groups, the surface topology, and the surface potential. For polymeric materials, the configurational flexibility of chains exposed a t the interface will depend on the type and processing of the material. Surface flexibility has been demonstrated to be an important factor in establishing the thrombogenicity of a material in contact with and appears to be an influential determinant, via steric effects, of the adsorption of platelets and serum proteins to polymeric materials.14J5 When protein adsorption data for a series of different materials are compared, the uncontrolled aspects of the surface chemistry are difficult to correlate. Thus, while the often observed correlation of protein adsorption and material hydrophobicity supports the importance of a hydrophobic driving force for adsorption, a rigorous demonstration that the hydrophobic interaction is the primary factor governing adsorption is lacking. We present an assessment of the role of the hydrophobic interaction in the adsorption of proteins to a nonpolar surface with invariant surface properties. This attractive force is examined in terms of the adsorption isotherms (investigated via a protein-radiolabeling technique), adsorption dynamics (via total internal reflection fluorescence, TIRF), and the lateral mobility of adsorbed proteins (via fluorescence recovery after pattern photobleaching, FRAPP). The hydrophobic interaction is the strong attractive force between nonpolar species interacting across an aqueous medium. The van der Waals force cannot account for the strength of this interaction.ls The hydrophobic ~~~~
(13)Reichert, W.M.; Filieko, F. E.; Barenberg, S. A. In Biomaterials: Interfacial Phenomenu and Applications; Cooper, S . L., Peppae, N. A., Hoffman, A. S., Ratner, B. D., Me.; American Chemical Society: Washington, DC, 1982;p 177. (14)Nagaoka, S.;Mori, Y.; Takiuchi, H.; Yokota, K.; Tanzawa, H.; Nishiumi, S. In Polymers as Biomoterials; Shalaby, S . W., Hoffman, A. S., Ratner, B. D., Horbett, T. A., Eds.;Plenum Prase: New York, 1984; p 361. (15)Mori, Y.;Nagaoka, S.; Takiuchi, H.; Kikuchi, T.; Noguchi, N.; Tanzawa, H.; Noishiki, Y. Trans.-Am. SOC.Artif.Intern. Organs 1982, 28,459. (16)Israelachvili, J. N.; Pashley, R. M. J. Colloid Interface Sci. 1984, 98,500.
0 1991 American Chemical Society
Langmuir, Vol. 7, No. 11, 1991 2711
Protein Adsorption interaction is closely related to the low solubility of nonpolar species in water, often referred to as the hydrophobic effect. The temperature dependence of the equilibrium partitioning of nonpolar solutes between aqueous and nonpolar phases demonstrates that the attraction is primarily entropic and requires the participation of solvent molecules in establishing the intersolute force." Further, hydrophobic interaction between nonpolar species is identified with a negative heat capacity ~ h a n g e . ' ~ The J ~ theory most consistent with experimental observation is that the attractive force results from the increased dynamic structuring of water in the vicinity of nonpolar species incapable of hydrogen bonding with water. Increased solvent structure in the interfacialregion leads to a large interfacial energy and a consequent thermodynamic driving force to reduce the total amount of structured water. This is accomplished by bringing the nonpolar surfaces into contact, thereby eliminating water-nonpolar interfaces. The resulting attractive force between nonpolar species in water, measured with the surface force apparatus, persists over a long range and decays exponentially with decay constants on the order of 1 nm.16" Claesson has demonstrated that the attractive hydrophobic force from a nonpolar surface is operative as far as 25-80 nm with two exponential decay constants of 2-3 and 13-16 nm, and it completely overwhelms the van der Waals force at small separations.21 As part of a thorough investigation of human serum albumin and bovine pancreatic ribonuclease A adsorption on polystyrene (made negatively charged by incorporation of styrenesulfonate comonomer), Norde and Lyklema determined that albumin adsorption to weakly charged, hydrophobic polystyrene proceeds with a negative heat capacity change (AdsCp), suggesting the participation of the hydrophobic interaction.% Ionic effects tend to favor and become prominent when the surface a positive AClhCp is highly charged and less hydrophobic. Ribonuclease adsorption is entropically driven under most conditions, but &d$p is influenced mostly by ionic effects. The dependence of A a d C p on the surface charge indicates, however, that while the hydrophobic interaction is not the primary determinant of AabCp, it does contribute significantly. The hypothesis that the hydrophobic interaction can dominate protein adsorption has yet to be verified. Currently, the best evidence to support this hypothesis is the correlation between surface hydrophobicity and protein adsorption. Given that the experimental evidence points to the solvent as the agent responsible for the hydrophobic interaction, we address its role in protein adsorption via manipulation of solvent properties. This approach is fundamentally different from, but complementary to, the many published investigations of protein adsorption on series of different materials (for example, refs 1-5). According to the construct provided by studies of the hydrophobic interaction, protein adsorption is affected by different degrees of solvent ordering due to the inherent incompatibility of a material with water. The usual experimental approach is to change this incompatibility by changing the surface; however, no account is (17) Israelachvili,J. N. Intermolecular and Surface Forces; Academic Press: New York, 1985. (18) Ben-Naim, A. Hydrophobic Interactions; Plenum Press: New York, 1980. (19) Nbmethy, G.; Scheraga, H. A. J. Chem. Phys. 1962,36,3401. (20) Israelachvili, J.; Pashley, R. Nature 1982,300, 341. (21) Claeeeon, P. M.; Blom, C. E.; Herder, P. C.; Ninham, B. W. J. Collord Interface Scr. 1986,114, 234. Claewon, P. M.; Christenson, H. K. J. Chem. Phys. 1988,92,1660. (22) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66,295.
taken of the possibility for different electrostatic or steric interactions with the new surface, or of the topology of the different surface. By manipulatingthe solvent properties, we force the protein adsorption mechanism to respond to the altered tendency of the solvent to participate in the hydrophobic interaction, while all surface properties remain invariant. This overcomes the uncertainties relating to inadequate control over all aspects of surface chemistry in the examination of adsorption onto a series of different materials. In the simplest terms, the preferred means of maintaining constant material properties is to not change the material. In an interesting approach to this issue, Elwing and co-workers measured the adsorption of several proteins to a surface with a continuous gradient of silane density, providing a hydrophobicity gradient.' Adsorption was greatest on the most hydrophobic end of the surface, but the issue of surface heterogeneities could not be dismissed. In this article we present an investigation of protein adsorption at the solid-liquid interface with constant protein and adsorbent surface chemistries, while systematically varying the strength of the lyophobic interaction through manipulation of solvent composition.29 We report the effects of alcohol cosolvents, known to alter the strength of the lyophobic interaction, on the mechanism of protein adsorption. Of course, this approach introduces new considerations for proper control of experimental variables that must be addressed in the careful selection of an experimental system.
Experimental Procedures Materials. Alcohol Cosolvents. Addition of methanol and other monohydric alcohols to aqueous solutions decreases the magnitude of the lyophobic interaction.l*.u-M This arises from preferential solvation of nonpolar species by the alcohol, disruptingthe tendency ofwatermolecules to orientin the interfacial region. Since they possess only a single hydroxyl group, these alcohols cannot enter into dynamic three-dimensionalhydrogenbonded networks with water. Currently, there are no direct quantitative measurements of the influence of these alcohols on the strength of the attractive force, but the effects may be quantified in terms of phenomena that are consequencesof the lyophobic interaction. The suppression of the lyophobic interaction by methanol is evident in the large negative free energies of transfer of nonpolar amino acid side chains and other nonpolar species from water to methanol-water solutions.%g The disruptionof thelyophobicinteraction by methanol is also evident in its inhibition of the dimerization of pyrene end labels on poly(ethylene glycol) in aqueous solution.2B.2a Polyhydric alcohols such as glycerol, on the other hand, form cooperativestructureswithwater in the liquid statem and enhance the magnitude of the lyophobic interaction. This is manifest in preferential exclusion of glycerol from nonpolar species in glycerol-watersolutions%and by nonpolar amino acid side-chain free energies of transfer from water to glycerol-water that are most often positive (but occasionally slightly negative).% The influence of these alcohols on the lyophobic interaction is consistent with their effects on the thermal stabilityof protein tertiary structure. Although discussionof the subject continues, (23) Note that the hydrophobic interaction is a particular type of l y e phobic, or soluent fearing, interaction. The more general term will be used ta describe the interaction in a solvent of altered compoeition. (24) Herskovita, T. T.; Gadegbeku, B.; Jaillet, H. J. Biol. Chem. 1970, 245,2588.
(25) Gekko,K. In Ions and Molecules in Solution; Tanaka,N., Ohtaki, H., Tamamushi,R., Me.; Ekevier Science PublishersB.V.: Amsterdam, 1983; Vol. 2, p 339. (26) Char, K.; Frank, C. W.; Gaet, A. P.; Tang, W. T. Macromolecules 1987,20,1833. (27) Roeeman, M.; Jencks, W. P. J. Am. Chem. SOC.1976,97,631. (28) Char, K.; Frank, C. W.; Gast,A. P. Macromolecules 1989, 22, 3177. (29) McDuffie, G . E., Jr.; Quinn, R. G.; Litovitz, T. A. J. Chem. Phys. 1962, 37, 239. (30)Gekko, K.; Timasheff, S. N. Biochemistry 1981,20,4677.
Tilton et al.
2712 Langmuir, Vol. 7, No.11, 1991
lyophobic interaction appears to be important in stabilizing tertiary structure. Spectroscopic studies indicate that the mediation of protein stability by alcohol cosolventsrelates to the type and concentration of alcohol, and the extent of the effect depends on the particular protein.a~m.~~ By decreasing the thermodynamic penalty for exposure of nonpolar groups to the solvent, methanol incrementally stabilizes contacts between nonpolar residues and solvent occurring in the unfolded protein conformation. In a relative sense, stabilization of the unfolded conformation destabilizes the native structure by shifting the equilibrium toward the unfolded state. As a result, the melting temperatures of small proteins in solution decrease with methanol addition.usal-@ Native tertiary structure is stabilized by glycerol due to an increase in the thermodynamic penalty for exposure of nonpolar species, thereby destabilizing the unfolded state in favor of the native state. Preferential exclusion of glycerol from the surface of a protein coincides with an increase in the protein chemical potential, causing it to slightly contract its volume to avoid contact with solvent.mM Stabilization is evident in the increase of protein melting temperatures accompanyingaddition of glycerol to the s o l ~ t i o n . ~ ~ ~ . ~ ~ @ In this study, methanol cosolvent selectively weakened the lyophobic interaction in some adsorption experiments, and glycerol cosolvent enhanced it in others. Control experiments were performed with aqueous solutions without cosolvents. All solutions were phosphate-buffered saline ([phosphate] = 10mM, [NaCl] = 150 mM) with constant ionic strength and adjusted to pH 7.4 witheither hydrochloric acid or sodium hydroxide. Water was deionized and Milli-Q filtered. Glycerol (analytical reagent grade, Mallinckrodt, Paris, KY) and methanol (spectrophotometricgrade, J. T. Baker, Phillipsburg,NJ) were usedas received. Protein adsorption involves a complicated interplay of van der Waals, steric, electrostatic, hydrophobic, and repulsive hydration forces. Given the dependence of van der Waals and electrostatic forces on the solvent dielectric constant, it was important to consider the change in dielectric constant that accompanied alcohol addition to the buffers. The dielectric constants of both glycerol-water and methanol-water mixtures were below that of water, the dielectric constant varying approximately linearly with composition.s Both alcoholswould then serve to enhance electrostatic and van der Waals interactions, while exerting opposing effects on the lyophobic interaction, leaving the consequences of lyophobic and dielectric phenomena separable. This was aided further by comparison of glycerolwater and methanol-water mixtures with equal dielectric constants. It is noteworthy that Singer detected no significant changes in counterion binding by ionizable groups on proteins accompanyingchanges in the solvent dielectric constant between 56 and 76, the range of dielectric constants encountered in this st~dy.3~ Protein. The goal of this research was to compare adsorption experiments with varying contributions from lyophobic attractions, in the presence of invariant protein and surface properties. Hence, it was essential to examine a protein whose structure is resistant to alcohol denaturation. If the protein were in its native conformation in glycerol-water and simple aqueous solutions, but in the denatured state in methanol-water, no reasonable comparison between experiments would be possible. The native structure of ribonuclease A (from bovine pancreas) is stabilized by glycerol, and it is quite resistant to methanol denaturation at acidic pH.25JC+3*3 Following standard procedures, ribonuclease A (Type IIA, Sigma, St. Louis, MO) was dialyzed against pH 6.5 phosphate buffer ([phosphate] = 0.2 M) for 2 days and then heated to 62 "C for 10min to irreversibly dissociate dimers in the commercial ~
(31) Biringer, R. G.;Fink, A. L. J. Mol. Biol. 1982,160, 87. (32) Gerlsma, 5.;Stuur, E. Int. J. Pept. Protein Res. 1972,4, 377. (33) Fink, A. L. Cryobiology 1986, 23, 28. (34) Gekko, K.; Timasheff, S. N. Biochemistry 1981,20,4667. (35) Back, J. F.; Oakenfull, D.; Smith, M. B. Biochemistry 1979,18, 5191. (36) Akerlhf, G.J. Am. Chem. SOC.1932,54, 4125. (37) Singer, S. J. Adv. Protein Chem. 1962, 17, 1. (38) Fink, A. L.; Grey, B. L. In Biomolecular Structure and Function;
Agris, P. F., Sykes, B., Loeppky, R., Eds.; Academic Press: New York, 1978; p 471.
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Figure 1. Melting temperature of RNase in pH 7.4 PBS. It is defined as the midpoint of the thermal transition from the native to the denatured state in the ultraviolet difference spectrum and decreased with increasing methanol concentration. Inset shows the UV absorbance temperature spectrum for RNase in 30% methanol PBS. The melting temperature was approximately 53
"C.
preparation."" Fractionation of one heat-treated protein sample on a Sephadex G-75 column (Pharmacia LKB Biotechnology, Pleasant Hill, CA) confirmed the protein preparation was free of dimers. Monomeric ribonuclease was covalently fluorescence labeled with eosin-5-isothiocyanate (Molecular Probes, Eugene, OR) or tritium labeled by reductive methylation using sodium [3H]borohydride (ICN Biomedicals, Costa Mesa, CA) according to procedures described e l s e ~ h e r e . ~Eosin-labeled ~,~~ ribonuclease A (E-RNase) had 0.45 mmol eosin bound/mmol of protein. The activity of the tritiated protein ([sHJRNase)was 243 mCi/ mmol. These labeling procedures do not affect the adsorption behavior of proteins." We verified the conformation1 stability of the monomeric RNase in pH 7.4 methanol-water phosphate-buffered saline (PBS) solutions via ultraviolet difference spectroscopy.u*mal* Briefly, this technique messes the conformational integrity of a small protein by monitoring the change in ultraviolet absorbance as a function of temperature. In the native state, where UV-absorbing aromatic amino acids are largely sequestered from the solvent, the absorbance is high. In the denatured state, the absorbance diminishes as these amino acids become exposed to solvent. Thus, any decrease in UV absorbance may be related to the fraction of proteins in the denatured state. The temperature where equal amounts of protein are in the native and unfolded state is termed the melting temperature. We performed UV difference spectroscopy on RNase in methanol-water PBS at pH 7.4 with varying amounts of methanol cosolvent. Each protein solution was tightly capped in a quartz cuvette to prevent evaporation and placed in the thermostated cuvette holder of a UV-vis spectrophotometer. The temperature was increased in 5 "C increments, and the absorbance at 278 nm was recorded at each temperature until the melting temperature had been exceeded and the absorbance remained constant over a 15 O C interval. Care was taken to ensure that the sample had equilibrated at each temperature. RNase melting temperature is plotted as a function of the PBS methanol content in Figure 1. Nuclear magnetic resonance (39) Crestfield, A. M.;Stein, W. H.; Moore, S.J.Biol. Chem. 1963,238, 618. (40) Fruchter, R. G.; Crestfield, A. M. J. Biol. Chem. 1965,240,3868. (41) Darst,S.A.;Robertaon,C. R.; Berzofsky,J. A. J . Colloidlnterface Sci. 1986, 111, 466. (42) Tilton, R. D.; Robertson, C. R.; Cast, A. P. J. Colloid Interface Sci. 1990, 137, 192. (43) Lok, B. K.; Cheng,Y. L.;Robertson, C. R. J . Colloid Interface Sci. 1983, 91, 87.
Protein Adsorption
Langmuir, Vol. 7, No. 11, 1991 2713
Table I. Mixed Buffer Properties RNase pwc, IO+D(RNase)ao.c, solub, PBS CP cmz/s mg/mL 100%H20 0.8386 1.34 f 0.03 225 78 255 MeOH 1.289 50% MeOH 1.394 0.73 0.03 >225 33% glycerol 2.748 0.39 0.01 a Values for salt-free mixtures from ref 36.
investigations,although at a buffer pH and ionicstrength different from those used in this study, have shown that the premelting conformation of RNase in methanol-water mixtures is the same as the aqueous native structure?l As 50% methanol was the buffer environment most antagonistic to RNase native structure in this study, we paid particular attention to this melting temperature, 45 OC. To ensure that RNase maintained its native structure in all experiments, we conducted each adsorption measurement at 30 "C, a temperature well below the onset of significant denaturation. This selection of protein and temperature ensured that any measured differences in adsorption behavior could not be attributed to conformational alterations of the protein in solution. The melting temperatures measured here at pH 7.4 are higher than those measured at more acidic pH by other investigators. This likely reflects the increased stability of the protein nearer its isoelectric point (pH 9.6), as suggested by Biringer and Fink.sl Surface. Besides maintaining consistencyof proteinstructure in the varying chemical environments, we needed to select a transparent hydrophobic material with surface properties insensitive to the solvent. Silanized surfaces were not considered due to concerns with uniformity and stability of surface characteristics. Three criteria were used for selection of a polymeric surface: (1)All solvents must be nonsolvents for the polymer. (2) The surface must not be able to reorient and present different functional groups in the presence of different solvents. (3) Steric forces due to the flexibility of the interfacial polymer chains must not differ in the presence of different solvents. The latter two criteria can be satisfied by a polymer with a high glass as this correlates with large kinetic transition temperature (T&, barriers to the sorts of chain reorientations required for a polymeric surface to accommodate changes in solvent type.4 Polystyrene has only nonpolar functionalities, and thus very little driving force for surface reconfiguration. Polystyrene is not soluble in the solvents used in this study, and with a TKof 100 OC, it satisfies all three of these criteria. As RNase and polystyrene surfaces were insensitive to addition of alcohol cosolvents, only changes in intermolecular interactions (distinguishable between dielectric and lyophobic phenomena) could explain any observed changes in adsorption behavior in different mixed solvents. Polystyrene was applied as a spin-cast film on glass slides in this study. The slides were cleaned according to the procedure of Cheng et al.8 Polystyrene (125 000-250 OOO molecular weight, Polysciences, Warrington, PA) was spin-cast onto the clean slides from 1w t 71 solutions in toluene (spectrophotometric grade, J. T. Baker) in a toluene-saturated atmosphere. After casting, the slides were dried under a heat lamp for 1 h and at room temperature for 24 h in the low-dust environment of a laminar flow hood. Scanning electron microscopy revealed that surfaces prepared in this way were defect-free on length scales as small as 100 nm. Hydrodynamic Considerations. Viscosities of the mixed buffers were measured at 30 OC by capillary viscometry using a Cannon-Ubelhode viscometer. RNase diffusion coefficients in various buffers were measured by dynamic light scattering at 30 O C . Table I contains these data, as well as measured solubility data for RNase in the mixed buffers and the dielectric constants of the alcohol-water mixtures. The measured diffusion coefficients reported in Table I are in excellent agreement with the Stokes-Einstein law, showing a linear correlation between the diffusion coefficient and the reciprocal solvent viscosity. This
supports the assertion that RNase resists denaturation in even the harshest solvent environment in this study, 50% methanol. Fluorescence Considerations. As two of the experimental techniques in this study were based on fluorescence, we investigated the response of the E-RNase fluorescence to changes in the composition of the buffer. The TIRF apparatus in this study used 514.5-nm-wavelength light to excite fluorescence of the adsorbed protein. Fluorescence emission at all visible wavelengths exceeding 550 nm was detected. Accordingly, we used a fluorescence spectrometer to integrate the emission from 1pg/cms solutions of E-RNase in the various buffers, from 550 to 600 nm, with 514.5-nm excitation. This range covered the emission that would be detected by the TIRF apparatus. The relative emission intensities were as follows: aqueous PBS, 1.00; 25% methanol PBS, 1.65; 50% methanol PBS, 11.6; 33% glycerol PBS, 1.37. Methods. Following the procedures of Darst et al.," we measured RNase adsorption isotherms for each of the buffers by adsorption of [SHIRNasefrom solutions in laminar flow at a wall shear rate ( 7 )of 66 s-'. Labeled protein solutions contacted the polystyrene surfaces for at least 8 h before rinsing for 10 min with protein-free buffer of the same composition used for the adsorption step. We verified that adsorption was completed in 8 h and that little adsorbed protein was lost during rinsing by independent TIRF experiments using E-RNase. As adsorption was slower in 50% methanol, RNase adsorbed for at least 12 h from this buffer. At the conclusionof each adsorption experiment, the slide was dried and immersed in 1 M nitric acid for 24 h a t 95 OC to completely remove surface-bound tritium. We then subjected an aliquot of the radioactive supernatant to liquid scintillation counting to determine the mass of protein adsorbed to the wetted area of the polystyrene surface. Total internal reflection fluorescence (TIRF) provided a continuous monitor of the adsorption dynamics of E-RNase on spin-cast polystyrene. Detailed descriptions of the theory and applications of the technique may be found elsewherePp The particular instrument used in this study was equipped with a frequency-stabilized argon ion laser as described in ref 42. The laser entered the prism at a 70' angle of incidencefor experiments with 100 vol % aqueous, 25 vol % methanol, and 50 vol % methanol PBS and at 78O for experiments with 33% glycerolPBS to ensure total internal reflection at the polystyrene surface. All experiments were conducted at 30 "C, and the buffers were purged by helium prior to the start of each experiment at this temperature to removedissolved oxygen. In eachsolvent mixture, TIRF fluorescencesignals were calibrated against absolute surface concentrations independently measured by adsorption of [3H]RNase. E-RNase adsorbed to polystyrene surfaces from dilute (510 pg/cmS) solutions in laminar flow, and examination of the dependence of adsorption rates on wall shear rates allowed discrimination between transport-limited and kinetic-limited adsorption processes." Initial adsorption rates were determined from the slope of the steady-state increase in fluorescence intensity, estimated by linear least-squares regression of the fluorescence data at the onset of adsorption. To calculate mass adsorption rates, we normalized the increasing fluorescence intensity by the calibrated fluorescence signal at the adsorption plateau. During adsorption of E-RNase to polystyrene, an overshoot in the TIRF signal occurs, suggesting a relaxation in the adsorbed layer. This occurs in 100% aqueous, 25% methanol, and 33% glycerol PBS, but is not observed in 50% methanol PBS over 7 h. An example of overshoot in 100% aqueous PBS is shown in Figure 2. The decay of the fluorescencesignal continues during extended periods with no laser illumination, confirming that it is not due to photobleaching. We normalized the TIRF signals by the fluorescence maximum in the calculation of initial adsorption rates, since the initial adsorption occurs before the apparent onset of this change in fluorescence signal. Use of the fluorescence maximum instead of the final steady fluorescence intensity would introduce no more than a 20 7% systematic error in the initial adsorption rates in 100% aqueous PBS. This error is not sufficient to change any of the conclusions of the following sections.
(44)van Damme, H. S.; Hogt, A. H.; Feijen, J. In Polymer Surface Dynamics; Andrade, J. D., Ed.;Plenum Press: New York, 1988; p 89.
(45) Lok, B. K.; Cheng, Y .L.; Robertson, C. R. J. Colloid InterfaceSci. 1983, 91, 104.
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Tilton et al.
2714 Langmuir, Vol. 7, No.11, 1991
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Time, h Figure 2. E-RNase adsorption in 100% aqueous PBS. Adsorption proceeds through an overshootin the fluorescencesignal. The continuation of the signal decay during extended periods without laser illumination demonstrates that photobleaching is not responsiblefor this observation. The concentration of protein is 1 pg/ml in 100% aqueous PBS in this experiment. We used the same TIRF apparatus, with an optional config uration for performing evanescentwave FRAPP, to measure the surface diffusion coefficient and mobile fraction of adsorbed E-RNase in the presence of different buffers. The apparatus and general procedures used in these experiments have been described previously.'z A flowing E-RNase solution contacted the surface for 5 h; then an unlabeled RNase solution displaced the labeled solution from the flow cell for 1 h prior to photobleaching adsorbed E-RNase. Lateral mobility was always measured in the presence of flowing unlabeled RNase solutions. This procedure removed any reversibly bound E-RNase from the polystyrene surface and from the tubing and other walls of the flow cell, ensuring that the measurements reflected the behavior of only irreversibly adsorbed protein molecules. We analyzed fluorescence recovery data according to the method described in ref 42. Results and Discussion Isotherms. RNase adsorption is irreversible with respect to dilution. Accordingly, one cannot interpret surface concentrations in terms of the Langmuir isotherm, as one foundation of this model is adsorption reversibility. We use the term isotherm in its most literal sense as the dependence of surface concentration on bulk concentration a t a constant temperature. In Figure 3A, we show adsorption isotherms for [3H]RNaseon polystyrene measured with 100% aqueous PBS and 33% glycerol PBS, and in Figure 3B we show the isotherms for 25 % methanol PBS and 5070 methanol PBS at a wall shear rate of 66 s-l. At high bulk concentrations, the amount of protein adsorbed from 33 % glycerol buffer is indistinguishable from the 100% aqueous buffer isotherm. The maximum coverage observed, 0.1 pg/cm2, corresponds to a [3H]RNase monolayer of area fraction 0.6 or 0.4, if all molecules are oriented side-on or end-on, respectively. This calculation is based on the unhydrated dimensions of the ellipsoidal molecule (25 X 25 X 40 A).* Adsorption from 25 5% methanol and 50 % methanol buffers is significantly reduced relative to adsorption from 100% aqueous buffer. This is consistent with the minimization of the lyophobic driving force for adsorption in methanol-containing buff(46) Richards, F. M.; Wyckoff, H. W. In The Enzymes; Boyer, P., Ed.; Academic Press: New York, 1971;Vol. 4, p 647.
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Wcm3)
Figure 3. (A) Adsorption isotherms for RNase on polystyrene at 30 O C in the aqueousand 33% glycerolbuffers. (B)Adsorption
isotherms for 25% and 50% methanol buffers. Note that 25% methanol and 33% glycerol have the same dielectric constant at this temperature. As the error bars in the surface concentration measurements were smaller than the symbol size, the scatter in the data reflects the variation inherent in the preparation of the surfaces. ers. Note that 33 % glyceroland 25 % methanol both have dielectric constants equal to 66 at 30 "C; thus the altered adsorption from the 25% methanol buffer is not a consequence of the altered dielectric environment around the protein and the surface. Similarly, since the native structure is not disrupted in any of the buffers a t 30 "C, the altered adsorption behavior does not arise from any differences in conformational integrity in solution. The effect of methanol on [3H]RNase solubility is also unable to account for this behavior. Whereas the adsorption isotherms for [3H]RNase in methanol-water buffers could be interpreted in a straightforward manner, the isotherm for [3H]RNase in 33% glycerol merits closer inspection. At high bulk concentrations, adsorption from 33 % glycerol PBS and 100% aqueous PBS are indistinguishable, but Figure 3A shows that a t bulk concentrations below 5 pglml, less [3H]RNaseadsorbs from 33% glycerol than from 100% aqueous PBS. This occurs in spite of the enhanced magnitude of the lyophobic interaction in this mixed solvent. Compared to the more typical appearance of the isotherm for the 1005% aqueous PBS,the apparent suppression of adsorption from dilute 33 % glycerol PBS is consistent with the formation of an adsorbed layer where the protein conformations, orientations, or lateral packing depend on the bulk concentration. Postadsorption molecular reconfigurations may result in barriers that prevent the adsorbed layer from attaining higher surface concentrations. If the protein experiences a conformational change causing it to spread on the surface, then the area occupied per protein would exceed the projected area of the native protein. A similar argument can be applied to reorientation of the oblate ellipsoidal RNase molecule, where the projected area of the native protein differs by a factor of 1.6 for the side-on and end-on orientations.
Protein Adsorption Such a molecular reconfiguration would occur to the greatest extent if the time scale for the reconfiguration were shorter than that for adsorption of proteins to the surface. If the protein were to experience such a reconfiguration at a rate slower than the rate of adsorption, spreading would be obstructed by crowding of the molecule by newly adsorbed neighboring proteins, and the expansion of the area occupied by a protein would be hindered. Thus, the rate of adsorption can influence the extent of reconfiguration and the specific area occupied per protein in the adsorbed layer. For the shear rate used, proteins in dilute solution apparently adsorb sufficiently slowly to permit reconfiguration, but at higher concentrations, proteins adsorb rapidly, crowding the surface before the surface-area-consumingreconfiguration can occur. This interpretation of nonequilibrium [3H]RNasecoverages in 33% glycerol PBS is reinforced by observations of the adsorption dynamics. Although we speculate that one possible area-consuming reconfiguration is the transition from end-on to side-on orientation relative to the polystyrene surface, it is noteworthy that Lee and Belfort observed a gradual reorientation of RNase from side-on to end-on orientation during the course of adsorption to charged mica in the surface force apparatus.47 Dynamics. 1. Initial Adsorption Rates. In combination with isotherms, the adsorption kinetics can be helpful in deducing the adsorption mechanism. Unfortunately, intrinsic protein adsorption kinetics are often so rapid that the adsorption is transport limited. Only lower bounds on the intrinsic rate of protein attachment can be obtained from studies of transport-limited adsorption. One approach taken to overcome transport limitations is to adsorb proteins from flowing solutions, where protein transport to the surface occurs by convective diffusion. By increasing the rate of transport to the surface, it is sometimes possible to enter the kinetic-limited regime of adsorption, where kinetic observationsmay be interpreted in terms of molecular events. Adsorption from wellcharacterized flows also facilitates the reproducibility of results from different investigations. Cheng et al. reported that the initial rate of albumin adsorption is transport limited on very hydrophobic poly(dimethylsi1oxane) and poly(diphenylsi1oxane)surfaces at wall shear rates between 25 and 4000 s-l, but it can be kinetic limited (slower intrinsic adsorption rates) on the less hydrophobic poly(methyl methacrylate) and poly(styrenesu1fonate) surfaces.9 Young et al. have ranked the intrinsic adsorption rates of a series of seven proteins on four polymeric surfaces, and with the exception of fibrinogen, intrinsic adsorption rates are higher for the more hydrophobic material.48 E-RNase was adsorbed from solutions in laminar flow in a rectangular slit in all experiments in this study. In this flow configuration, the LBvgque solution for the initial rate of convective-diffusion-limited adsorption is45349
where c8 is the surface concentration, r is the gamma function, y is the wall shear rate, D is the protein diffusion coefficient in solution, 1 is the distance traveled by the protein front to the point of TIRF observation, and co is the protein concentration in solution. If adsorption were transport limited in all cases, the LBveque solution could (47) Lee, C. S.;Belfort,G.Proc. Natl. Acad. Sci. U.S.A.1989,86,8392. (48) Young, B.R.;Pitt, W. G.; Cooper, S. L. J . Colloid Interface Sci.
1988, 125, 246.
(49)L6v&que,M.Ann. Mine8 1928, 13, 284.
Langmuir, Vol. 7, No. 11, 1991 2715 Table 11. Prediction of Relative Adsorption Rates for Transport-LimitedAdsorption According to the Uvbque Solution buffe9 100% HzO 25% MeOH 50% MeOH 33% glycerol
re1 viscosb
1.00 1.53 1.66 3.28
OaiWIDP 1.00 0.75d 0.71 0.45
a 30 OC. Capillary viscometry. Dynamic light scattering. from Stokes-Einstein law.
D
predict the relative rates of E-RNase adsorption from the different mixed buffers, based on their different viscosities (differentprotein diffusion coefficients). The relative rates would equal the ratio of the diffusion coefficients raised to the 2/3 power, as reported in Table 11. Figure 4A-D shows the initial steady-state adsorption rates for E-RNase on polystyrene in each of the four buffers, plotted as a function of the parameter y1/3c0. The solid lines in each of the plots show the adsorption rates predicted with the LBv6que solution, accounting for the differences in the RNase diffusion coefficient that correspond to the different buffer viscosities. Adsorption from the 100% aqueous buffer is well described as a transport-limited process. Adsorption is intermediate between transport and kinetic limited for 25% methanol, and is independent of y, or kinetic limited, in 50% methanol. It is apparent that addition of methanol weakens the attractive force for adsorption to the extent that the intrinsic adsorption kinetics become rate limiting. Unlike the methanol-containing PBS, where RNase adsorption isotherms and kinetics are both suppressed, adsorption from 33 % glycerol is consistently faster than the LBv6que solution predicts for transport-limited adsorption, regardless of the final surface concentration attained. Since proteins cannot adsorb to the surface any faster than they are transported to that surface, no change in the intrinsic kinetics of the adsorption process can account for adsorption rates exceeding the rate of transport-limited adsorption. The inability of the LQv6que solution to account for these rapid adsorption rates implies that the transport mechanism in 33% glycerol PBS deviates from simple convective diffusion. Perhaps, the unusually rapid initial adsorption rates result from the emergence of an additional component of protein transport toward the surface due to the enhanced lyophobic attractive force. This additional velocity component would provide another way, besides diffusion, for proteins to cross fluid streamlines. 2. Enhanced Adsorption Rates in the Presence of a Strong Lyophobic Attraction. Enhancement of adsorption rates in the presence of 33% glycerol PBS probably is a consequence of the strong attractive force between the polystyrene surface and the E-RNase molecules. Israelachvili and Pashley measured the hydrophobic force between layers of hexadecyltrimethylammonium bromide (CTAB)adsorbed to mica.l63 According to these surface force measurements, the attractive hydrophobic force is of the form
F = -&VIh
(2)
where the amplitude B = (140dyn/cm)a, a is the particle radius, and the decay length h is 1nm. While this force profile was measured for the interaction between macroscopic nonpolar species, it appears to be a valid description of the hydrophobic force between single nonpolar molecules as small as methane.16 We assume the exponential force law remains valid for the lyophobic attraction between the nonpolar polystyrene surface and
Tilton et al.
E-RNase in the 3376 glycerol buffer. Although Claesson et al. found that the hydrophobic attraction is better described by a double-exponential force the singleexponential suffices for the semiquantitative calculations that follow. We estimate the contribution of the attractive lyophobic force to the initial rate of protein adsorption by considering that a protein under the influence of this force will move at a velocity determined by the balance between the attractive force and the viscous drag force. This velocity, V,(y), is directed in the -y direction (bulk fluid flow is parallel to the surface in the +x direction, and the normal vector directed away from the surface defines the +y direction). Protein solutionsare in laminar flow during the adsorption process, and transport to the surface occurs by convective diffusion. A rigorous treatment of this transport problem would call for numerical integration of the full convective-diffusion partial differential equation in time and two spatial dimensions. Full calculations for transient adsorption from solutionsin laminar flow without attractive or repulsive forces have been performed by Lok et al.& and recently extended by Shibata and LenhoffeM At the low protein concentrations examined in the current experiments, however, adsorption occurs predominantly at steady state.4s We consider the coupling between diffusion and the attractive force to calculate an effective diffusion coefficient,D*,that will besubstituted into the LBv4quesolution. The attractive force, and V,(y),are significant within 10 nm of the surface, where V,(y) is negligible. In order to calculateD*, we consider the simple case of mass transport across a region bounded by two surfaces where the concentration is held at zero on one surface and at c,,, the bulk protein concentration, at the other surface. In the presence of the lyophobic attractive force, mass transfer occurs via convective diffusion
-
C: 50%methanol
V, dc/dy = D d2c/dy2
(3)
We replace V,(y) by -(B/o)exp(-ylh), where w is the drag coefficient for a sphere, Gupa, p is the viscosity, and a is now the radius of the protein. In this simple treatment, we do not consider either orientation-dependent forces or a distance-dependent diffusion coefficient. Defining the dimensionlessvariables Y = y/h and C = c/co, eq 3becomes
D:3396 glycerol c
In
N
-4
5
4
5
3
e-'dC/dY = -H d 2 C / d p (4) The dimensionless group H = Dw/Bh = kT/Bh provides a measure of the relative importance of thermal and lyophobic forces in protein transport. This equation is subject to the boundary conditions
X
C(Y = 0) = 0
1
0
0
10
20
30
40
ylBc0(~g/cm~s'B)
Figure 4. Initial adsorption rates of E-RNase on polystyrene from various mixed buffers at 30 "C.The LBvQue solution (solid lines) predicts that initial adsorption rates should show a linear dependence on y*/3c0. Adsorption from 25% methanol (B)is intermediate between kinetic and transport limitation, while hindrance of adsorption from 50% methanol (C) renders the process kinetic limited. Adsorption rates exceed the LBvbque where the dashed line represents solution in 33 % glycerol (D), a fit of the data for analysis accordingto calculationsof adsorption via enhanced diffusion. This fit is weighted by the size of the error bars on each point. Concentrations of E-RNase are as follows: 10071 aqueous buffer, 0.1 rg/cm3; 25%methanol, 5 c(g/ cm3;50%methanol, 1.0c(g/cm3;33 % glycerol,0.5 (closedcircles) and 5.0 pg/cma (open circles).
C( Y 6/h) = 1 (5) The boundary condition at the surface assumes that proteins adsorb instantaneously upon contacting the surface, in other words, that adsorption is transport limited.46 The dimensionless concentration is held constant at unity, a distance 6 from the surface. Solving eqs 4 and 5 provides the dimensionless flux of proteins to the surface +
(6)
We express the extent of diffusion enhancement as the (50) Shibata,C. T.;Lenhoff,A.M. J. ColloidlnterfaceSci.,submitted.
Langmuir, Vol. 7, No. 11,1991 2717
Protein Adsorption
2.0
-
1.5
-
1.0
-
0.5
-
. 0
n n
0
0
0.2
0.4
0.6
1/H
Figure 5. Increase in the effective diffusion coefficient, D*,as the lyophobicinteraction becomes increasinglyimportant relative to thermal forces. Table 111. Adsorbed E-RNase Lateral Mobility on
Polystyrene
c,, mobile dcmz fractn 0.05 0.66 f 0.1 0.05 0.44 0.04 100% HzO 33% glycerol 0.07 0.89 0.08 33% glycerol 0.02 0.38f 0.05
buffer 25% MeOH
~~
*
IO'D, cmZ/s lOgDp,pcm/sz 0.34 f 0.1 0.4f 0.1 1.7 f 0.6 1.4f 0.5 0.83 f 0.03 2.3 0.1 2.5 f 0.5 0.9f 0.2
ratio D*/Do, where Do is the bulk diffusion coefficient with no attractive force. This is equal to the ratio of the flux by coupled attraction and diffusion and the flux by simple diffusion. The diffusion enhancement is plotted in Figure 5 as a function of 1/H and fitted with a quadratic equation. Increasing the strength of the lyophobic attraction relative to thermal forces (increasing 1/H) increases the effective diffusion coefficient. For 6/h >> 1, the results are insensitive to the choice of 6. Using D*as a fitting parameter in the LBvbque solution, we determine that the initial adsorption rate data in Figure 4D are consistent with a value of D* equal to 2.500. This suggests that a lyophobic attraction parameter of value H = 1.4 describes the adsorption of E-RNase to polystyrene in 33% glycerol. One would predict H = 0.01 by quantitatively applying the hydrophobic force law of Israelachvili and Pashley for pure water and the hydrodynamic properties of RNase in 33% glycerol a t 30 "C.The surface of RNase, consisting of both hydrophilic and hydrophobic residues, probably represents a considerably less hydrophobic surface than a pure CTAB monolayer. Accordingly, the higher value of H obtained from this semiquantitative treatment is not surprising. This first-order treatment supports the premise that the magnifying effect of glycerol on the lyophobic attraction strength can result in enhanced rates of protein adsorption. Surface Diffusion. The lateral mobility of E-RNase adsorbed to polystyrene exhibits an unexpected dependence on the buffer composition. The mobile fractions, raw surface diffusion coefficients, and the surface diffusion coefficients corrected for viscosity by assuming StokesEinstein behavior are presented in Table 111. One might expect that the lateral mobility should decrease as the strength of the lyophobic interaction increases. In other words, lateral mobility should be greatest in methanolcontaining buffers and least in glycerol-containing buffer.
In fact, the corrected surface diffusion coefficients increase in the order 25% methanol (c, = 0.05 pg/cm2) < 100% aqueous PBS (c8 = 0.05 pg/cm2) < 33% glycerol (c, = 0.02 pg/cm2; cs = 0.07 pg/cm2). Differences in surface concentration cannot account for this ordering. The mobile fraction increases in the order 33% glycerol (c, = 0.02 pg/cm2) < 100% aqueous PBS < 25% methanol < 33% glycerol (c, = 0.07 pg/cm2). The conflict between expectation and experiment illustrates the complexity of the surface diffusion process. It does not depend in a simple way on the strength of lyophobic interactions. The longrange lyophobic interaction may primarily determine the rate and extent of adsorption, but shorter range forces may be important determinants of the surface dynamics of the adsorbed proteins. An alternative hypothesis is that the increase of the surface diffusion coefficient with increasing lyophobic interaction strength may be related to the unknown effect of this force on the conformation of the adsorbed protein. A strong lyophobic force that stabilizes the protein in solution may increase ita tendency to alter its structure on the surface to maximize the contacts between nonpolar amino acid residues and the polystyrene. While the number of contact points between each protein and the surface would likely increase in this manner, the protein might diffuse more rapidly due to increased molecular flexibility. The lateral mobility of E-RNase adsorbed from 33% glycerol PBS at high and low surface concentrations, shown in Table 111, complements the interpretation of the isotherm shape (Figure 3), in that it indicates differences in adsorbed protein packing in the high- and lowconcentration regimes. The uncertainty surrounding the effect of solvent composition on lateral mobility is obviously not a factor in a comparison of the lateral mobility of proteins adsorbed from the same solvent a t different concentrations. Lateral interactions among diffusing adsorbed proteins hinder self-diffusion,and the area fraction coverage is the important determinant of the extent of hindrance (ref 51 and references cited therein). In spite of the unequal number of E-RNase molecules migrating and interacting on the polystyrene surface, the surface diffusion Coefficients are approximately equal at surface concentrations of 0.07 and 0.02 pg/cm2, and the mobile fraction at the lower concentration is less than half that at the higher concentration. Recall that the isotherm shape is consistent with a greater area occupied per protein in the low-concentration regime than in the high-concentration regime and that this could be due to adsorptionrate-dependent packing, conformation, or orientation in the adsorbed layer. If the area occupied per protein is indeed larger at the lower concentration, the area fraction coverage is larger than that calculated from the protein dimensions and surface mass concentration. As a result, the diffusion hindrance operative a t a particular mass concentration is disproportionately large in this regime. The diffusion hindrance at low concentrations is magnified further by the smaller mobile fraction, due to the greater effectiveness of immobile species as obstacles to diffusion. The large difference in mobile fraction in the high- and low-concentration regimes strongly suggests that the protein packing varies significantly with concentration. In one simple construct of the adsorbed layer, illustrated in Figure 6, vertically oriented proteins occupy the majority of the high-concentration, rapidly adsorbed layer and are mobile. In the low-concentration, slowly adsorbed layer, most of the proteins are oriented horizontally and im(51) Tilton, R.D.;Gast, A. P.;Robertson, C. R.Biophys. J. 1990,58,
1321.
2718 Langmuir, Vol. 7, No. 11, 1991
DILUTE
CONCENTRATED
Figure 6. Protein packing of the adsorbed layer. If slow, area-
consuming protein reconfigurations occur after adsorption, an adsorbed layer formed from a dilute, slowly adsorbing, solution will be characterized by a larger area occupied per protein than a layer formed from a concentrated,rapidly adsorbing, solution. The reconfiguration illustrated is a reorientation of RNase molecules of aspect ratio 1.6,but conformational changes could have an equivalent effect. mobilized; the proteins adsorbing in the later, kineticlimited stages of adsorption fill in the gaps on the surface by orienting vertically. Different proportions of vertically and horizontally oriented proteins could thus determine the mobile fractions in the two regimes. A similar scenario based on different distributions of conformational states in the two regimes is also consistent with the experimental results.
Conclusions We introduce a novel test of the importance of the lyophobic interaction in the adsorption of proteins to solidliquid interfaces. The results of this investigation confirm the primary importance of the lyophobic interaction in protein adsorption. This test, employing cosolventsknown to alter the strength of the lyophobic interaction in independent investigations, provides information that is complementary to the more common investigations of adsorption to a series of materials of varying hydrophobicities. This approach offers improved control over surface chemistry variables; however, to achieve this control, careful selection of experimental materials is essential. The conclusions of this work are based on phenomenological aspects of the hydrophobic interaction and of the effects of alcohols on its magnitude. Although prior experimental evidence supports the role of dynamic solvent ordering in the hydrophobic interaction, the conclusions are not linked to this mechanism. Both the extent and rate of RNase adsorption from aqueous solution to polystyrene are impaired by addition of methanol, an alcohol known to weaken the lyophobic interaction, at constant pH and ionic strength. Alterations in solvent dielectric properties or protein solubility accompanying alcohol addition are not responsible for these effects. The results indicate that the diminished lyophobic interaction in methanol-containing buffers accounts for the diminished adsorption affinity. As a cosolvent with water, glycerol magnifies the lyophobic interaction, and it influences the mechanism of RNase adsorption at the level of both fluid-phase protein transport and protein binding to the surface. Development of adsorption-rate-dependentstructure in the adsorbed layer can explain the shape of the adsorption isotherm and the unintuitive surface dynamics. This depends on the occurrence of a slow, surface-area-consuming molecular reconfigurationof the adsorbed proteins that is a direct consequence of the strength of the surface forces. This hypothesized reconfiguration is consistent with the distribution of mobile and immobile proteins in the adsorbed layer in the high- and low-concentration regimes of the isotherm. Similar reconfigurations may explain the atypical behavior of some proteins that appear to defy the correlation between adsorption affinity and material
Tilton et al.
hydrophobicity. In the glycerol-containingbuffer, proteins adsorb at initial rates exceeding the Uvhque solution for transport-limited adsorption. A simple calculation supports the assertion that this results from the strong attractive force between the RNase molecules and the surface in the presence of glycerol. The glycerol-induced mode of enhanced protein transport may be a new mechanism not operative in either purely aqueous or methanolcontaining protein solutions, or it may be an amplification of a usually insignificant feature of the normally operative mechanism. The lateral mobility of adsorbed RNase cannot be interpreted in a straightforwardmanner. While a decrease in the attractive force between the surface and the adsorbed proteins may increase the mobile fraction, it decreases the surface diffusion coefficient. This unexpected dependenceof the adsorbed protein lateral mobility on buffer composition clearly demonstrates the complexity of the dynamics of adsorbed proteins.
Acknowledgment. This material is based upon work supported by the National Science Foundation under Grants CBT-8813517and BCS-9007866.We thank Ms. Daniella Evans for important technical contributions to this work, Professor C. W. Frank for the use of his fluorescence spectrometer, and Ms. F. Thomas for her assistance with electron microscopy. Glossary a protein radius b half-depth of flow chamber amplitude of hydrophobic force B protein concentration C bulk protein concentration co surface protein concentration CI dimensionless concentration C heat capacity CP D diffusion coefficient diffusion coefficient in the absence of attractive Do force D* effective diffusion coefficient in the presence of attractive force mobile fraction of adsorbed proteins f hydrophobic force F decay length of hydrophobic force h dimensionless group relating lyophobic and H thermal forces k Boltzmann constant distance traveled by protein front to point of TIRF 1 observation t time glass transition temperature TB X coordinate parallel to surface coordinate normal to surface Y velocity in y direction VY velocity in x direction vx arbitrary distance for mass transport 6 wall shear rate Y r gamma function viscosity CC Stokes drag coefficient w Registry No. RNase A, 9001-99-4; methanol, 67-56-1;polystyrene, 9003-53-6;glycerol, 56-81-5.
