Macromolecular Crowding Modifies the Impact of Specific Hofmeister

28 Jul 2015 - Macromolecular crowding alters many biological processes ranging from protein folding and enzyme reactions in vivo to the precipitation ...
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Macromolecular Crowding Modifies the Impact of Specific Hofmeister Ions on the Coil−Globule Transition of PNIPAM Kenji Sakota,* Daiki Tabata, and Hiroshi Sekiya Department of Chemistry, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan S Supporting Information *

ABSTRACT: Macromolecular crowding alters many biological processes ranging from protein folding and enzyme reactions in vivo to the precipitation and crystallization of proteins in vitro. Herein, we have investigated the effect of specific monovalent Hofmeister salts (NaH2PO4, NaF, NaCl, NaClO4, and NaSCN) on the coil−globule transition of poly(N-isopropylacrylamide) (PNIPAM) in a crowded macromolecular environment as a model for understanding the specific-ion effect on the solubility and stability of proteins in a crowded macromolecular environment. It was found that although the salts (NaH2PO4, NaF, and NaCl) and the macromolecular crowder (polyethylene glycol) lowered the transition temperature almost independently, the macromolecular crowder had a great impact on the transition temperature in the case of the chaotropes (NaClO4 and NaSCN). The electrostatic repulsion between the chaotropic anions (ClO4− or SCN−) adsorbed on PNIPAM may reduce the entropic gain of water associated with the excluded volume effect, leading to an increase in the transition temperature, especially in the crowded environment. Furthermore, the affinity of the chaotropic anions for PNIPAM becomes small in the crowded environment, leading to further modification of the transition temperature. Thus, we have revealed that macromolecular crowding alters the effect of specific Hofmeister ions on the coil−globule transition of PNIPAM.

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INTRODUCTION More than a century ago, Hofmeister reported the effect of specific salts on the precipitation of certain proteins in an aqueous solution, called the Hofmeister series.1 The salts that have a tendency to stabilize or precipitate proteins are referred to as kosmotropes, whereas the salts that denature or solubilize proteins are referred to as chaotropes. The typical order of monovalent anions in the Hofmeister series is

(PEG) have been used as precipitants for proteins in their purification and crystallization from salt solutions.8 Because the reliability of the biochemical experiments strongly depends on the purity of the biomaterials used, the effectiveness of the purification process is important.9 In addition, as the solubility of the proteins seems to correlate with their stability, understanding the effect of macromolecular crowding on the solubility of proteins may provide some insights into its effect on the stability of the proteins in the salt solutions. Poly(N-isopropylacrylamide) (PNIPAM) is one of the most popular thermoresponsive polymers.10,11 It exhibits a lower critical solution temperature (LCST) at approximately 32 °C in an aqueous solution. Because the coil−globule transition occurs at the LCST, PNIPAM has been regarded as a model system for the folding and denaturation of proteins.12−14 So far, the Hofmeister effect on the LCST of PNIPAM has been extensively studied by Cremer and co-workers.15−17 They found that the modifications of LCST induced by kosmotropes and chaotropes have different physical origins. In particular, the chaotropic anions, such as SCN− and I−, have shown a Langmuir-type binding behavior onto PNIPAM, leading to an increase in the LCST. Furthermore, the effect of macromolecular crowding on the LCST of PNIPAM has been reported by Ding and Zhang.18 They have revealed that adding PEG as the macromolecular crowder lowers the LCST, obviously suggesting that macromolecular crowding modifies

H 2PO4− > F− > Cl− > Br − > NO3− > I− > ClO4− > SCN−

in which the anions on the left of Cl− are kosmotropes, whereas those on the right of Cl− are chaotropes. Such a specific-salt effect has also been found in many other kinds of physical behaviors, such as interfacial phenomena and phase transitions of surfactants.2−4 Although some of the salts in the Hofmeister series are ubiquitous chemical compounds, the effects of the salts on physical behavior remain unknown, to a large extent owing to a lack of molecular-level understanding of their mechanism of action. A characteristic feature of the interior of living cells is the crowded macromolecular environment. Although many biochemical experiments have been conducted in dilute solutions (in vitro) to understand biochemical processes such as protein folding and enzyme reactions, these do not adequately reflect the processes in vivo because macromolecular crowding in cells modifies the rates and the equilibria of the biochemical processes.5−7 Furthermore, a study of the effect of macromolecular crowding on the solubility of proteins in salt solutions has practical significance in biochemical experiments because macromolecular crowders such as polyethylene glycol © XXXX American Chemical Society

Received: February 6, 2015 Revised: July 21, 2015

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DOI: 10.1021/acs.jpcb.5b01255 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B the coil−globule transition of PNIPAM. Because macromolecular crowding alters the biochemical processes, the effect of salts on the coil−globule transition of PNIPAM is also expected to be influenced by macromolecular crowding. To the best of our knowledge, the influence of macromolecular crowding on the effect of specific ions on the coil−globule transition has not been studied for PNIPAM. In this article, we report the effect of specific monovalent Hofmeister salts on the coil−globule transition of PNIPAM in a crowded macromolecular environment. By studying the effect of the ions on the coil−globule transition of PNIPAM, an attempt has been made to understand the effect of the ions on the solubility and stability of proteins in a crowded macromolecular environment. We have revealed that macromolecular crowding alters the impact of specific monovalent ions on the coil−globule transition of PNIPAM.

Figure 1. Temperature dependence of the turbidity curves for PNIPAM (red) and PNIPAM + PEG (blue). The concentrations of PNIPAM and PEG were 5 and 300 mg/mL, respectively.

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absorbance in the curves. As seen in the figure, the values of Tcp in the solutions with and without PEG are 17.0 and 31.1 °C, respectively. The values of Tcp for the PNIPAM solutions containing salts of various monovalent ions were measured, and the differences in Tcp (ΔTcp) between the PNIPAM solutions in the presence and absence of the salts were calculated for the solutions with and without PEG. Hence, the relationships ΔTcp = Tcp - 17.0 (°C) and ΔTcp = Tcp - 31.1 (°C) were used for the PNIPAM solutions with and without PEG, respectively. The values of ΔTcp for the solutions with and without PEG are plotted in Figure 2 as a function of salt concentrations. In the cases of NaH2PO4, NaF, and NaCl (Figure 2), the solutions without PEG (indicated with circles) show a linear decrease in the transition temperature as a function of salt concentration. This behavior is consistent with the previous study.15 Furthermore, the values of ΔTcp in the solutions containing PEG (indicated with squares in Figure 2) also show a similar dependence on salt concentration. In the cases of chaotropes such as NaClO4 and NaSCN (Figure 2), ΔTcp shows a nonlinear dependence on salt concentration. In NaClO4 solutions without PEG, ΔTcp decreases steeply as the NaClO4 concentration increases. However, in NaSCN solutions, the dependence of ΔTcp on the NaSCN concentration is gradual for solutions without PEG (indicated by circles) in the concentration range from 0 to 1.0 M. Beyond a NaSCN concentration of 1.0 M, however, ΔTcp decreases steeply. We note that the trends for ΔTcp observed in the present study in the cases of NaClO4 and NaSCN solutions without PEG agree with the trend reported in the literature, where the behavior of ΔTcp was studied in a limited concentration range from 0 to 1.0 M.15 In addition, a similar steep decline in ΔTcp was reported for KSCN solutions without PEG at KSCN concentrations exceeding 1.0 M.19 It is also found that adding PEG into the NaClO4 and NaSCN solutions (indicated by squares in Figure 2) greatly influences the coil−globule transition. In both cases, the difference in ΔTcp between solutions with and without PEG increases with an increase in the salt concentration. For example, the differences in ΔTcp for the NaClO4 and NaSCN solutions with and without PEG are 3.7 and 0.4 °C, respectively, at 0.5 M and 23.3 and 17.4 °C, respectively, at 2.0 M. Cremer and co-workers demonstrated that the experimental values of ΔTcp for PNIPAM measured as a function of salt concentration can be fitted well with the equation shown below eq 1.15

EXPERIMENTAL METHODS PNIPAM (Mn ≈ 104) was purchased from Sigma-Aldrich. PNIPAM was purified by first dissolving it in acetone and then precipitating it in n-hexane three times. The purified PNIPAM was dried for 2 weeks under vacuum. NaH2PO4, NaF, NaCl, NaClO4, NaSCN, and polyethylene glycol 6000 (PEG 6000) were purchased from Wako Pure Chemical Industries and used as received. Ultrapure water (Wako) was used for preparing all of the solutions. The stock solution of PNIPAM (50 mg/mL) was stored for 2 days in a refrigerator (approximately 5 °C). For salt solutions without PEG, the salt and stock solutions were dissolved at the desired concentration. For salt solutions with PEG, the salt solution was prepared at the desired concentration, and then PEG and the stock solution were dissolved in the salt solutions. The final concentrations of PNIPAM and PEG were 5 and 300 mg/mL, respectively, for all of the experiments. A spectrophotometer (JASCO, V-650) was used for measuring the cloud-point temperature (Tcp) of PNIPAM during the heating process. The temperature of the solutions was controlled by a cell holder with a thermostat (JASCO, PAC-743R). The heating rate was 0.2 °C/min in all of the experiments. The temperature stability was within ±0.1 °C. 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS) was purchased from Tokyo Chemical Industries. The fluorescence spectra of 1,8-ANS were measured in PNIPAM solutions containing the salts (NaClO4 and NaSCN) or PEG by a fluorometer (JASCO, FP-750). The solutions were prepared with 1,8-ANS aqueous solutions (1 × 10−5 M) instead of pure water. Excitation wavelengths for the solutions are listed in Table S1. All of the fluorescence spectra in the PNIPAM solutions were measured at 13 °C. The fluorescence spectra of 1,8-ANS in 13 different kinds of solvents were also measured at 20 °C to examine the dielectric constant values of PNIPAM solutions containing the salts and PEG. The concentration of 1,8-ANS was 1 × 10−5 M in the solutions. The solvents used for the measurements are listed in Table S2.

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RESULTS AND DISCUSSION Although the PNIPAM solution was transparent below the LCST, it was clouded above the LCST due to the collapse and aggregation of PNIPAM. Figure 1 shows a comparison of the turbidity curves obtained as a function of temperature for 5 mg/mL PNIPAM aqueous solutions in the presence and absence of PEG. The value of Tcp was defined as the temperature corresponding to the onset of increase in B

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Figure 2. Difference in cloud-point temperatures (ΔTcp) of PNIPAM in salt solutions as a function of salt concentration. The squares and circles represent experimental data in the solutions with and without PEG, respectively. Dotted and solid lines are the results of fitting the experimental data to eq 1. The concentrations of PNIPAM and PEG are 5 and 300 mg/mL, respectively.

ΔTcp = c[M ] + Bmax [M ]/(KD + [M ])

Table 1. Values of c, Bmax, and KD Obtained by Fitting ΔTcp Measurements to Eq 1

(1)

In the above equation, c is a constant having the units of temperature/molarity, [M] is the molar concentration of the salts, Bmax has the unit of temperature, and KD is the equilibrium dissociation constant. Note that the KD value in eq 1 is an apparent dissociation constant because it is obtained from nonisothermal measurements. The first term shows a linear dependence of ΔTcp on the salt concentration, and the second term expresses the adsorption of the salts to PNIPAM, which is similar to that of the Langmuir isotherm. Bmax is, therefore, the coefficient that represents the change in ΔTcp induced by the salt adsorption. The experimental data shown in Figure 2 fit well with eq 1, and the fitting results are displayed by dotted and solid lines for the solutions with and without PEG in Figure 2. We found that the ΔTcp values for the NaH2PO4, NaF, and NaCl solutions could be fit well to the equation shown in eq 1 by assuming an infinitely large KD value, essentially implying that ignoring the second term in eq 1 allowed the experimental data to be fit well. Note that in the previous study, the first term was used to fit the data for the NaH2PO4, NaF, and NaCl solutions without PEG.15 The fit parameters obtained are summarized in Table 1. From Table 1, it can be seen that the values of c are very similar to each other for the NaH2PO4, NaF, and NaCl solutions with and without PEG, respectively. From these results, it can be concluded that macromolecular crowding had little effect on the linear dependence of ΔTcp on NaH2PO4, NaF, or NaCl concentrations in the solutions. These similarities imply that the salts (NaH2PO4, NaF, and NaCl) and the macromolecular crowder lower the temperature of the coil− globule transition almost independently. It can also be seen from Table 1 that the constant Bmax showed positive values for the chaotropic solutions (NaClO4

agenta

c (°C/M)

Bmax (°C)

KD (M)

NaH2PO4 NaH2PO4 + PEGb NaF NaF + PEGb NaCl NaCl + PEGb NaClO4 NaClO4 + PEGb NaSCN NaSCN + PEGb

−29.5 −30.6 −23.7 −22.3 −12.2 −12.9 −22.8 −27.0 −20.4 −22.7

― ― ― ― ― ― 24.5 554 56.4 559

― ― ― ― ― ― 1.33 21.6 1.81 19.8

a

PNIPAM concentration was 5 mg/mL. bPEG concentration was 300 mg/mL.

and NaSCN), implying that the adsorption of the chaotropes on PNIPAM raises the transition temperature (Figure S1). Such a behavior has been well-established for chaotropic anions previously.15 Table 1 also suggests that macromolecular crowding exerted a great influence on the adsorption properties of the chaotropes on PNIPAM, leading to a drastic modification in the transition temperature. The KD values for the NaClO4 and NaSCN solutions with PEG were approximately 16 and approximately 11 times larger than those without PEG, respectively. In addition, the Bmax values for the NaClO4 and NaSCN solutions with PEG were approximately 23 and approximately 9.9 times larger than those without PEG, respectively. The affinity of the chaotropes for PNIPAM was, therefore, smaller in the crowded environment. However, once the chaotropes were bound to PNIPAM, their influence on the transition temperature became significant. In contrast, the values of constant c for the NaClO4 and NaSCN solutions with C

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The Journal of Physical Chemistry B PEG were 1.2 and 1.1 times smaller than those without PEG, respectively. Compared to those in the KD and Bmax values, the differences in the c values were not significant, implying that the impact of macromolecular crowding on the linear dependence of ΔTcp is small. Recently, Okur et al. carried out attenuated total reflection Fourier transform spectroscopy measurements and reported a shift of the amide I band for d-butyramide in salt solutions.20 They found that Na+ was excluded from the oxygen atom of the amide group. Pluhařová et al. also performed ab initio molecular dynamics (MD) simulations to understand the binding behavior of Na + to the amide group of Nmethylacetamide21 and found that Na+ shows weak interactions with the amide oxygen. Accordingly, state-of-the-art experimental and theoretical studies demonstrate that the affinity of Na+ to the amide group is negligibly small. Indeed, Langmuirtype binding behavior appears on the ΔTcp curves only in the case of the chaotropic solutions (Figure 2). This finding implies that, as shown in the previous study,15 the adsorption of chaotropic anions (ClO4− and SCN−) on PNIPAM brings about the binding behavior on the ΔTcp curves. It has been proposed in previous studies that chaotropic anions are bound to the nitrogen atom of the amide group of PNIPAM because its resonant structure imparts a partial positive charge to the amide nitrogen.15,17,19 In another study, thermodynamic LCST measurements, proton NMR experiments, and molecular dynamics simulations have been employed to investigate the ion binding sites on elastin-like polypeptides.22 According to this study, chaotropic anions such as SCN− and I− interact with the amide nitrogen and the adjacent α-carbon on the peptide backbone. Furthermore, in this study, it was found that the chemical shifts of the methyl proton in poly(N, N-dimethylacrylamide) vary in a nonlinear fashion as a function of NaSCN concentration, whereas the nonlinearity in chemical shifts is much less prominent for the amide proton in poly(acrylamide). Such a result clearly indicates that the binding of SCN− on polymers that contain amide groups involves an aliphatic group adjacent to the amide nitrogen. These findings may also be valid for other kinds of polymers that contain amide groups. Thus, chaotropic anions such as ClO4− and SCN− may also be bound to the amide nitrogen and the adjacent carbon sites on PNIPAM. To elucidate the major factors that modify the adsorption properties of ClO4− and SCN− in a crowded macromolecular environment, we measured the fluorescence spectra of 1,8-ANS in various solutions. It has been well established that in hydrophobic environments, the fluorescence quantum yield of 1,8-ANS increases.23 Figure 3 shows the fluorescence spectra of 1,8-ANS in PNIPAM solutions. We note that in PNIPAM solutions, PNIPAM-rich or PNIPAM-poor microdroplets emerge above the LCST, owing to liquid−liquid phase separation.24 It has been shown that in some protein solutions containing PEG, the two inhomogeneous phases caused by liquid−liquid phase separation contain different amounts of PEG, leading to PEG-rich and PEG-poor phases.25−27 Therefore, in PNIPAM solutions, PEG may preferentially partition to one phase over the other above the LCST when PEG is added to the solutions, leading to an inhomogeneous PEG concentration in the solution. To examine hydrophobicity in homogeneous solutions, we measured the fluorescence spectra at 13 °C, which is below the LCST of all the solutions. As shown in Figure 3, although the addition of NaClO4 or NaSCN hardly changed the fluorescence of 1,8-ANS in

Figure 3. Fluorescence spectra of 1,8-ANS in PNIPAM solutions (black), PNIPAM and PEG solutions (green), and PNIPAM and salt solutions with and without PEG (red and blue, respectively). All spectra were measured at 13 °C, which is below the LCST of PNIPAM for all the solutions. Raman scattering from water is observed around 410−420 nm.

comparison with the results from PNIPAM solutions without the salts (blue and black curves), the fluorescence intensity of 1,8-ANS in the PNIPAM solutions became much stronger in the presence of PEG (green and red curves). Indeed, the fluorescence intensity was strong even in a solution containing only PEG without PNIPAM and salts (Figure S2). This observation clearly suggests that the addition of the macromolecular crowder renders the solutions hydrophobic. The fluorescence spectra of 1,8-ANS were also measured in 13 different kinds of solvents at 20 °C. In Figure 4, the

Figure 4. Dielectric constants of solvents as a function of the wavelength corresponding to the 1,8-ANS fluorescence peak. The solid line is the result of a linear fit of the experimental data.

dielectric constants of the solvents are plotted as a function of the wavelength corresponding to the fluorescence peaks of 1,8ANS. Each data point on the plot corresponds to a certain solvent. From the plot, it can be seen that the peak wavelength is blue-shifted linearly as the dielectric constant decreases. The fluorescence peaks of 1,8-ANS are observed at approximately D

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activity coefficient becomes small in solutions having low dielectric constant values.30 This is consistent with the fact that macromolecular crowding decreases the dielectric constant of the solutions (Figure 4). We note, however, that the magnitudes of decrease in the dielectric constant values for NaClO4 and NaSCN solutions containing PEG are not so pronounced (31−34%) to make the activity coefficient a dominant factor causing the larger apparent KD values. In a PEG solution without salts, PEG lowers the LCST of PNIPAM from 31.1 to 17.0 °C (Figure 1), probably due to the excluded volume effect, i.e., the excluded volume around PNIPAM, where the centers of PEG are excluded, decreases so as to increase the translational entropy of PEG.31 Previous studies have demonstrated that Na+ is bound to the oxygen atoms of PEG in salt solutions.32−36 The adsorption of Na+ creates a net positive charge on PEG, leading to an expansion or collapse of PEG depending on the salt concentration. Therefore, in the salt solutions containing PEG, the effect of PEG on the LCST of PNIPAM would be modified because the change in the PEG size modifies the excluded volume. This would lead to a modification of the ΔTcp curve in the NaClO4 and NaSCN solutions with PEG. A similar modification would also be expected for the ΔTcp curve in the NaH2PO4, NaF, and NaCl solutions containing PEG because Na+ is bound to PEG in these solutions also. However, the ΔTcp curves obtained in their solutions with and without PEG are very similar to each other (Figure 2), implying that the adsorption of Na+ on PEG causes only a negligible change in the effect of PEG on the LCST. According to a previous study, the decrease in the LCST of PNIPAM is dependent on the molecular weight of PEG.18 However, the difference in LCST is very small for solutions containing PEG with molecular weights of 3750 and 10750. Thus, the expansion and collapse of PEG induced by the adsorption of Na+ may have a minor effect on the LCST. As mentioned above, PNIPAM and PEG can be regarded as apparent polyelectrolytes in the chaotropic solutions because the anions (ClO4− or SCN−) and cations (Na+) are adsorbed on PNIPAM and PEG, respectively. The polymers having opposite charges may interact with each other on electrostatic grounds, leading to the modification of the LCST. Unfortunately, at the present stage, it is difficult to address the effect of the electrostatic interactions among PNIPAM and PEG on the LCST quantitatively. The difference in the electrostatic interaction in the coil and globular states of PNIPAM may modify the enthalpy difference between them. In addition, the electrostatic interaction between PNIPAM and PEG may reduces the translational entropy of PEG because the electrostatic interaction attracts PEG to PNIPAM, which may restrict the volume in which PEG can move. The subtle balance of the enthalpy and entropy differences induced by the electrostatic interaction may modify the LCST of PNIPAM. The physical origins of the coil−globule transition in PNIPAM have not been fully understood yet. One of the most important findings is, however, that the transition is endothermic.18,37 Therefore, it is evident that the transition is driven by entropy even though the globular states of PNIPAM obviously lose the conformational entropy. The next obvious question would be to identify the major contributor to the entropic gain and determine how it gains its entropy. Recently, Kinoshita and co-workers have developed the angle-dependent integral equation theory combined with the morphometric approach to clarify the driving force of protein folding.38,39 Proteins have excluded volumes within which the centers of

500 and approximately 499 nm for the NaClO4 and NaSCN solutions containing PNIPAM and PEG (Figure 3), and the corresponding dielectric constant values of the solutions are approximately 55 and approximately 53, respectively. These values are 31−34% lower than that of water (see also Table S2). On the basis of the fluorescence experiments, we hypothesize that the reasons for the larger KD values observed in the NaClO4 and NaSCN solutions containing PEG compared to those of the corresponding solutions without PEG are as follows. Large anions such as ClO4− and SCN− are weakly hydrated because of their small charge densities. In fact, large anions have a certain amount of hydrophobicity.28,29 Owing to their hydrophobic nature, the large anions prefer a hydrophobic environment. On the basis of the results of the fluorescence experiments, it is evident that macromolecular crowding makes the solutions hydrophobic. Therefore, the crowded environment may lower the free-energy values of the solvated large anions that do not bind to PNIPAM, leading to larger KD values in the crowded environment. Furthermore, the fluorescence experiments also indicate that macromolecular crowding decreases the dielectric constants of the solutions. In a solution with a low dielectric constant, electrostatic interactions among charges are not efficiently shielded. PNIPAM resembles a polyelectrolyte when a certain amount of anions (ClO4− or SCN−) is adsorbed onto it. Solvated anions, which do not bind to PNIPAM, experience more prominent electrostatic repulsion from the charged PNIPAM in solutions with PEG than in the solutions without PEG. Therefore, it is difficult for the solvated anions to bind to the charged PNIPAM in the crowded environment. This results in a free-energy cost for the adsorption of the anions on PNIPAM and consequently, larger KD values in the crowded environment. Note that the KD values are related to a free-energy difference (ΔG) caused by the adsorption and desorption of the anions (ΔG = kBT·ln(KD), where kB is the Boltzmann constant). As mentioned above, the KD values for the NaClO4 and NaSCN solutions with PEG were approximately 16 and approximately 11 times larger than those without PEG, respectively (Table 1). Although the KD values are the apparent dissociation constants obtained from the nonisothermal measurements, the differences in ΔG (i.e., ΔΔG) for the NaClO4 and NaSCN solutions in the absence and presence of PEG is estimated to be approximately 2.8kBT and approximately 2.4kBT, respectively, from the apparent KD values. This implies that the free-energy cost for the adsorption of the anions in the crowded environment is a few times larger than thermal energy. Another plausible explanation for the larger KD values in the crowded environment is as follows. In eq 1, the molar concentration of the salts ([M]) is used to fit the experimental data. However, it is well-known that strong electrolyte solutions exhibit more significant nonideal behaviors than nonelectrolyte solutions, owing to the strong interionic interactions.29 The nonideality of the electrolyte solutions is usually expressed by the mean ionic activity coefficient. When we take into account the nonideality of the salts in the solutions, the apparent KD value in eq 1 may be expressed by the ratio of the thermodynamic equilibrium dissociation constant to the mean ionic activity coefficient. Therefore, the larger apparent KD values in the crowded environment suggest that the activity coefficients of the salts in the solutions containing PEG may be smaller than those in the solutions without PEG. In fact, on the basis of the Debye−Hückel theory or its extended ones, the E

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networks is filled by water molecules whose hydrogen bonds are disrupted or weakened. Therefore, the entropic gain by the excluded volume effect is favorable in this region because the number of water molecules gaining the translational entropy increases due to the larger water density, which causes the transition temperature to be lowered. According to this scenario, macromolecular crowding may not alter the effect of salts on the water structure so much, resulting in similar c values in the solutions with and without PEG. Obviously, further study has to be carried out to study the validity of the scenarios proposed in this study.

water molecules cannot enter. When protein folding occurs, the excluded volumes overlap, giving rise to the reduction of the excluded volumes for water. Therefore, the total volume in which the water molecules can move increases, leading to a gain in the translational entropy of water. The theory shows the primary importance of the effect of the excluded volume for water on the protein folding. This scenario may be applied to the explanation for the coil− globule transition of PNIPAM and, in particular, the difference in the Bmax values in the chaotropic solutions with and without PEG (Table 1). In this scenario, PNIPAM minimizes the excluded volume for water to gain the translational entropy when the transition occurs. In the chaotropic solutions, however, ClO4− or SCN− adsorbs on PNIPAM, causing electrostatic repulsion among the anions when the transition occurs. Thus, when the transition occurs, PNIPAM reduces the excluded volume with the constraint that the electrostatic repulsion is also small. In this case, the decreased amount of the excluded volume is small due to the constraint. Raising the transition temperature compensates for the unfavorable entropic penalty, leading to the positive Bmax values. Moreover, in the crowded environment, the electrostatic repulsion among the anions is more prominent because the dielectric constant of the solution is small. This makes the constraint of reducing the excluded volume strict, giving rise to a further increase in the transition temperature, and a larger Bmax value in the crowded environment. A free-energy gain arising from the entropy term is expressed as TΔS. The Bmax values correspond to an increase in the transition temperature when the adsorption of the anions is saturated. Based on the Bmax values (Table 1), the transition temperature rises to 844 and 849 K for the NaClO4 and NaSCN solutions with PEG, respectively. These values are approximately 2.6 and approximately 2.4 times larger than those for the solutions without PEG (329 and 360 K, respectively). Accordingly, the entropic gains (ΔS) and the decreased amount of the excluded volumes for the NaClO4 and NaSCN solutions with PEG may be approximately 2.6 and approximately 2.4 times smaller than those for the solutions without PEG when the adsorption of the anions is saturated. Another intriguing finding in our study is that the impact of macromolecular crowding on the values of constant c is not so prominent (Table 1). In the previous study, a relationship between the values of constant c and the surface tension increase of anions was found.15 In line with this finding, macromolecular crowding may have little influence on the surface tension increase of anions, leading to the similar c values in the salt solutions with and without PEG. In addition, recent experimental and theoretical findings may provide a complementary explanation. There is an ongoing debate on whether the Hofmeister ions alter the structure of water beyond their first solvation shell.4 Some of experimental results insist that the effect of the ions on the water structure is limited to their first solvation shell.40 However, recently, some research groups have shown experimental and theoretical evidence that suggest that the effect spans beyond the first solvation shell.3,41 In particular, Tsubouchi and co-workers have published an experimental work on terahertz time-domain spectroscopy.42 They found experimental evidence showing that dissolved ions exhibit the structure breaking effect beyond the first hydration shell, in which the hydrogen bonds of water are disrupted (the so-called region B of the Frank−Wen model).43 In this region, the density of water may increase because the void space formed by the hydrogen-bonded

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CONCLUSIONS In summary, we found that macromolecular crowding alters the effect of specific monovalent ions on the coil−globule transition of PNIPAM. Although adding the salts (NaH2PO4, NaF, and NaCl) and the macromolecular crowder (PEG) lowers the transition temperature independently, macromolecular crowding modifies the influence of chaotropes (NaClO4 and NaSCN) on the transition temperature significantly. The adsorption of chaotropic anions (ClO4− or SCN−) on PNIPAM raises the transition temperature because the electrostatic repulsion between the adsorbed anions reduces the entropic gain for water associated with the excluded volume effect. The influence of electrostatic repulsion on the excluded volume is more prominent in a crowded environment having low dielectric constants, giving rise to a further increase in the transition temperature of the chaotropic solution. In addition, the affinity of the anions for PNIPAM is much smaller in the solutions with PEG than in the solutions without PEG (i.e., larger apparent KD values in the crowded environment), suggesting that the hydrophobic environment of the solutions containing PEG stabilizes the solvated chaotropic anions. This results in further modification of the transition temperature. An understanding of the effect of the Hofmeister ions on the coil−globule transition of PNIPAM in the crowded macromolecular environment has provided new insights into the solubility and stability of proteins under physiological conditions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b01255. Figures and supplementary information regarding the residual parts of ΔTcp after the removal of the linear portion of ΔTcp for the NaClO4 and NaSCN solutions in the presence of PEG and the fluorescence spectrum of the 1,8-ANS solution with PEG. Tables and supplementary information regarding the excitation wavelengths for the fluorescence spectra and 13 different solvents used for measuring the fluorescence spectra of 1,8-ANS. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by Grants-in-Aid for Scientific Research C (no. 25410022), Scientific Research B F

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The Journal of Physical Chemistry B

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(no. 26288010), and Scientific Research on Innovative Area (no. 26104527).

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DOI: 10.1021/acs.jpcb.5b01255 J. Phys. Chem. B XXXX, XXX, XXX−XXX