Hofmeister Anion Effects on Protein Adsorption at an Air–Water

Article pubs.acs.org/Langmuir

Hofmeister Anion Effects on Protein Adsorption at an Air−Water Interface Yohko F. Yano,*,† Yuki Kobayashi,† Toshiaki Ina,‡ Kiyofumi Nitta,‡ and Tomoya Uruga‡ †

Department of Physics, Kindai University, 3-4-1 Kowakae, Higashiosaka City, Osaka 577-8502, Japan Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cyo, Sayo-gun, Hyogo 679-5198, Japan

‡

S Supporting Information *

ABSTRACT: Hofmeister anion effects on adsorption kinetics of the positively charged lysozyme (pH < pI) at an air−water interface were studied by surface tension measurements and time-resolved X-ray reflectometry. In the salt-free solution, the protein adsorption rate increases with decreasing the net positive charge of lysozyme. When salt ions are dissolved in water, the protein adsorption rate drastically increases, and the rate is following an inverse Hoffmeister series (Br− > Cl− > F−). This is the result of the strongly polarized halide anion Br− being attracted to the adsorbed protein layer due to strong interaction with local electric field, while weakly polarized anion F− having no ability to penetrate the protein layer. In X-ray reflection studies, we observed that the lysozyme molecules initially adsorbed on the air−water interface have a flat unfolded structure as previously reported in the salt-free solution. In contrast, in the concentrated salt solutions, the lysozyme molecules begin to refold during adsorption. This protein refolding as a result of protein−protein rearrangements may be a precursor phenomenon of crystallization. The refolding is most significant for Cl−, which is a good crystallization agent, whereas it is less observed for the strongly hydrated F−. It is widely known in the bulk state that kosmotropic anions tend to precipitate proteins but at the same time stabilize proteins against denaturing. On the other hand, at the air−water interface where adsorbed proteins usually unfold, we observed chaotropic anions strongly bound to proteins that reduce electrostatic repulsion between protein molecules, and subsequently they induce protein refolding whereas the kosmotropic anions do not.

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lysozyme isoelectric point (pI ≈ 11). They found an inverse anion series at low salt concentrations and a direct series at high salt concentrations. As reported by Schwierz et al.,11 a partial or total inverse in the series can be ascribed to the surface polarity and the charge, i.e., the direct anionic Hofmeister series takes place at negatively charged hydrophobic surfaces, whereas reversal takes place at positively charged hydrophobic surfaces or at negatively charged hydrophilic surfaces. Proteins generally adsorb on an air−water interface as amphipathic molecules, and they dramatically reduce the surface tension.15 The surface adsorption of amphipathic molecules is essentially caused by the strong hydrogen bond network in the solvent water, i.e., covering the water surface with solute having weaker interactions to reduce the surface energy of the bare water. Previously, we found the assembly of amphipathic molecules at the air−water interface strongly correlates with that in the bulk state.16 On the other hand, adding salt ions to water increases the surface tension of water. The magnitude of the surface-tension increment of the salt has been known to follow the rank order

INTRODUCTION Protein solubility is a macroscopic property resulting from various molecular interactions lying in the bulk solution and depends on temperature, pressure, pH of the solution, and additives. The effect of salts on the solubility of proteins is classified by the well-known Hofmeister series, defined by the concentration of various ions needed to precipitate a mixture of egg-white proteins.1−6 Hofmeister series for the relative efficiency of sodium salts in precipitation of egg-white protein HPO4 3 − > SO4 2 − > F− > Cl− > Br − > NO3− > I− > ClO4 − > SCN−

The Hofmeister ions are usually categorized as chaotropes or kosmotropes based on their perceived influences on water structure.7−9 The ions on the left side of the series, defined as kosmotropes, exhibit strong interactions with water molecules, whereas the ions on the right side of the series, defined as chaotropes, are weakly hydrated by water molecules. Although the relative position of the species in the series has been demonstrated to be almost universal, inverse Hofmeister sequences have been observed in many systems.10−14 Zhang and Cremer10 measured the cloud point temperature of oversaturated lysozyme suspensions at pH 9.4 below the © XXXX American Chemical Society

Received: June 24, 2016 Revised: August 21, 2016

A

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Langmuir of the Hofmeister series.17−19 Several recent studies20−22 looking at interfaces have revealed that the propensity for salt anions to adsorb at an air−water interface follows an inverse Hofmeister series, i.e., strongly hydrated kosmotropic anions effective in precipitating proteins do not adsorb at the air− water interface and vice versa. These results suggest that studying the surface adsorption of proteins in a salt solution could provide valuable insight into the protein precipitation. Previously, we examined the adsorption process of hen eggwhite lysozyme in a concentrated NaCl solution at various pH using time-resolved X-ray reflectometry to investigate the salting-out phenomenon at the air−water interface.23,24 We observed that the lysozyme initially adsorbed on the air−water interface with unfolded configuration is gradually refolding during adsorption. Such refolding is not observed either at the isoelectric point or in the salt-free solution, suggesting that it is induced by screening of the positive charges in the lysozyme by chloride ions. In the present study, we examine the protein adsorption process in various Hofmeister salt solutions to elucidate interactions between positively charged lysozyme and Hofmeister anions.

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Figure 1. Time dependence of surface tension changes. Lysozyme injected into salt-free solution at various pH values (a) and into 1 M NaX (X = F, Cl, Br, I) solutions at pH 7 (b).

the air−water interface was saturated to form an interfacial layer during this period. When the interface was fully covered, LSZ molecules try to penetrate into the interfacial layer. They affect the surface tension less. The rate of the adsorption process can be analyzed by29 ⎛ Δγ(t ) ⎞ ⎟⎟ = −kt ln⎜⎜1 − Δγe ⎠ ⎝

EXPERIMENTAL METHODS

(1)

where Δγ(t) and Δγe are the surface tension changes at time t and in the equilibrium state, respectively, and k is the rate constant. The rate constant k, therefore, can be estimated from the slope of ln(1 − Δγ(t)/Δγe) versus t. Two linear parts are observed in this plot as shown in Figure 2. We derived the rate constant of the diffusion kd from the slope in the region of t < 100 s and the rate constant of the penetration kp from the slope in the region of t > 100 s.

Materials. The globular protein, lysozyme (hereafter, LSZ), is elliptical in shape with approximate dimensions 30 × 30 × 45 Å3. It is regarded as a rigid molecule due to the presence of four disulfide bridges. The isoelectric point is 11.35. Three-times crystallized and lyophilized hen egg-white lysozyme was purchased from Sigma (prod. no. L6876) and used as supplied. Protein solutions were made using phosphate buffer solutions (0.02 M NaH2PO4/Na2HPO4) of pH 3, 7, or 11 and a sodium acetate buffer solution of pH 4 using UHQ-grade water. Protein solutions were made to concentrations of 43 mg/mL, from which 1 cm3 portions were added to a 42 cm3 buffer solution with NaX (X = F, Cl, Br, I) in a Langmuir trough25 to give final concentrations of 1 mg/mL. Note that the saturated solution of NaF (0.97 M) will be denoted as 1 M in the following section. Methods. The X-ray reflectivity measurements were performed using a liquid-interface reflectometer at the undulator beamline BL37XU of SPring-8.26 The time-resolved X-ray reflectivity measurements were started when a LSZ solution in a syringe was automatically injected into a buffer solution contained in a Langmuir trough. The Langmuir trough was mounted on a heat sink, and the temperature was controlled at 25 ± 1 °C. The position of the X-ray beam on the sample was changed horizontally at intervals of 1 s to avoid radiation damage. The surface tension was monitored throughout using a Wilhelmy plate made of platinum mounted on the Langmuir trough. Data Analysis. The X-ray reflectivity data were fitted using a threeslab (or four-slab for the 1 M NaI solution) model with Parrat32 software,27 taking parameters of the film thickness, the electron densities, and the roughness of the three slabs. As shown in the Supporting Information, at least three slabs are necessary to explain the time evolution of the X-ray reflectivity data.

Figure 2. ln(1 − Δγ(t)/Δγe) as a function of time for lysozyme injected into 1 M NaX (X = F, Cl, Br, I) solutions at pH 7.

Figure 3 shows the pH dependence of rate constants of kd and kp. The rate constants for the salt-free solution increase

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RESULTS Surface Tension. Figure 1(a) shows the dynamic surface tension in the absence of added salt at various pH values. The surface tension decay increased with increasing pH. The rate of protein adsorption can be inferred from these tension decays. The adsorption rate is found to be strongly affected by the net positive charge of LSZ, faster at lower net charge. When a salt is dissolved in water, the surface tension decays much faster (Figure 1(b)) as a result of salt anions shielding the positive charge of the LSZ.28 The surface tension becomes steady state within 100 s, suggesting that the adsorbed amount of LSZ at

Figure 3. Rate constants vs pH for lysozyme in buffer solutions of 1 M NaX (X = F, Cl, Br, I). (a) Diffusion rate kd. (b) Penetration rate kp. B

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Langmuir with increasing pH. For the salt solutions, kd and kp decrease with increasing pH, indicating that the number of ions associated with LSZ decreases as the pH approaches the isoelectric point. The magnitude of these rate constants almost follows an inverse Hofmeister series. For NaI at pH 4, the smallest kd is obtained because a white precipitate was formed instantaneously after LSZ injection. Furthermore, the value of kp is much larger than those of other anions. Unlike the other anions, the iodide ion attracts the hydrophobic residues of LSZ.30−32 The intermolecular interaction of LSZ molecules with iodide ions becomes much more attractive, and subsequently the diffusion rate decreases due to the LSZ aggregates. On the other hand, the penetration rate increases drastically because the LSZ molecules can penetrate into the interfacial layer more easily. The rate constants increase with an increase in the concentration of the salts as shown in Figure 4.

protein−anion interaction for NaI is different from that of other anions. X-ray Reflectivity. Figures 5−8(a,b) show the time dependence of the intrinsic structure factor |Φ(qz)|2 for LSZ injected into buffer solutions at pH 7. They are given by the Xray reflectivity data divided by the Fresnel reflectivity and capillary wave contribution.33−35 The time dependence intrinsic structure factors have the following features: the broad peak at around qz = 0.2 Å−1 observed every |Φ(qz)|2 originates from a LSZ monolayer 20 Å thick, the small peak at qz < 0.1 Å−1 which is increased during adsorption originates from a LSZ second layer. Furthermore, the decrease in intensity at qz > 0.3 Å−1 indicates that the density of the topmost layer decreases during adsorption as already observed previously.36 Such intensity decreases are observed significantly for the solutions with higher salt concentrations. Figures 5−8(c,d) show the time dependence of the electron density profiles obtained by the refinement. The gray area in each figure corresponds to the profile of the native configuration for which the LSZ molecule is oriented with its long axis parallel to the air−water interface (side-on).37 In all conditions, the electron density profiles for the initially adsorbed LSZ are highly distorted as compared to the native configuration. This is the result of the interface-induced denaturation occurring at hydrophobic interfaces such as the air−water interface.24 For the 0.5 M salt solutions, the LSZ molecules initially unfold and lay flat on the surface to form a monolayer, and subsequently, LSZ molecules gradually adsorb underneath the first layer to form a multilayer, with a lower density molecular configuration. However, the density profiles of the topmost layer are almost unchanged during the adsorption process. For the 1 M salt solutions, however, the electron density profile for the topmost layer gradually decreases during the adsorption process (Figures 6−8(d)) and approaches that of the native structure 2 h after injection. This behavior was also previously observed for 2 M NaCl

Figure 4. Rate constants vs salt concentration dependence of lysozyme in buffer solutions of 1 M NaX (X = F, Cl, Br, I) at pH 7. (a) Diffusion rate kd. (b) Penetration rate kp.

At a low salt concentration, the rate constants of NaI are the largest, whereas at a high salt concentration, the rate constants of NaBr are the largest, also supporting that the origin of the

Figure 5. Left: Time dependence of the intrinsic structure factor |Φ(qz)|2 for LSZ injected into NaF buffer solutions at pH 7. Refined |Φ(qz)|2 profiles are shown as continuous lines. Right: Time dependence of electron density profiles obtained by the refinement. The gray area in each figure represents the profile of native LSZ with a side-on orientation taken from ref 37. C

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Figure 6. Left: Time dependence of the intrinsic structure factor |Φ(qz)|2 for LSZ injected into NaCl buffer solutions at pH 7. Refined |Φ(qz)|2 profiles are shown as continuous lines. Right: Time dependence of electron density profiles obtained by the refinement. The gray area in each figure represents the profile of native LSZ with a side-on orientation taken from ref 37.

Figure 7. Left: Time dependence of the intrinsic structure factor |Φ(qz)|2 for LSZ injected into NaBr buffer solutions at pH 7. Refined |Φ(qz)|2 profiles are shown as the continuous lines. Right: Time dependence of electron density profiles obtained by the refinement. The gray area in each figure represents the profile of native LSZ with a side-on orientation taken from ref 37.

solutions at pH 3 and 7,23 whereas it was not observed for 2 M NaCl solutions at pH 12 and 1 M salt solutions at pH 11 (see Figure S2). Note that the adsorption amount of LSZ becomes constant when the unfolded lysozyme molecules are refolding (see Figure S3). Time dependence of the excess maximum electron densities, in which the bulk electron densities are subtracted from the maximum electron densities, are plotted in Figure 9. The values for 0.5 M salt solutionss decrease les than those for 1 M. Except for NaF, the excess maximum electron densities in the 1 M salt

solutions approach that of the native structure. We conclude that the LSZ molecules are refolding in the concentrated salt solutions except for NaF. The LSZ refolding is a result of anions shielding the positive charge of LSZ,38,39 highly influenced by the protein−salt interactions. The degree of LSZ refolding increases in the order F− < Br− ≈ I− < Cl−.

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DISCUSSION The surface tension, i.e., the surface free energy per area, can be regarded as the work in bringing a molecule from the interior of D

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Figure 8. Left: Time dependence of the intrinsic structure factor |Φ(qz)|2 for LSZ injected into NaI buffer solutions at pH 7. Refined |Φ(qz)|2 profiles are shown as the continuous lines. Right: Time dependence of electron density profiles obtained by the refinement. The gray area in each figure represents the profile of native LSZ with a side-on orientation taken from ref 37.

Figure 9. Time dependence of excess maximum electron densities for LSZ injected into salt solutions at pH 7. For the concentration of 1 M, the excess maximum electron densities, except for NaF, approach that of the native structure (denoted by the dashed-dotted line). Figure 10. Schematic diagram of the observed surface tension of pure water (A), salt solutions (B), LSZ in the salt-free solution (C), and LSZ in the salt solutions (D).The parameter zp is the net positive charge of LSZ.

a liquid to the surface and is strongly affected by molecular interactions. The observed surface tension can be divided into water−water (ww), salt−water (sw), protein−water (pw), protein−salt (ps), salt−salt (ss), and protein−protein (pp) contributions: γ = γww + Δγsw + Δγpw + Δγps + Δγss + Δγpp

where the enzymatic activity of LSZ reaches its maximum.41 It is known that a protein with higher conformational stability exhibits lower adsorption rate.15 Additionally, the repulsive protein−protein interaction inhibits the protein adsorption, and consequently it contributes to suppress the surface tension decrease (Δγpp > 0 (C)). On the other hand, when protein molecules are dissolved in salt solutions, the surface tension decrease is enhanced as a result of binding anions to produce net neutral proteins (Δγps < 0 (D)). This is analogous to cationic surfactant solutions in the presence of salt anions in which strongly polarized halide anions such as Br− or I− are attracted to the interface due to strong interaction with the local electric field, while weakly polarized ones (F−, Cl−) have no ability to penetrate the interfacial layer.42 Subsequently, the attractive interaction

(2)

Figure 10 shows a schematic diagram of the observed surface tension of pure water (A), salt solutions which are estimated using the previous data19 (e.g., the largest surface tension increment of 1.8 mN/m for 1 M NaF solution and the smallest value of 1.0 mN/m for 1 M NaI solution) (B), a protein in the salt-free solution (C), and a protein in the salt solutions (D). As we know, salt ions increase the surface tension of water (Δγsw > 0 (B)), whereas proteins decrease (Δγpw < 0 (C)). However, the protein adsorption rates are found to be strongly affected by the protein net charge, faster at lower net charge. This is opposite to the diffusion rate of LSZ in the bulk solution which increases with decreasing pH.40 It is known that the conformation of LSZ is most stable in the range of pH 4−6, E

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concentrated NaX (X = F, Cl, Br, I) solutions, the initial surface tension decay became much faster. The rate constants of LSZ obtained from the initial decay followed an inverse Hoffmeister series. This is the result of the binding of anions to produce net neutral LSZ. Weakly hydrated anions bind directly to proteins. This effect is attributed to the formation of insoluble LSZ−anion complexes, which crystallize. For the concentrated salt solutions, the LSZ molecule initially unfolds and lays flat on the surface and subsequently refolds during the adsorption process. The degree of LSZ refolding increases in the order F− < Br− ≈ I− < Cl−. This protein refolding may be a precursor phenomenon of the crystallization.

between anion-binding proteins (denoted by p′) contributes to enhance the surface tension decrease in contrast to those without added salt (Δγp′p′ < 0 (D)). The protein−water interaction in the salt solutions is weaker than that in the saltfree solution because the solubility of LSZ decreases with the increase in salt concentration.43 The weakened protein−water interaction enhances the protein adsorption (Δγp′w < Δγpw < 0). Ultimately, the diffusion rate kd is mostly affected by the protein−water interaction (the weaker interaction increases kd), whereas the penetration rate kp is affected by the protein− protein interaction (the stronger interaction increases kp). Both of the rate constants follow an inverse Hofmeister series. In the X-ray reflection studies, we observed the growth and rearrangement of the interfacial protein layer. The LSZ molecule initially adsorbed on the air−water interface has a flat unfolded structure. In contrast, in the concentrated salt solutions, the LSZ molecules begin to refold during adsorption. Although the protein refolding does not affect the surface tension, the degree of LSZ refolding estimated from the electron density profile increases in the order F− < Br− ≈ I− < Cl−. The strongly hydrated F− ions do not assist LSZ refolding. It is widely known that kosmotrope anions tend to precipitate proteins at the same time stabilizing proteins against denaturation in the bulk state (salting-out).8 At the air−water interface, we observed that chaotropic anions strongly bound to proteins reduce the electrostatic repulsion between protein molecules, and subsequently they induce protein refolding associated with a decrease in the solvent-accessible surface area of protein (Figure 11(b)), whereas the kosmotropic anions do not (Figure 11(a)).

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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.langmuir.6b02352. Details of the profile fitting of X-ray reflectivity, the electron density profiles for LSZ injected into 1 M salt solutions at the isoelectric point, and the surface excess of LSZ (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone +81-6-6721-2332, ext. 4088. Fax +81-6-6727-4301. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The synchrotron radiation experiments were performed at the BL37XU beamline at the SPring-8 facility with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal nos. 2012A1252, 2012B1197, 2013A1278, 2014A1195, 2014B1216, and 2015B1191). Y.Y. acknowledges financial support from a Grant-in-Aid for Scientific Research (no. 22018028) in Priority Area “Molecular Science for Supra Functional Systems” and a Grant-in-Aid for Scientific Research (C) (no. 24540444) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Figure 11. Anion adsorption onto LSZ at the air−water interface.

Lysozyme often crystallizes in the presence of weakly hydrated anions such as Cl− or thiocyanate but only with difficulty from sulfate or phosphate.32,39 The protein refolding observed at the air−water interface may be a precursor phenomenon of the crystallization. Protein crystallization is widely predicted by protein−protein osmotic second-virial coefficient B22, which is a direct measure of the protein−protein pair potential.39,44 A necessary condition for protein crystallization is that B22 lies in a crystallization parameter window. Jia et al. proposed that protein crystallization is also predicted using surface-tension measurements.29 They showed that the diffusion rate of LSZ increases with increasing NaCl concentration, but an amorphous aggregation appears when transport of protein is much faster than the molecular rearrangement. That is, the protein crystallization condition does not simply obey the Hoffmeister series as we have seen in the degree of the refolding LSZ at the air−water interface.

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REFERENCES

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CONCLUSIONS We investigated adsorption kinetics of the positively charged LSZ (pH < pI) at an air−water interface to study the effect of Hofmeister anions. In salt-free solution, the protein adsorption rate was strongly dependent on pH. The adsorption rate showed a maximum at the isoelectric point, suggesting that it is strongly affected by the net charge of LSZ. For the F

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