Adsorption of RNase A on Cationic Polyelectrolyte Brushes: A Study

Article pubs.acs.org/Biomac

Adsorption of RNase A on Cationic Polyelectrolyte Brushes: A Study by Isothermal Titration Calorimetry Alisa L. Becker, Nicole Welsch, Christian Schneider, and Matthias Ballauff* Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Hahn-Meitner-Platz 1, 14109 Berlin, Germany Department of Physics, Humboldt University Berlin, Newtonstr. 15, 12489 Berlin, Germany S Supporting Information *

ABSTRACT: We present a study of the adsorption of a positively charged protein to a positively charged spherical polyelectrolyte brush (SPB) by isothermal titration calorimetry (ITC). ITC is used to determine the adsorption isotherm as a function of temperature and of salt concentration (at physiological pH 7.2). At low ionic strength, RNase A is strongly adsorbed by the SPB particles despite the fact that both the SPB particles and the protein are positively charged. Virtually no adsorption takes place when the ionic strength is raised through added salt. This is strong evidence for counterion release as the primary driving force for protein adsorption. We calculated that ∼2 counterions were released upon RNase A binding. The adsorption of RNase A into like-charged SPB particles is entropy-driven, and protein protonation was not significant. Temperature-dependent measurements showed a disagreement between the enthalpy derived via the van’t Hoff equation and the calorimetric enthalpy. Further analysis shows that van’t Hoff analysis leads to the correct enthalpy of adsorption. The additional contributions to the measured enthalpy are potentially sourced from unlinked equilibria such as conformational changes that do not contribute to the binding equilibrium.

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of the nanoparticles has become a key issue in the safety assessment of various nanosystems.8 Attaching long polymeric chains to surfaces (“polymer brush”) is the state-of-the-art method to avoid protein adsorption2 and also for protein immobilization.9 However, the driving forces for attraction or repelling of proteins to polymers are not yet understood and present an extremely complex problem.10 The desire to understand what drives protein adsorption has increasingly led to the use of isothermal titration calorimetry11 (ITC) to characterize the process. ITC is the method of choice to measure the quantity of protein adsorbed (n), the affinity of the protein for the surface (K), and the thermodynamics of the adsorption process in a single experiment. It has been extensively used for investigating the interaction of proteins with nanoparticles.11−20

INTRODUCTION

The adsorption of proteins onto macroscopic surfaces or nanoparticles is important for biomedical devices, for diagnostic devices, and in food processing.1,2 In these applications the surfaces must deal with the high protein concentrations in blood or milk. In particular, blood plasma contains 50−70 g L −1 of protein, which can be adsorbed onto the surfaces after incubation. This conceals the original surface chemistry so that the body “sees” only the adsorbed protein corona.1,3,4 Proteins adsorbed on the surface of particles can act as opsonins and alter the way the body processes the material.5,6 Nanoparticles may lead to unfolding of proteins, e.g., fibrinogen, and promote inflammation.7 Thus, adsorbed proteins are of central importance when discussing issues related to the toxicity of nanoparticles.8 Moreover, the denaturation of adsorbed proteins on solid surfaces may lead to biofouling, causing major problems in biotechnological applications.2 Therefore, modeling of the interactions between proteins and the interface © 2011 American Chemical Society

Received: July 9, 2011 Revised: August 25, 2011 Published: October 4, 2011 3936

dx.doi.org/10.1021/bm200954j | Biomacromolecules 2011, 12, 3936−3944

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Figure 1. Interaction of proteins with polyelectrolyte chains attached to a colloidal particle. Left: representation of a positively charged SPB particle and the chemical structure of the cationic polymer in the brush: MAETA. The core-to-shell ratio for the SPB is depicted to scale. Right: electrostatic interaction of a positively charged protein with a like-charged SPB particle. The surface of a negatively charged protein is not homogeneous and contains patches of positive charge. This positively charged patch (blue) can replace the counterions associated with the negatively charged polymer, causing the release of the counterions associated with both the polymer and the protein. Top right: structure of RNase A (bovine, PDB: 1FS3). The amino acids are colored as follows: positive residues are blue, negative residues are red, polar residues are yellow, and hydrophobic residues are gray. The image was generated using the Swiss PDB Viewer.

The role of charge−charge interactions onto protein adsorption can be assessed by attached long highly charged macromolecules to the surface of the nanoparticles (spherical polyelectrolyte brush (SPB);21−24 see Figure 1). SPB particles are ideal model surfaces because they consist of well-defined, simple polyelectrolytes with large surface-to-volume ratios. Therefore, they can be investigated using a wide range of techniques including small-angle X-ray scattering (SAXS),25 FTIR,26 and calorimetry.11 Moreover, these systems are ideal for thermodynamic studies as the system is closer to a true equilibrium than other surfaces that result in irreversible protein adsorption. Indeed, the adsorption is nearly completely reversible when the ionic strength of the solution is increased.21,27 All findings reported so far demonstrate that there is a strong interaction between negatively charged SPB particles and proteins even above the isoelectric point, that is, even when both the SPB particle and the protein carry an overall negative charge.23,24 This “polyelectrolyte-mediated protein adsorption” (PMPA) could be related to the release of counterions bound to the polyelectrolyte layer and to the surface of the protein.21,24 Hence, the origin of the PMPA is entropic which could be shown directly by ITC.11 Counterion release can also be invoked to explain the protein adsorption on a similarly charged polyelectrolyte in the case of multilayered films assembled by the layer-by-layer technique28 and for the assembly of charged macromolecular systems in general. 29 All of the systems studied so far consisted of negatively charged components.24 Here, we investigate the adsorption of RNase A at physiological pH (i.e., well below its isoelectric point) onto positively charged SPB particles. RNase A has a molecular weight of 13 700 g mol−1 and an isoelectric point around pH 9.5.30 The adsorption of RNase A near its isoelectric point onto a negatively charged SPB was previously investigated. 26,31 After

adsorption it showed a loss of only 5% of α-helix and β-sheet secondary structure.26 SAXS measurements confirmed that the RNase A was adsorbed to the polyelectrolyte chains of SPB particles and not to the surface of the particle core.32 All previous studies point to the fact that the adsorption of proteins onto SPB particles is accompanied by only small or negligible changes of their secondary or tertiary structure.33 All previous work has mainly considered the role of the ionic strength for the PMPA. Here, we examine the role of temperature for this process for the first time. The binding constant K as well as the enthalpy of adsorption is measured by ITC within a small but sufficient range of temperatures (283− 310 K) for different ionic strengths. Thus, the interaction of RNase A with a cationic SPB particle can be examined as the function of the most important parameters, namely temperature and salinity. In particular, these data allow us to discuss the temperature dependence of K and the enthalpy of adsorption ΔHITC. In principle, the dependence of K on temperature should be related directly to ΔHITC; that is, ΔHITC should coincide with the enthalpy ΔHVH derived from application of the van’t Hoff equation to K(T). However, recent investigations by ITC on related problems such as protein−protein interaction or protein unfolding have revealed serious discrepancies between the two enthalpies.34,35 This finding has led to a number of investigations36,37 which are summarized in ref 35. The present set of data allows us to take a fresh look onto this problem and to discuss the analysis of protein adsorption by ITC based on a set of data from a wellcontrolled model system. Analysis of Adsorption Equilibria by ITC. ITC measures the heat change upon injection of a protein aliquot into a solution of SPB particles compared to a reference cell filled with water. Integration of this raw output gives the total heat change after each injection (see the discussion of Figure 2 3937


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Figure 2. Raw ITC data for RNase A adsorption onto MAETA_ SPB particles at 298 K in 10 mM MOPS buffer (pH 7.2) (a−c) and in 5.7 mM Tris buffer (pH 7.2) (d−f). (a, d) Raw ITC output, (b, e) integrated heat peaks, and (c, f) corrected for protein heat of dilution; the solid black line is the fit of the single set of independent binding sites model.

below). After correcting for the protein and SPB heat of dilution, the total heat change after each injection can be fit with an equilibrium model to determine the parameters K, n, and ΔHITC. Since the analysis of these data is at the center of the present study, we briefly describe and discuss the derivation of the equilibrium model. For a more detail description see ref 38. The simplest model applied to ITC data is a simple equilibrium with a set of n identical binding sites. In this model, the equilibrium constant is described by the fraction of sites containing bound protein, Θ:

Its solution is described in eq 3.

(3)

This expression can then be related to the heat measured in ITC by the relationship between Θ and the total heat Qi of the solution in volume V after i injections.

(1)

(4)

As the concentration of unbound protein [protein] u is unknown, it must be related to the known total protein and SPB particle concentrations [protein]t and [SPB]t by eq 2.

To fit the ITC isotherm, we need to understand the heat change of the solution ΔQ, which must also take into account the displaced volume ΔVi.

(2)

The fraction of sites containing bound protein, Θ, can be solved by combining eq 1 and eq 2 and solving the resulting quadratic.

(5) 3938


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of entropy ΔS caused by the release of counterions: 11

Finally, the parameters K, n, and ΔHITC are extracted via fitting of eq 5, which also incorporates eq 4 and eq 3, to the ITC isotherm. Equation 5 is solved for each injection using initial guesses for the parameters and then compared to the experimental data. Standard Marquardt methods are used to improve the values and the process is iterated until there is no further improvement in the fit. Equation 6 is then used to calculate the remaining values ΔG and ΔS.

(8)

Hence, the first term in eq 8 refers to the release of the counterions condensed onto the patches of the protein. The second term in eq 8 is related to the partial decrease in the Donnan pressure within the brush layer by counterion release. It must be noted that this model is directly related to the surface structure of the protein under consideration (see the discussion of Figure 1 below). Thus, the interaction of polyelectrolytes with proteins is specific. Evidently, a model that proceeds from the net charge of the protein as expressed through the zeta-potential would come to erroneous conclusions. Such a model would predict strong repellence of proteins by the SPB particles below the isoelectric point, which is in opposition to the findings reported here and elsewhere.21,24

(6)

Hence, adopting this analysis leads in essence to two enthalpies: the directly measured enthalpy ΔHITC and the enthalpy of binding ΔHbind that follows from the temperature dependence of the measured ΔGbind. Both quantities need not necessarily agree inasmuch as ΔHITC provides a measure for the full caloric effect of mixing the SPB particles with the protein, which will contain other contributions as well (see the discussion of this point in ref 18). Hence, it may contain contributions that are not related to the binding process. Since ITC conducted at different temperatures allows one to obtain both quantities, a thorough comparison of ΔHITC and ΔHbind may lead to these additional contributions. However, we note that this is only true for completely independent, “unlinked” equilibria. For linked equilibria the specific occupation of individual states plays a role.36 Counterion Release. The analysis of protein−polyelectrolyte interactions must start from the fact that the surface charge of proteins is not distributed at random but assembled into patches. These patches can act as multivalent counterions for linear polyelectrolytes, thus releasing the counterions of the patches as well as from the polyelectrolyte upon binding (Figure 1). 22 Evidently, the strength of this interaction is directly related to the gain of entropy of the respective counterions. It must vanish if the ionic strength is raised in the system (“counterion release force”, see ref 39). In the present case we consider the adsorption of RNase A below its isoelectric point with a cationic brush. Hence, both systems carry an overall positive charge and the adsorption takes place on the “wrong side” of the isoelectric point. 22,24 Therefore, we need to consider the interaction of the negatively charged patches on the surface of the protein (see Figure 1) with the positively charged polyelectrolyte chains of the brush layer. We assume that the negatively charged patches carry a total of N− charges. The solution is a low concentration of a monovalent salt, csalt. Two concentrations are important within the model: (i) The surface concentration, csi, is the concentration of counterions near the negatively charged patch on the protein. As discussed in ref 11, this surface concentration can be approximated through csi ≈ σ 2lB, where σ is the charge density of the negatively charged patch and lB the Bjerrum length. (ii) The counterion concentration within the brush cbrush, which is given by the Donnan equilibrium (eq 7) 11

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MATERIALS AND METHODS

Materials. RNase A, sodium azide, 3-(N-morpholino)propanesulfonic acid (MOPS), tris(hydroxymethyl)aminomethane (Tris) buffer, [2-(methacryloyloxy)ethyl]trimethylammonium (MAETA), α,α′-azodiisobutyramidin dihydrochloride (>97%, V-50), and styrene were purchased from Sigma-Aldrich (Munich, Germany) and used as received. NaCl was obtained from VWR (Darmstadt, Germany). The synthesis of the photoinitiator 2-[p-(2-hydroxy-2methylpropiophenone)] ethylene glycol methacrylate (HMEM) was performed according to the method used by Guo et al. and purified chromatographically.40 In all experiments we used water obtained from a Millipore ion exchange apparatus. Synthesis of SPB. The MAETA_SPB was synthesized as described previously,41 with the exception of using MAETA as the comonomer and emulsifier in the emulsion polymerization of the core particles. Thus, the synthesis of the core particles is an emulsifier-free emulsion polymerization. In a 5 L three-neck round-bottom flask with a mechanical stirrer and a reflux condenser 2450.44 g of water was mixed with 21.78 g of 70 wt % MAETA solution and 350.04 g of styrene at 320 rpm. The stirring speed was kept constant during the whole synthesis procedure. After heating the reaction mixture to 80 °C in 30 min the polymerization was started by the addition of 1.38 g of the initiator V-50. The polymerization was allowed to proceed at 80 °C for 1 h. The addition of 39.27 g of 62 wt % HMEM solution was started after cooling of the reaction mixture to 70 °C in 8 min. The reaction was allowed to proceed further for 5 h at 70 °C. After cooling the reaction mixture to 40 °C, the suspension was filtered with glass wool. Before and after the photoemulsion polymerization of the poly(MAETA) shell, the suspension was cleaned by extensive serum replacement with water. It should be noted that we avoided the use of detergent solution for the cleaning of instruments and equipment during the entire synthesis process of the SPB. Instead, we used pure organic solvents and a mixture of isopropanol and potassium hydroxide. In this way we avoided contamination of the system by surfactants. Characterization of SPB. The hydrodynamic radius of the core particles was 82 ± 1 nm, which was measured by dynamic light scattering (DLS) in 10 mM KCl via cumulant analysis using the third cumulant and the Einstein−Stokes relationship. The hydrodynamic radius of the SPB particles at very low ionic strength was 108 ± 5 nm, which includes the hydrodynamic shell thickness of 26 ± 5 nm and the hydrodynamic radius of the core particles. For the DLS measurements the suspensions were filtered through a 1 μm PES filter. Transmission electron microscopy (TEM) gave a radius of 76.9 nm and a polydispersity of 1.001 for the core particles. After cleaving off the chains from the core particles, we determined the molecular weight distribution by gel permeation chromatography (GPC). Because of the strong basic conditions during the cleaving

(7)

where cp is the concentration of charged monomers in the brush. Based on these considerations, the driving force for protein adsorption can be estimated in terms of the change 3939


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process, the poly(MAETA) chains were completely hydrolyzed to PMAA. The number-averaged molecular weight Mn of the cleaved chains was 6910 g/mol with a polydispersity index (PDI) of 1.97. This PDI value is typical for a free radical polymerization. The contour length of the chains was determined using Mn from the GPC measurements. This yielded 20 nm for the contour length where the monomer size has been estimated to 0.25 nm. The mass ratio between the core and the shell of the SPB was 15.5 ± 1.9 as determined by gravimetry and conductometric titration with AgNO3. Considering the mass ratio and Mn of the polyelectrolyte chains, we calculated a grafting density of 0.04 ± 0.01 chains per nm2. Using the mass ratio, we also calculated the number of charged units per SPB particle to 371 000 ± 57 000. The absolute number concentration of the SPB suspension after the photoemulsion polymerization was (8.46 ± 0.80) × 1018 m−3. We calculated the particle concentration using the solid content of the suspension determined by gravimetry and the size of the core particles determined via TEM. We also accounted for the weight of the shell layer of the SPB using the core−shell mass ratio. For calculating the weight of the core particles we assumed a uniform density of 1.054 g/cm3, which is the polystyrene bulk density. ITC Measurements. ITC experiments were performed with a Microcal VP_ITC. Approximately 300 μL of RNase A (1.095 mM; 15 g L−1) was titrated into a buffer-matched MAETA_SPB suspension (1.4 mL; 0.8−1.5 g L−1). Experiments were performed at 283, 291, 298, and 310 K. All solutions were degassed at 1 K below the experimental temperature prior to use. Control experiments were performed by substituting the appropriate buffer for the protein in the syringe and for the SPB suspension in the cell. The buffer systems used were MOPS_7 (10 mM MOPS, 2 mM NaN3, pH 7.2), MOPS_12 (10 mM MOPS, 2 mM NaN3, 5 mM NaCl, pH 7.2), MOPS_22 (10 mM MOPS, 2 mM NaN3, 10 mM NaCl, pH 7.2), and Tris_7 (5.67 mM Tris, 2 mM NaN3, pH 7.2). SPB particles were transferred into the appropriate buffer via ultrafiltration; proteins were dialyzed in buffer before use. ITC Analysis. The integrated heats from dilution experiments were subtracted from the integrated heat of adsorption experiments. The data were fitted using the supplied ITC module for Origin 7.0 software (Microcal, Northampton MA). A single site model was used to fit the RNase A data, using n and K as variable parameters. ΔHITC was measured by single titrations of dilute RNase A (1.5 g L−1) into a SPB suspension (4.8 g L−1) and fixed during fitting of the model, except for the case of Tris buffer, where K was fit using the average value from the MOPS data and ΔHITC and n and were variable parameters. ΔS and ΔG were calculated using eq 6. n was converted to τ using the molecular weight of RNase A (13 700 g mol−1) and the molecular weight of the SPB particles (1.45 × 109 g mol−1). Complete thermodynamic parameters are listed in the Supporting Information.

for the concentration of titrate (Figure 2b). All titrations were corrected for the heat of dilution of the protein by subtracting the heat released when RNase A was titrated into buffer (Figure 2b, black line). The dilution of SPB particles when buffer was titrated into the solution was negligible (Figure 2a, blue line) and therefore not considered. Because of the high concentrations used in these titrations, the heat of dilution of RNase A is significant compared to the measured heat of interaction. Moreover, the concentration of the particles could not be further increased to allow the measurement of the entire sigmoid binding curve, so a model-independent method was used to measure the enthalpy ΔHITC by a single titration at a low protein/particle ratio. This was achieved by selecting a protein concentration in the region where all of the injected protein was adsorbed onto the SPB particles. These measurements were repeated and averaged to determine the ΔHITC. This value was then fixed during the model fitting process, where n and K were treated as variable parameters. Moreover, the ΔHITC was measured in the lowest salt concentration by the model independent technique for each of the temperatures examined and was assumed to remain constant with increasing salt to allow fitting of the data and reduction of errors in the other parameters. The only exception was in the case of Tris buffer, where K was fit using the average value from the MOPS data and the ΔHITC and n were treated as variable parameters. The similarity in the binding curves for the MOPS and Tris measurements is clear (Figure 2c,f). The offset of data points observed in the uncorrected integrated heats (Figure 2b) occurs because of a slight discrepancy between the different injection volumes. However, it is corrected when the heat of protein dilution is subtracted from the value and no offset is observed in the final integrated heats (Figure 2c). It is clear from the isotherm that the positively charged RNase A does indeed interact with the positively charged SPB at low ionic strength (Figure 2), as expected. A single binding site model was sufficient to achieve a good fit for the heat released in the interaction between RNase A and MAETA_ SPB particles (Figure 2). Summarizing this analysis leads to the conclusion that ITC is capable of analyzing the process of protein adsorption even in the presence of large additional effects such as a considerable dilution enthalpy. The data thus derived have good precision and provide a good basis for an in-depth discussion of the thermodynamic parameters deriving from this analysis. Dependence of Protein Binding Equilibrium on Temperature. We have examined RNase A binding to MAETA_SPB in 7 mM ionic strength MOPS buffer pH 7.2 using van’t Hoff analysis (see Figure 3). ITC allows us to obtain the equilibrium constant K as well as the enthalpy ΔHITC as a function of temperature. As shown in Figure 3b, the equilibrium constant does not depend on temperature within the present limits of error. Hence, the enthalpy of binding ΔHbind derived from K through the van’t Hoff equation (eq 9) is zero under these conditions, and the binding was determined to be entropy driven (Table 1).

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RESULTS AND DISCUSSION Analysis of RNase A Binding by ITC. Figure 1 displays the surface structure of RNase A. These patches are clearly visible and provide the basis for the electrostatic model of counterion release (see above). In salt-free solutions the SPB particles have a 20 nm thick shell consisting of a methacrylate polymer bearing positively charged trimethylammonium functional groups (Figure 1). The grafting density of the brushes is 0.04 ± 0.01 nm−2. The MAETA_SPB is referred to as a quenched brush because the charged functional groups are not sensitive to pH (Figure 1). All adsorption studies of RNase A on MAETA_SPB were performed at pH 7.2, i.e., well below the isoelectric point of the enzyme. To study the binding of RNase A onto MAETA_ SPB particles, a solution of RNase A was titrated into a suspension of MAETA_ SPB particles and the change in heat measured over the course of the experiment (Figure 2a, red line). The change in heat was integrated with respect to time and adjusted

(9)

ΔSbind can also be extracted from the temperature dependence of the free energy as follows: (10) 3940


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Figure 3. (a) Adsorption isotherms measured using ITC at different temperatures. (b) van’t Hoff analysis of RNase A adsorption onto MAETA_SPB in 7 mM ionic strength MOPS buffer pH 7.2.

Table 1. Thermodynamic Parameters from Adsorption of RNase A onto MAETA_SPB Particles at 298 K ionic strength/buffer 7 mM/MOPS 12 mM/MOPS 22 mM/MOPS 7 mM/Tris a

τ (mg g−1) 364 625 175 450

± ± ± ±

56 92 7 132

ΔGbind (kJ mol−1) −23.8 −21.1 −19.9 −24.1

± ± ± ±

ΔHbind (kJ mol−1)

ΔSbind (kJ mol−1 K−1)

−2 ± 8 0±8 −4 ± 37

0.09 ± 0.01 0.08 ± 0.01 0.05 ± 0.04

0.7 1.2 0.2 0.1

ΔHresa (kJ mol−1) 29 32 32 24

± ± ± ±

8 8 37 7a

ΔCp,res (kJ mol−1 K−1) −0.18 ± 0.04 −0.18 ± 0.04 −0.18 ± 0.04

In Tris buffer only this value represents ΔHITC not ΔHres.

Here we present another example of a discrepancy between van’t Hoff (ΔH VH ) and calorimetric (ΔH ITC ) values. However, unlike many of the discrepancies discussed in the past,34 the two values are not easily reconciled by considering, e.g., experimental errors. Over the years, there has been much discussion about the disparate results obtained by calorimetric and van’t Hoff analysis.34−37 Heat capacity changes ΔCp were observed in calorimetric experiments that were not apparent in van’t Hoff analysis and could account for a portion of these inconsistencies.34,35 This is not relevant to this discussion, as the ΔCp is not compatible with the constant K. Previous studies determined that coupled conformational changes did not contribute to differences between ΔHVH and ΔHITC,36 but coupled protonation equilibria could contribute under specific conditions. 37 ΔHVH and ΔHITC discrepancies are also frequently observed in differential scanning calorimetry (DSC) protein unfolding experiments. In fact, the ratio of ΔHVH/ΔHITC has been used as a “calorimetric criterion” for the applicability of two-state unfolding models. 42 In our research it is clear that the K and ΔHITC are not directly related to the binding equilibrium, but the latter quantity must contain additional contributions that derive from other sources. Evidently, K is derived from the equilibrium of interest, i.e., the formation of the protein/SPB complex. ΔHITC, on the other hand, includes all contributions ranging from protein binding to other unrelated processes, such as mixing, conformational changes, etc. In the course of the analysis by ITC, ΔHITC can hence be treated as a “marker enthalpy” that allows us to observe the binding and with this the binding constant Kbind. However, ΔHbind and ΔSbind must be calculated via van’t Hoff analysis (eq 9) of the temperature dependence of Kbind.

In our calculations we assume that ΔCp = 0, which seems to be justified within the small temperature window and error of the measurements although this is not necessarily a requirement of the analysis. The remaining enthalpy is denoted ΔHres and its associated heat capacity ΔCp,res. Because ΔHbind as derived from the analysis of K(T) was negligible, in this case the entire ΔHITC is sourced from processes other than protein binding, i.e., ΔHITC = ΔHres. Therefore, the ΔHres acts as a marker enthalpy in the analysis by ITC. In the following we discuss the various possible sources for ΔHres. Analysis of Protonation Changes upon RNase A Binding. ΔHres is approximately equal to the bond energy expected from one hydrogen bond in water (10−40 kJ mol−1).43 Additionally, protonation may be a linked equilibrium, which can complicate thermodynamic analysis. 37 Many protein binding events are accompanied by protonation or deprotonation.44 A change in protein ionization in the presence of an SPB particle’s high electrostatic potential is expected from theoretical models45 and has been proposed as a mechanism for adsorption on the wrong side of the isoelectric point, known as charge regulation.46,47 Additionally, proton effects were determined to be important in protein binding to α-Zr(PO4) nanoplates17 and poly(styrene) nanoparticles.48 The source or sink of these protons is generally the buffering compound; thus, the ionization of the buffer often contributes to ΔHITC. Thus, we investigated the possibility that the enthalpy is the result of the deprotonation of one of the positively charged residues on the protein. By performing the adsorption titration in an additional buffer with a different enthalpy of ionization (ΔHi), it is possible to determine if the protonation state of the protein changes upon binding. RNase A adsorption was performed in two buffers with the same pH and ionic strength, but different ΔHi: MOPS, ΔHi = 21.1 kJ mol−1; and Tris, ΔHi = 47.45 kJ mol−1.49 The 3941


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Figure 4. (a) Adsorption isotherms measured using ITC at increasing salt concentrations. (b) van’t Hoff analysis of Kbind at increasing salt concentrations. (c) Temperature dependence of ΔG at different salt concentrations. (d) Salt dependence of ΔSbind (the linear fit has been forced to an intercept of 0).

ΔHITC was endothermic and essentially the same in both MOPS and Tris buffers (Figure 2c,f), even though the uncorrected heat changes are exothermic in Tris and endothermic in MOPS (Figure 2a,d). Consequently, it is clear that protonation does not contribute significantly to ΔHITC in RNase A binding to like-charged SPB particles and can therefore be neglected. This provides further evidence for counterion release as the main driving force and the subsequent analysis of adsorption as the function of ionic strength will fully corroborate this conclusion. Analysis of the Dependence of RNase A Binding on Ionic Strength. The analysis of the temperature dependence of Kbind indicates that the adsorption of the positively charged protein onto the positively charged SPB particles is entropy driven. This is strong evidence for counterion release as the adsorption mechanism.11,21 If the binding of RNase A is indeed driven by counterion release, increasing the salt concentration should weaken that process. That is what we observe when salt is added to the buffer solution during RNase A adsorption. To analyze the salt data, we assumed that the “marker enthalpy” of the secondary process ΔHres is unchanged with salt and fix the value during fitting. This assumption is required because the concentrations required for single point measurements are not accessible at the higher salt concentrations. Figure 4a shows the obvious change in the interaction between RNase A and the SPB with increasing ionic strength that we expected from previous studies.11,22 Even small increases in ionic strength (e.g., 5 mM) cause a significant decrease in the measured Kbind.

Van’t Hoff analysis of Kbind was used to determine ΔHbind and ΔSbind at higher salt concentrations (Figure 4b). The values at 298 K are listed in Table 1, and the complete thermodynamic parameters are listed in the Supporting Information. The ΔSbind decreases with increasing salt concentration (Table 1). The entropy calculated from the intercept in the van’t Hoff plots (Figure 4b) and in the temperature dependence of ΔG (Figure 4c) are in agreement. The counterion release model for proteins in polymer brushes (eq 8) can be used to estimate the total number of counterions (i.e., N− + N+ = 2N−) released from the adsorption of RNase A in MAETA_SPB particles. The negatively charged patch on the surface of RNase A was approximated to be 2 negative charges in an area of 1.5 nm2, thus σ = 1.3 × 1016 dm−2 and csi = 2.1 M. Other parameters for the lowest salt concentration were csalt = 0.007 M, cp = 0.3 M, and cbrush = 0.3 M. At this salt concentration the value of N− was ∼1; thus, the number of counterions released for RNase A adsorption was determined to be ∼2. We extended this calculation to the higher salt concentrations to determine if there are any contributions to the entropy not depending on ionic strength. Thus, eq 8 was plotted with ΔSbind against the known constants (Figure 4d). The linear fit of the data points is best when the intercept is fixed through the origin. This means that counterion release is the sole contributor to ΔSbind. Therefore, it must be the driving force for protein adsorption. The total number of counterions released upon RNase A adsorption from the slope of Figure 4d was determined to be ∼1 (i.e., total 3942


Biomacromolecules

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counterion release of 2). This is in good agreement with the small size of the negatively charged patch on RNase A. It rests to discuss the potential sources of the “marker enthalpy”. The marker enthalpy does not contribute to the binding equilibrium, but it must vanish as binding reaches completion. Thus, we propose that ΔHres is associated with changes of the protein when entering the brush layer. This change may be related to its conformation or solvation but is unrelated to the interaction of the protein with the polyelectrolyte chains of the brush layer. Therefore, it leads to a difference between ΔHbind (being negligible in the present case) and ΔHITC. This is in opposition to previous examples where binding is always accompanied by conformational change, and it is strongly linked, or even essential, to the binding equilibrium.36

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CONCLUSION We have analyzed the adsorption of RNase A onto cationic SPB particles by ITC. We found that the equilibrium constant was unchanged with temperature but that ΔHITC decreased with increasing temperature. This led to the conclusion that the ΔHITC and K were not related to the same equilibrium process. We assumed that K was from the protein adsorption equilibrium (K = Kbind), whereas the ΔHITC was a marker enthalpy that contained additional contributions from an unlinked equilibrium (ΔHITC = ΔHbind + ΔHres). By performing van’t Hoff analysis on the equilibrium constant, we determined that ΔHbind was negligible and that the adsorption process was entropy driven. Data taken at different salt concentrations indicated that entropy sourced from counterion release is the driving force for protein adsorption on likecharged polyelectrolyte brushes. We calculated that ∼2 counterions are released for each RNase A molecule adsorbed to the SPB. Moreover, there was no significant change in the protonation state of the RNase A upon adsorption. ΔHres may be sourced from small changes in the conformation of the protein or its solvatation in the presence of the brush as part of an unlinked equilibrium process.

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ASSOCIATED CONTENT

S Supporting Information *

Tables of thermodynamic parameters. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

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ACKNOWLEDGMENTS The authors thank J. Dzubiella for helpful discussions. REFERENCES

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Biomacromolecules

Article

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Adsorption of RNase A on Cationic Polyelectrolyte Brushes: A Study

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