Article pubs.acs.org/Langmuir
Kosmotrope-like Hydration Behavior of Polyethylene Glycol from Microcalorimetry and Binding Isotherm Measurements Wen-Yih Chen,*,† Min-Yen Hsu,† Ching-Wei Tsai,† Yung Chang,‡ Rouh-Chyu Ruaan,† Wei-Hung Kao,† E-Wen Huang,† and Hsiao-Yeh-Tzu Chung Chuan† †
Department of Chemical and Materials Engineering, National Central University, Jhong-Li Taoyuan 320, Taiwan Department of Chemical Engineering, Chung Yuan Christian University, Jhong-Li Taoyuan 320, Taiwan
‡
ABSTRACT: Polyethylene glycol (PEG) at various molecular weights (MWs) has been regarded as a wonder molecule in biomedical applications. For instance, PEG serves as a unique moiety for pegylation of “biobetter” drug development, PEG provides controlled-release and preserved activity of biologics, and PEG modified surface works as an antibiofouling surface. The primary characteristics of PEG molecules used in relevant applications have been attributed mainly to the hydration behavior in aqueous solutions. However, the effects on the solvation of solutes in solution caused by presenting PEG molecules as a cosolvent, as well as the thermodynamics aspect of the hydration behavior of PEG in solution, have not been well documented. The solvation behavior of solutes, such as protein, with PEG as a cosolvent, indicates the success of PEG applications, such as biofouling and controlled release. In this investigation, we examined the effects of a buffer solution containing PEG molecules on the solution behavior of solute and the interactions between solid surfaces with solutes. We adapted the study by selecting a lysozyme as a solute in a buffer solution with either ammonium sulfate (kosmotrope) or sodium chloride (chaotrope) and anionic resin (SP-Sepharose) as solid surfaces. The experiments primarily involved binding equilibrium measurements and thermodynamics analysis. The results revealed that, in both saline buffers, adding PEG increases the binding affinity between the lysozyme and the resin, similar to kosmotropic salt in the examined salt concentrations. The thermodynamics analyses involving microcalorimetric measurements show that the bindings are mainly driven by enthalpy, indicating that electrostatic interaction was the primary binding force under these experimental conditions. The variations of the enthalpy and entropy of the binding thermodynamics when adding PEG to different salt types in the buffer solution showed opposite behavior, and the results support the concept of kosmotrope-like behavior of PEG. The equilibrium and thermodynamics data demonstrate that PEG has a kosmotrope-like hydration behavior, and the extent of kosmotrope-like behavior depends on the molecular weight of PEG with the outcomes of various molecular weights of PEG being added to the binding solution. The results of this study provide essential knowledge for PEG as an additive (or cosolvent) in various research applications.
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INTRODUCTION Polyethylene glycol (PEG), a polymer composed of various −(CH2CH2O)n− (ethylene glycol, EG) monomers, is nontoxic, nonimmunogenic, nonantigenic, and highly water soluble and has been approved by the FDA for internalization in the human body.1,2 PEG has also been applied to surface coatings for antibiofouling materials, pegylated drugs, and biologics.3,4 The conformation of low-molecular-weight PEG involves a random coil that can form strands for higher molecular weight PEG in aqueous solutions. PEG has high solubility (e.g., 50% weight of PEG8000) in water. The hydration-free energy of 1,2dimethoxyethane (the lowest MW of PEG) is −5.14 kcal/ mol and −4.8 kcal/mol by performing molecular modeling and by conducting experiments.5 To obtain the molecular structure of PEG in solution, the general approach is using nuclear magnetic resonance (NMR) and infrared spectrum (IR) spectroscopies.6 Results show that © 2013 American Chemical Society
each EG monomer can bind approximately three water molecules in an aqueous solution. When PEG dissolves in a water solution, EG monomers have a hydrogen-bonding provider that can bind water molecules by the H-bond, and the C−C bond of EG also aligns three bound water molecules. The hydrogen bonding and water alignment cause PEG to have higher water solubility. However, in high concentrations of chloroform or benzene solution, PEG molecules aggregate by intermolecular hydrogen bonding.7 Aqueous two-phase (ATP) systems, which are formed by mixing PEG with various salts in certain concentrations, have been adapted for the purification of biomolecules by establishing a partition between the two aqueous phases. The formation of ATP can be easily Received: November 11, 2012 Revised: January 6, 2013 Published: January 18, 2013 4259
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Measurement of Fluorescence Spectroscopy. Fluorescence spectroscopy (FP) spectra were recorded using a Jasco FP-6500 spectropolarimeter (Japan) and a cell with a 1 mm optical path length. To investigate the structural difference of the lysozyme in different environments, the lysozyme was diluted in 10 mM of a phosphate buffer (pH = 7) and the mentioned stock solutions by using a series of different salt or PEG concentrations to a final lysozyme concentration of 1 mg/ mL. The samples were excited by the UV light at the wavelength of 280 nm, and the measurement of fluorescence emission was detected from 300 to 400 nm. The reported spectra were the average of five scans, and the buffer baselines were subtracted from the spectra. Adsorption Isotherm of Lysozyme with SP Sepharose FF Resin. All adsorption isotherm experiments were performed at 25 °C. After washing the SP Sepharose FF resin by distilled water using fivefolds of gel solution volume, we equilibrated the resins in various species and concentrations of salts (NaCl and AS) in 10 mM of a phosphate buffer. The adsorption experiments were conducted by mixing the various concentrations of the lysozyme and 0.1 mL of resin (sulfonic ligand density is 6 mmol/mL-resin) for 4 h. The final concentrations of protein adsorption on the resins ranged from 0.01 to 4 mg/ mL by the presence or absence of the PEG additives. The salt concentration of ammonium sulfate (AS) ranges from 14 mM to 1.2 M, whereas that of NaCl ranges from 42 mM to 1.8M. To control the same monomeric number of EG, the PEG additives including the PEG 400, PEG 3000, and mPEG 5000 were prepared at 10 wt %. After adsorption equilibrium, the samples were centrifugated at 3000 rpm, and the amounts of lysozyme adsorption on the SP Sepharose FF resin were quantified by measuring the supernatant absorbance of 280 nm using the Synergy H1 BioTek microplate reader (USA). Measurement Using Isothermal Titration Calorimeter. We used an isothermal titration calorimeter (ITC) (Microcal Omega, MCS and/or VP-ITC, Microcal Inc.) to analyze the binding thermodynamics of the lysozyme and strong anionic resins under the conditions of various salt species and PEG additives. All of the samples and buffer solutions were degassed using the AS-3 NEWLAB aspirator (Taiwan). The chamber volume of 1.44 mL contains 0.1 mL of resins that are preequilibrium at respective buffer or PEG additives buffer solution. The titration syringe was filled with 20 mg/mL of lysozyme solution. The titrations were performed by first injecting 2 μL and then injecting 10 μL of lysozyme solutions into the cell at a 5 min interval for each injection. The power from each injection was recorded. To analyze the ITC data, the dilution heat was subtracted, and the adsorption enthalpy change (ΔH) throughout the titration was integrated. A simple one-site binding model was used to fit the titration curve; thus, the Gibb’s free energy change (ΔG) and the adsorption binding affinity constant (Ka) could be further obtained. On the basis of these two thermodynamic parameters, the entropy change (ΔS) of adsorption could also be obtained.
determined when salt is added to the PEG/aqueous solution. Dissolved ions compete for the water molecules (hydration) and reduce the PEG cloud point (aggregation point) and then form phase separation, as in the ATP system. Experimental results from a previous study showed that the cations listed as kosmotropes (salting out) in Hofmeister series (lyotropic series) have higher inclinations of forming ATP with PEG.8 These results indicate the hydration characteristics of PEG and the competition for PEG hydration with that of cations in resolving ATP formation. However, anions have no such tendency as listed in Hofmeister series. Because of the formation of PEG and anions complex in solution, the hydration behavior of the ions cannot be solely considered for the possibility of ATP formation.9 Moreover, Pullara et al. examined that PEG molecules play a crucial role in lysozyme crystallization arising from entropic contributions.10 On the basis of the mentioned PEG properties, we investigated the kosmotrope-like hydration behavior of PEG from the aspects of thermodynamics and hydration that involves binding PEG water molecules. Hence, we measured the binding isotherm and binding thermodynamics (enthalpy and entropy) of a lysozyme as a model protein (solute) with PEG presented into both kosmotropic and chaotropic salts in the binding solution. The equilibrium and thermodynamics data, as indicated by the presence of PEG as a cosolvent, were used to demonstrate that the hydration behavior of PEG is similar to that of a kosmotrope-like salt. Furthermore, various MWs of PEG were also added to the salt solution to reveal the MW effects of the kosmotrope-like behavior of PEG.
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MATERIALS AND METHODS Chemical Components. The lysozyme (MW = 14 320 Da) used in this study was purchased from Sigma-Aldrich (USA). Polyethylene glycol 5000 monomethyl ether (mPEG5000, MW = 5000 g/mol) and polyethylene glycol 3000 (PEG3000, MW = 3000 g/mol) were purchased from Fluka (Switzerland). Polyethylene glycol 400 (PEG400, MW = 400 g/mol) was provided by En Hou Polymer Chemical (Taiwan). Sodium phosphate dibasic 12 hydrate was purchased from Shimakyu (Japan). Sodium dihydrogen phosphate, ammonium sulfate (AS), sodium chloride (SC), and sodium fluoride were all purchased from Sigma-Aldrich (USA). SP Sepharose Fast Flow (FF) resin (particle diameter: 90 μm) were purchased from GE Healthcare Life Sciences (USA). Measurement of Circular Dichroism. Circular dichroism (CD) spectra were recorded using a Jasco J-810 spectropolarimeter (Japan) using a cell with an optical path length of 1 mm. The lysozyme was dissolved in 10 mM of a phosphate buffer (pH = 7) to a final concentration of 20 mg/mL. Stock solutions of ammonium sulfate and sodium fluoride was prepared in 10 mM of a phosphate buffer (pH = 7) to final concentrations of 4 and 3.6 M, respectively. PEG of three molecular weights (400, 3000, and 5000 g/mol) were dissolved in 10 mM of a phosphate buffer at the concentration of 200 mg/mL. To investigate the structural difference of the lysozyme in different environments, the lysozyme was diluted in 10 mM of a phosphate buffer and the mentioned stock solutions by using a series of different salt or PEG concentrations to a final lysozyme concentration of 1 mg/mL. The samples were scanned at a wavelength range of 200−350 nm under a nitrogen flush. The reported spectra were the average of five scans, and the buffer baselines were subtracted from the spectra.
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RESULTS AND DISCUSSION Effects of PEG on Lysozyme Conformation. In aqueous solutions, hydrogen bonding between EG and water molecules, as well as the alignment of water molecules on the C−C bond, may create surface tension11,12 and result in different interaction (binding) behaviors and mechanisms between the protein and the resin. Furthermore, exposure of each EG moiety to an aqueous solvent of PEG macromolecules is 4260
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dependent on the molecular weight of PEG because of possible secondary conformations of PEG molecules that may exist in the solution. In addition, the excluded volume effect of PEG must be accounted for when high-molecular-weight PEG is present as a cosolvent in the binding solution. As discussed, the effects of presenting PEG molecules on the structure of the dissolved protein must be illuminated for the binding mechanism deliberations. Thus, circular dichroism (CD) and fluorescence spectroscopy were adapted to reveal any possible conformational changes in various solutions. The CD results revealed that the secondary structures of lysozyme do not discernibly change (data not shown). While undergoing fluorescence detection, a blue shift of 340 nm was detected, and the intensity was reduced in both the ammonium sulfate and sodium chloride solutions when various MWs of PEG were added (data not shown). The blue shift indicated inward structural shrinkage of tryptophan residues, and the intensity reduction is due to the quenching effect of PEG molecules adsorbed on a hydrophobic patch on the lysozyme. Jiang et al. revealed in a molecular modeling study that a compact lysozyme structure with smaller SQMD was obtained in the presence of PEG in solution.13 A similar phenomenon was observed by Svergun et al. in conducting a model analysis of small-angle X-ray scattering, revealing that the quaternary structure of PEG-conjugated hemoglobin appears compacted, resulting from the part of the PEG chain that enters the cavities between the hemoglobin subunits.14 Batch Isotherm Measurements. In 2012, Werner et al. investigated the PEGylated lysozyme and PEG on a midhydrophobic interaction resin, observing that the adsorption of PEG on the resin and the binding constant of the derived lysozyme may have been affected by the PEG adsorption.15 On the basis of these arguments, we measured the binding isotherm of the lysozyme on SP Sepharose FF strong cation (SP) exchange resin in kosmotropic (ammonium sulfate, AS) and chaotropic (sodium chloride, SC) salt solutions, with or without PEG as a cosolvent. We varied the salt concentrations and added PEG at different MWs for the batch isotherm and binding affinity constant measurements between the lysozyme and SP ion-exchanger resin. The binding affinity constants were employed in the binding thermodynamics data derivation, based on the results of ITC experiments. All of the batch isotherm experiments were performed at pH 7 with 10 mM of a phosphate buffer, and the binding affinity (Ka) was obtained by a simple Langmuir isotherm fitting. The lysozyme had a pI of 10.7 and the SP Sepharose FF is a cation exchanger resin; therefore, attractive electrostatic interaction dominated the binding between the lysozyme and SP in the pH 7 solution. The measured binding affinity was 1.61 × 106 M−1 in the phosphate buffer solution. Two types of salts with the same ionic strength, ammonium sulfate (AS) (14 mM, 30 mM, 45 mM, 0.1 M, 0.6 M, and 1.2 M) and sodium chloride (SC) (42 mM, 90 mM, 135 mM, 0.3 M, and 1.8 M), were added to the 10 mM phosphate buffer for systematic binding isotherm measurements. We have also performed the adsorption of PEG on the SP Sepharose resins; it is found that the PEG molecules (PEG 400, PEG 3000, and mPEG 5000) are not adsorption on to anionic resin under our buffer condition. On the basis of the isotherm data (Figures 1 and 2), at the higher salt concentrations examined in this investigation (0.6 and 1.2 M of AS and 0.3 and 1.8 M of SC), the binding affinities were greatly reduced because of the screening effect
Figure 1. Adsorption isotherm of lysozyme binding to the SP Sepharose resins in pH 7.0, 10 mM phosphate buffer containing 90 mM SC (NaCl) with 100 mg/mL various molecular weight PEG (PEG-400, PEG-3000, and mPEG-5000) at 25 °C. The fitting curve is fitted by Langmuir model.
Figure 2. Adsorption isotherm of lysozyme binding to the SP Sepharose resins in pH 7.0, 10 mM phosphate buffer containing 30 mM AS with 100 mg/mL various molecular weight PEG (PEG-400, PEG-3000, and mPEG-5000) at 25 °C. The fitting curve is fitted by Langmuir model.
caused by the high concentration of ions in the binding solution, as listed in Tables 1 and 2. For the other salt concentrations with added buffer solutions, the Ka variations of Table 1. Association Constant (Ka) and Maximum Adsorption Amount (qmax) of Lysozyme Adsorption to SP Sepharose along with Various Salt Concentrations of Salts (SC and AS) sodium chloride
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ammonium sulfate
[SC] (mM)
Ka × 10 (M−1)
qmax (M × 10−4/gel mL)
[AS] (mM)
Ka× 104 (M−1)
qmax (M × 10−4/gel ml)
42 90 135 300 1800
121.00 28.80 6.44 0.31 1.39
81.45 69.55 47.50 18.16 14.81
14 30 45 100 600
140.85 132.45 44.72 0.86 0.09
114.25 83.21 71.15 60.97 89.30
4
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increasing the amount of protein that can be bounded on the resin. However, when the MW of PEG increases, a shielding effect of PEG between protein and SP resin and/or the excluded volume effect reduce the binding amount. These results demonstrate that PEG with different MWs has structural effects when the PEG is a cosolvent in this binding mechanism. Different extents of hydration conduct of each EG monomer as residue of PEG polymer caused by the degree of exposure make the MW of PEG selection a profound factor that should be considered in various applications. As shown in Table 2, the values of Ka increase slightly in the binding solution of AS with or without PEG. Similar results were also observed by Werner et al., the binding affinity of PEGylated lysozyme or when lysozyme adsorbs on the mildly hydrophobic resins increases in the binding solution containing AS. On the other hand, similar behaviors were also observed in the binding solution containing SC salt; in particular, the Ka values significantly increase as the PEG addition. In summary, PEG can be considered as a kosmotrope-like molecule because salting-out effects were demonstrated in the binding isotherm measurements. The conformational effect of different MWs of PEG has also been recognized because the hydration of each EG monomer behaves differently at different MWs of PEG polymer, thus resulting in different levels of binding performance between protein and resin. Binding Thermodynamics by Isothermal Titration Calorimetry. The presented isotherm data did not divulge the effects of the binding mechanism when using PEG as a cosolvent. ITC, however, is a powerful tool to reveal the binding mechanism through the information of binding enthalpy and entropy.19−21 We, therefore, applied ITC on the mentioned binding conditions and report the binding thermodynamics as follows. With use of the binding equilibrium data from the binding isotherm, the enthalpy of adsorption is obtained. Detailed derivations and methods are reported in our previous publications.19−21 As shown in Table 3, the binding enthalpy of the lysozyme on the SP resin in 10
Table 2. Effect of Molecular Weight of PEG Additive on the Association Constant (Ka) and the Maximum Adsorption Amount (qmax) of Lysozyme Adsorption to SP Sepharose with 90 mM SC and 30 mM ASa 90 mM sodium chloride Ka × 10 (M−1)
qmax (M × 10−4/gel mL)
28.80 383.14 102.00
69.55 103.85 47.46
50.20
51.95
4
additives w/o PEG-400 PEG3000 mPEG5000 a
30 mM ammonium sulfate additives w/o PEG-400 PEG3000 mPEG5000
Ka× 104 (M−1)
qmax (M × 10−4/gel mL)
132.45 183.49 170.00
83.21 91.40 95.36
188.00
91.65
Each PEG concentration is 100 mg/mL.
the lysozyme in both the AS and SC solution are the same. In other words, Ka decreases as the AS concentration increases, so does SC salt. Furthermore, it is clearly observed that the Ka values of lysozyme adsorption on SP resin in the AS solution is higher than those in SC solution under the same ionic strength. These binding behaviors between the lysozyme and SP by the existence of the electrolytes matched those of the Hofmeister series. AS is classified as a kosmotrope, whereas SC is a chaotrope. Regarding the hydration conduct, AS has a saltingout effect and makes lysozymes separate from solutions and may adsorb on the solid surfaces of resins. Thus, the salting-in effect of SC dissolves lysozymes and less adsorption occurs. These phenomena have been observed in various applications and reported in relevant literature.16−18 The main purpose of this investigation was to elucidate how PEG affects the binding isotherm of lysozymes. The question remains whether PEG acts in the manner of a kosmotrope or chaotrope in aqueous solutions when the hydration behavior of PEG in aqueous solution is of concern. Furthermore, how does the presence of PEG affect the binding thermodynamics between protein and resin? This has not been effectively addressed and the answers to these questions would be beneficial for chromatographic bioseparation applications. Furthermore, does this kosmotrope-like or chaotrope-like behavior of PEG depend on the molecular weight of PEG? To clarify these questions, for lysozymes and SP systems, 30 mM AS or 90 mM SC with or without various MWs of PEG in 100 mg/mL as binding solutions were designated. The same weight of different MWs of PEG in solution indicates the same amount of EG monomers in solution, and the conformational effects caused by the MW of PEG can then be revealed. We measured the binding isotherms and derived the binding affinities. Most important, we quantified the binding enthalpy and binding entropy by performing isothermal titration calorimetry, and molecular level aspect descriptions were hypothesized. First, the binding affinity (Ka) increases with the addition of PEG400, PEG3000, and mPEG5000 in 90 mM SC binding solution, and the smallest MW of PEG400 has the highest increase of Ka, as shown in Table 2. The maximum binding amount also increases with PEG400, but decreases when the MW of PEG becomes larger. Our explanations are as follows. As the results from the fluorescence measurements, the presence of PEG in solution that was examined in this investigation may be confined on the lysozyme surface because of the hydrophobic effect, thus causing the lysozyme to become shriveled, reducing the protein−protein repulsive force, and
Table 3. Thermodynamic Parameters of Lysozyme Binding to SP Sepharose with Different Salt Types Ka × 104 (M−1) ΔG (kJ/mol) ΔH (kJ/mol) −TΔS (kJ/mol)
buffer
30 mM AS
90 mM SC
161.29 −35.41 −716.84 681.42
132.45 −34.93 −963.10 928.18
28.80 −31.15 −750.92 719.74
mM of a phosphate buffer solution and the buffer solution with 30 mM AS or 90 mM SC is an exothermic reaction, indicating that strong electrostatic interactions dominate the binding. Adding AS or SC causes ΔG to be less negative (lower Ka) and reduces ΔS (less chaos in the system), indicating that the hydration behavior of ions from the salts compete and remove hydrated water molecules from the protein surface, and that the system chaos created by the water molecules released from the protein surface in the binding process is reduced. From this aspect, the reduction of the system chaos by kosmotropic ions is more noticeable than that of chaotropic ions. This phenomenon is documented in Table 3 and was also reported by Cremer et al.11,12 These data serve as a reference for discussions on the binding thermodynamics changes by adding PEG in binding solutions. 4262
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solute (protein) surface, thereby reducing the surface tension of the solute and promoting the solubility of solute into the solution. Although PEG competes for water molecules, the subsistence of SC ions also helps dissolve PEG in the solution. In addition, the small MW of PEG has the binding capability with the protein through the hydrophobic effect, as mentioned in the Binding Isotherm Measurements section. Therefore, the binding thermodynamics of the protein with SP resin in SC in the presence of PEG show higher binding affinity with less exothermic binding enthalpy and higher system chaos generated (Table 4), unlike PEG in the AS solution. Furthermore, we investigated the thermodynamics information variation by examining the PEG MW effect. The results are plotted in Figures 3 and 4. We added the same weight (10 mg/ mL) of PEG at various MWs (400, 3000, and 5000) to the binding solution and found that little variation of ΔG was observed among the PEG in both the AS and SC solutions. The changes of the ΔH and ΔS to the variations of PEG MW in both AS and SC solutions behave contrarily. The same amount of EG monomers (same amount of weight of PEG at different MWs) in solution may not show linearly the behavior of EG quantity because of the conformation of the PEG molecules in the solution. This phenomenon was clearly indicated in the binding thermodynamics data. In the AS solution, ΔH decreases (less exothermic) as the MW of PEG increases and ΔS decreases (less system chaos); we referred that the higher molecular weight of PEG has lower the number of PEG molecules (under the same weight of EG in solution) and with the spatial conformation effect, the hydration capacity (number of total hydrated water molecules) reduced in higher MW of PEG and fewer kosmotropic characteristics, as compared with a lower MW PEG at the same weight, resulting in less binding enthalpy and entropy. By contrast, in the SC solution, fewer kosmotropic characteristics of higher MW PEG creates higher exothermic binding enthalpy and system chaos, as shown in Figure 3.
Similar to the isotherm measurements, 100 mg/mL of PEG400, PEG3000, and mPEG5000 were added to the 30 mM AS or 90 mM SC solutions for the ITC studies of binding thermodynamics between protein and SP resin. First, adding low MW PEG (PEG400) to the two types of salt solution creates different effects on the binding thermodynamics conduct. As shown in Table 4, more exothermic enthalpy and Table 4. Effect of the PEG 400 Addition on the Thermodynamic Parameters of Lysozyme Binding to SP Sepharose with the Different Salt Types 30 mM AS Ka × 104 (M−1) ΔG (kJ/mol) ΔH (kJ/mol) −TΔS (kJ/mol)
30 mM AS + 100 mg/mL PEG400
90 mM SC
90 mM SC + 100 mg/mL PEG400
132.45
183.49
28.80
383.14
−34.93
−35.73
−31.15
−37.56
−963.10
−4059.85
−750.92
−533.16
928.18
4024.13
719.77
495.60
less chaos were observed when PEG was added to the kosmotropic AS solution, whereas contrasting data were perceived for the chaotropic SC solution. As discussed in the subsection on binding isotherm measurements, PEG is a kosmotrope-like molecule in aqueous solution, and EG monomer has a C−C bond and hydrogen-bonding provider. These structural characteristics compete for the bound water molecules on the solute (protein) surface and create surface tension of the solute in the solution. Furthermore, the characteristics provide hydration capability and reduce the freedom of water molecules, thus suppressing the system’s chaos after binding. The binding thermodynamics results (less negative ΔG (binding affinity increase), less negative ΔS (system chaos decrease), and ΔH more negative (exothermic binding)) shown in Table 4 demonstrate the PEG effects on the binding thermodynamics. However, the presence of PEG400 molecules in the SC solution generates opposite thermodynamics behaviors. As discussed, SC acts as a chaotrope (salting in) in aqueous solutions. Both cations and anions hydrate with water molecules and tend to dissolve in the
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CONCLUSION This study elucidates the kosmotrope-like hydration behavior of PEG by determining the role of PEG in solution regarding protein binding with ion-exchanger resin (SP-Sepharose FF).
Figure 3. Thermodynamic parameters of the interaction of lysozyme and SP Sepharose with the addition of various molecular weights of PEG (PEG-400, PEG-3000, and mPEG-5000) at 25 °C; the buffer condition is pH 7.0, 10 mM phosphate buffer, 90 mM SC. 4263
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Figure 4. Thermodynamic parameters of the interaction of lysozyme and SP Sepharose with the addition of various molecular weights of PEG (PEG 400, PEG 3000, and mPEG 5000) at 25 °C; the buffer condition is pH 7.0, 10 mM phosphate buffer, 30 mM AS. (7) Castellanos, I. J.; Crespo, R.; Griebenow, K. Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres. J. Controlled Release 2003, 88 (1), 135−45. (8) Ananthapadmanabhan, K. P.; Goddard, E. D. Aqueous biphase formation in polyethylene oxide-inorganic salt systems. Langmuir 1987, 3 (1), 25−31. (9) Omta, A. W.; Kropman, M. F.; Woutersen, S.; Bakker, H. J. Negligible effect of ions on the hydrogen-bond structure in liquid water. Science 2003, 301 (5631), 347−349. (10) Pullara, F.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U. Protein crystallization: universal thermodynamic vs. specific effects of PEG. Faraday Discuss. 2008, 139, 299−308; discussion. 309−25, 419− 20. (11) Zhang, Y. J.; Cremer, P. S. Interactions between macromolecules and ions: the Hofmeister series. Curr. Opin. Chem. Biol. 2006, 10 (6), 658−663. (12) Zhang, Y. J.; Furyk, S.; Bergbreiter, D. E.; Cremer, P. S. Specific ion effects on the water solubility of macromolecules: PNIPAM and the Hofmeister series. J. Am. Chem. Soc. 2005, 127 (41), 14505− 14510. (13) Shao, Q.; He, Y.; White, A. D.; Jiang, S. Different effects of zwitterion and ethylene glycol on proteins. J. Chem. Phys. 2012, 136 (22), 225101−225106. (14) Svergun, D. I.; Ekstrom, F.; Vandegriff, K. D.; Malavalli, A.; Baker, D. A.; Nilsson, C.; Winslow, R. M. Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic. Biophys. J. 2008, 94 (1), 173−81. (15) Werner, A.; Blaschke, T.; Hasse, H. Microcalorimetric study of the adsorption of PEGylated lysozyme and PEG on a mildly hydrophobic resin: influence of ammonium sulfate. Langmuir 2012, 28 (31), 11376−83. (16) Muller, E.; Josic, D.; Schroder, T.; Moosmann, A. Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins. J. Chromatogr. A 2010, 1217 (28), 4696−703. (17) Heinitz, M. L.; Kennedy, L.; Kopaciewicz, W.; Regnier, F. E. Chromatography of proteins on hydrophobic interaction and ionexchange chromatographic matrices: mobile phase contributions to selectivity. J. Chromatogr. 1988, 443, 173−82. (18) Chen, J.; Sun, Y. Modeling of the salt effects on hydrophobic adsorption equilibrium of protein. J. Chromatogr. A 2003, 992 (1−2), 29−40. (19) Huang, H. M.; Lin, F. Y.; Chen, W. Y.; Ruaan, R. C. Isothermal Titration Microcalorimetric Studies of the Effect of Temperature on
We measured the binding isotherm and binding thermodynamics in the presence of various MWs of PEG molecules in both kosmotropic and chaotropic salt solutions. The thermodynamics information was obtained by performing ITC. Binding equilibrium and thermodynamics suggest that PEG has kosmotrope-like characteristics as a cosolvent in aqueous binding solution. Furthermore, conformation of PEG caused by a higher PEG MW in solution also influences each EG monomer hydration capability and results in different binding conducts of protein on resin. These results provide instructive information of PEG and MW of PEG in various applications.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel.: +886-3-4227151, #34222. Fax: +886-3-422-5258. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the National Science Council, Taiwan (NSC-101-2221-E-008-089-MY3 and NSC-101-2811-E-008010) for financial support of this project.
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REFERENCES
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dx.doi.org/10.1021/la304500w | Langmuir 2013, 29, 4259−4265
