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Cryopreservation of Proteins Using Ionic Liquids: A Case Study of Cytochrome c Takahiro Takekiyo, Yuka Ishikawa, and Yukihiro Yoshimura J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/ acs.jpcb.7b05158 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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The Journal of Physical Chemistry

ILs

77 K Cryo preservation

Native

Refolding

Denature Dialysis

Cyto c

Liquid N2

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Native Cyto c


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Cryopreservation of Proteins Using Ionic Liquids: A Case Study of Cytochrome c Takahiro Takekiyo*, Yuka Ishikawa, and Yukihiro Yoshimura Department of Applied Chemistry, National Defense Academy, 1-10-20, Hashirimizu, Yokosuka, Kanagawa, 2398686 Japan. *Corresponding author: e-mail: [email protected]

Abstract : Aqueous ionic liquid (IL) solutions form a glassy state at 77 K over a wide concentration of ILs. They have potential as novel cryopreservation/refolding solvents for proteins. However, even if proteins in glass-forming concentrations of ILs are preserved at 77 K, the recovery of activity and the structure of the proteins after cryopreservation are still unclear. To achieve high recovery of protein activity and structure by removal of ILs after cryopreservation at 77 K, we studied the recovery of activity and structural stability after cryopreservation of bovine heart cytochrome c in aqueous solutions with ILs, including ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazolium thiocyanate ([bmim][SCN]) over wide IL concentrations using UV-Vis, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy. On the whole, although the addition of both ILs induced a decrease of activity and unfolding of the secondary structure of cytochrome c before and after cooling to 77 K, EAN, a weak denaturant, showed a reduction in protein damage (decrease of activity and unfolding of secondary structure) during the reheating process from 77 K (protection ability). In contrast, [bmim][SCN], a strong denaturant, did not have this protective ability. A remarkable result is that although the addition of both ILs caused cytochrome c denaturation, >90% of activity and structure after cryopreservation (X > 10 mol%IL) was recovered after the removal of both ILs by dialysis. These recoveries after the removal of ILs are slightly higher than the results for dimethyl disulfide (DMSO), another cryoprotectant. The present results indicate that concentrated aqueous IL solutions have potential as one-pot (i.e., solubilization/preservation/refolding) solvents for proteins, which easily aggregate after purification, with comparable results to DMSO.

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1. Introduction Ionic liquids (ILs) can be tailored by varying their constituent cations and anions to obtain solvents with different chaotropic and kosmotropic properties, which are crucial for high solubility in water and control of the protein stability and are being used in bioscience and biomaterial applications.1-3 Examples include protein storage, buffers, and biocatalyst activity.1-3 An intriguing feature of aqueous IL solutions with or without proteins, previously shown by differential thermal analysis (DTA) and Raman spectroscopic studies, is that aqueous IL solutions easily form a glassy state at low temperature (77 K) over a wide IL concentration range, up to X (mol%IL) > 10, and the glass transition temperatures (Tg) were about 160 ~ 180 K in X = 10 ~ 100.4,5 This glass formation ability of aqueous IL solutions is an advantage for cryopreservation of biomolecules. In relation to this, as a recent intriguing bioscience topic using ILs, Fujita et al.6 demonstrated the solubilization of aggregated recombinant proteins (CcCel6A: 52.2 kDa) expressed in Escherichia coli (E.coli) using concentrated ILs, such as chloline dihydrogen phosphate ([Chl][dhp]), an ammonium-based IL.6 They revealed that the use of [Chl][dhp] may facilitate the development of an improved and convenient method for renaturation of aggregated recombinant proteins expressed in E.coli. Generally, it is difficult to preserve recombinant proteins expressed in E.coli without a suppressive agent for protein aggregation, such as guanidine hydrochloride (GdnHCl) and urea, at room temperature over a long period because these proteins, after purification, easily aggregate.7-10 Thus, as a condition for preservation of recombinant proteins, the glassy state is useful.11 However, aqueous GdnHCl and urea solutions do not have glass-forming abilities and form ice nuclei, playing a major damaging role during cryopreservation with a low content (X < 10) of GdnHCl or urea. Cryoprotectants, such as dimethyl sulfoxide (DMSO), glycerol, or sugars, have been used to inhibit ice formation and reduce protein structural changes. In fact, DMSO has been widely used as an additive in the cryopreservation of biopolymers.12,13 Concentrated solutions of DMSO often induce protein aggregation and protein damage during the heating process from the cryogenic storage temperature. 5, 2 ACS Paragon Plus Environment


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However, aqueous solutions with certain imidazolium-based and ammonium-based ILs, which

can form glassy state, induced intriguing structural transitions of proteins without protein aggregation, specifically, the folded state → unfolded state → partial globular state (α-helical formation disrupted tertiary structure).17,18 Moreover, it has been reported that imidazolium-based and ammonium-based ILs with shorter alkyl-chain length have the effective refolding enhancer,19-22 and certain aqueous IL solutions have a suppressive ability for amyloid formation in concentrated IL solutions.23 Thus, glass-forming ability at 77 K and the suppression and solubilization ability for protein aggregation of aqueous IL solutions might be useful for cryoprotection of recombinant proteins. In a preceding study,5 we reported the structure and activity of a chicken lysozyme in aqueous solutions with ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazolium chloride ([bmim][Cl]) over a wide IL concentration range by cooling to obtain information on lysozyme stability at cryogenic temperatures. The effect of cooling to 77 K on lysozyme activity and structure is small. However, the activity and structure recovery of proteins after removal of ILs after cryopreservation at 77 K are still unclear. Another problem is the technique for removal of ILs in concentrated IL solutions with proteins, though there have been investigated using dilution, dialysis, and column chromatography in dilute aqueous IL solutions.6,22,24,25 It is difficult to remove ILs from concentrated IL―protein solutions and obtain high functional recovery (activity and structure) for over 90% of the proteins. Therefore, the recovery of protein activity and structure in concentrated aqueous IL solutions above X = 10, forming a glassy state at 77 K after cooling, is necessary to evaluate the potential of ILs as cryopreservation agents. This information might be useful for the application in the long-storage cryopreservation technique for membrane and valuable proteins. In this research, to achieve high recovery of protein activity and structure after removal of ILs after cryopreservation at 77 K, we investigated the recovery of activity and structural stability after cryopreservation of bovine heart cytochrome c in aqueous IL solutions, with glass-forming 3 ACS Paragon Plus Environment

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concentrations of ILs (X > 10), using UV-Vis, FTIR, and CD spectroscopy. Cytochrome c with heme iron (Fe) contains 104 amino acid residues and is a helical-rich, globular heme protein wherein the strong fields of His18 and Met80 coordinate at axial positions to stabilize its low spin sets.26 Cytochrome c activity,27 using the reduction reaction (Fe3+→Fe2+) of heme Fe by xanthine (XA) and xanthine oxidase (XOD), can be measured without influence by the ILs. Further, the effects of ILs on the activity and structural stability of cytochrome c have been well investigated.28-31 For example, Fujita et al.28,29 reported that the addition of [Chl][dhp] to aqueous proteins solutions induces an increase in thermal stability of cytochrome c and also an increase of protein activity. Bihari et al.30 showed the secondary structure and peroxidase activity of cytochrome c is maintained in 1-ethyl-3methylimidazolium ethyl sulfate ([emim][EtSO4]). Fluorescence correlation spectroscopy by Monjumdar et al.31 showed that the addition of 1-butyl-3-methylimidazolium bromide ([bmim][Br]) to cytochrome c denatured by GdnHCl induced refolding. Thus, we used cytochrome c as a model protein for the cryopreservation and refolding of proteins in this study. For ILs, we selected EAN and 1-butyl-3-methylimidazlium thiocyanate ([bmim][SCN]). EAN is a weak denaturant of protein activity and structural stability and has suppressive ability for protein aggregation.18,23 Summer et al.22 reported that the use of dilute EAN solutions as a protein storage medium allowed lysozyme to be stored and reclaimed with 75% – 90% of its original activity, which was higher than that obtained when stored in 1-alkyl-3-methylimidazolium-based ILs. On the contrary, [bmim][SCN] is a stronger denaturant, compared to EAN. Both ILs have a suppressive ability for insulin amyloid formation23 and have the reverse property for protein stability with the Hofmeister series,3 which ranks the relative influence of ions on the physical behavior of a wide variety of aqueous processes, ranging from colloidal assembly to protein folding. The present finding is that > 90% of the original activity and structure of cytochrome c in aqueous solutions with both ILs after cryopreservation was recovered after the removal of ILs. The cryoprotectant ability of ILs is discussed. 4 ACS Paragon Plus Environment


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2. Experimental Section 2.1 Samples. Bovine heat cytochrome c (Sigma-Aldrich), chicken lysozyme (Wako junnyaku Ind. Co.), xanthine sodium (XA) (Wako junnyaku Ind. Co.), xanthine oxidase (XOD) from butter milk (Nakarai tesqu Co.), and Micrococcus lysodeikticus (Sigma-Aldrich) were used without further purification. As ILs, EAN (Iolitec GmbH) and [bmim][SCN] (Kanto Chemical Co.) were used without further purification. As a reference additive, DMSO (Wako Junyaku Co.) was used without further purification. All mixtures containing different concentrations of ILs and DMSO were prepared by mixing the required amount of additives and D2O (99.9%, Aldrich Co.) to desired concentrations (mol%IL or mol%DMSO). The limited solubility of cytochrome c in aqueous solutions with additives are X = 50 for EAN, X = 40 for [bmim][SCN], and X = 30 for DMSO. All pH and pD values in aqueous solutions with ILs and DMSO throughout the studied concentrations X were 5.4―6.8 for pH and 5.8 ― 7.2 for pD. The pD values were estimated by adding 0.4 to the reading values taken from a pH meter.32 All samples were prepared in a dry box to avoid contamination by atmospheric H2O and CO2. For UV-Vis measurements, protein concentration in the solution was adjusted to 1 mg/mL using a stock solution (10 mg/mL). For FTIR measurements, that in the solution was adjusted to 10 mg/mL using a deuterated aqueous solutions with ILs and DMSO. The entire sample solution was directly immersed into liquid nitrogen and kept at 77 K for ~ 30 min. Heating from the temperature of liquid nitrogen to the room temperature was performed by natural heating. Before and after removing the sample from the liquid nitrogen, cytochrome c activity and FTIR spectra were recorded.

2.2. Cytochrome c Activity. We tested cytochrome c activity in aqueous solutions with ILs and DMSO, according to the published procedure,27 which is based on the increase at 550 nm (Q-band) due to heme’s π-π* transitions,30 which indicate the change from Fe3+→Fe2+ of heme within XA and XOD. Cytochrome c in aqueous solutions with ILs and DMSO was mixed with aqueous XA solutions in 50 mM phosphate 5 ACS Paragon Plus Environment

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buffer (pH 6.8), and XOD solutions were added to these solutions. Cytochrome c activities were measured by the increase in absorbance at 550 nm using Nicolet GENESYS 10S UV-Vis spectroscopy (Thermo SCIENTIFIC). The increase in absorbance at 550 nm (Abs550nm) was measured from 0 to 180 s at 3-s intervals. The activity was determined by the linear slope of Abs550nm for up to 60 s. When the increase in Abs550nm in water was 100%, the activity in the aqueous solutions of ILs and DMSO was evaluated. We also tested lysozyme activity according to the published procedure.5

2.3. Dialysis. Dialysis was used the float A-lyzer G2 dialysis-kit with MWCO = 3.5~5 kD (Funakoshi Co.). Dialysis was performed using distilled water over eight hours (exchanging every 2 h), after 10-fold dilution of the cytochrome c solutions containing ILs with 50 mM phosphate buffer (pH 6.8), avoiding the amount lost due to cytochrome c aggregation and the dissolution of cellulose membranes by the ILs.

2.4. FTIR and CD Spectral Measurements. Fourier transform infrared (FTIR) spectra were recorded using a Nicolet 6700 FTIR spectrometer equipped with an MCT liquid nitrogen-cooled detector. Typically, 512 interferograms were collected to obtain a spectrum with a resolution of 4 cm−1. The sample was loaded into a cell with CaF2 windows and a 50-μm Teflon spacer for spectral measurements. However, we could not observe the FTIR spectrum of cytochrome c in the aqueous IL solutions above X = 30 because of the strong peak from imidazolium and ethylammonium cations.17,18 Circular dichroism (CD) spectra were measured over the wavelength range from 200 to 300 nm on a JASCO J-820 spectropolarimeter. Typical spectra were accumulated at a scan rate of 20 nm/min with 0.1 nm steps. Five scans were averaged for each spectrum. The obtained spectra were converted to mean residue ellipticity units using [θ] = θobs/(10ncl), where θobs is the observed ellipticity, l is the path length, c is the concentration of proteins, and n is the number of residues. Solvent spectra were also measured under the same conditions used for the protein solution 6 ACS Paragon Plus Environment


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measurements; the solvent spectra were then subtracted from the protein solution spectra.

3. Results and Discussion 3.1. Cryopreservation and Refolding of Cytochrome c in Aqueous IL Solution. First, the cytochrome c state in aqueous solutions with EAN and [bmim][SCN], after cooling to 77 K in glass-forming concentrations above X = 10 and removing ILs using dialysis, were measured to reveal the effect of the removal of ILs on the recovery of cytochrome c activity and its secondary structure. Figure 1a shows representative UV-Vis spectra of cytochrome c in aqueous EAN solutions at X = 50 before and after dialysis, along with those of EAN only and cytochrome c only. The strong peak at 300 nm due to EAN disappeared after dialysis. Similar UV-Vis spectra were also obtained at other EAN concentrations (X = 10 and 30) and in aqueous [bmim][SCN] solutions (X = 10 and 30). Moreover, the soret band at 409 nm, corresponding to the heme-Fe3+ oxidized-form of native cytochrome c,24,30,33 is in good agreement after dialysis with that for cytochrome c only. These results indicate that both ILs in concentrated IL solutions can be removed by dialysis. Next, cytochrome c activity in aqueous IL solutions was measured after cooling and dialysis. Figure 1b shows the time dependence of absorbance at 550 nm (Abs550nm) due to the heme Fe of cytochrome c in aqueous EAN solutions with XA and XOD at X = 50 before and after dialysis, along with the result for native cytochrome c. Although an increase at Abs550nm was barely observed in aqueous EAN solutions at X = 50 before dialysis, an increase of Abs550nm was observed after dialysis. The determined cytochrome c activities using the slopes in Fig. 1b are shown in Figure 1c. Interestingly, although the activities in aqueous IL solutions forming the glassy state above X = 10 were reduced to below 40% for EAN and 2.0% for [bmim][SCN] before dialysis, > 90% of the cytochrome c activity in the aqueous solutions with EAN (X = 10, 30, and 50) and [bmim][SCN] (X = 10 and 30) was recovered after the removal of ILs. To determine whether the present dialysis method can be applied to other proteins after cooling, we tested chicken lysozyme in aqueous [bmim][SCN] solutions using 7 ACS Paragon Plus Environment

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the same dialysis conditions. Lysozyme activity in the media at X = 20 after cooling was 0.8%. On the contrary, lysozyme activity was 93.8% after the removal of [bmim][SCN]. Based on these results, we succeeded to recover protein activity after cooling and using dialysis. In relation to this, we investigated the refolding of secondary and tertiary structures of cytochrome c using far-UV and near-UV region of CD spectra, combined with the results of the folded and unfolded states by 8 M GdnHCl, as shown in Figures 2a and b. On the whole, far-UV and near-UV CD spectra of cytochrome c after dialysis are close to those of the folded states. The helical contents determined by far-UV CD spectra are 50.7% for the folded state and 49.8% and 48.2% after the removal of EAN (X = 50) and [bmim][SCN] (X = 30), respectively. Since near-UV CD spectra, due to the Trp residues at 282 and 289 nm,34 after dialysis were also similar to those in the folded state, this suggests that the tertiary structure of cytochrome c is close to the folded state. Thus, the secondary and tertiary structures of cytochrome c after dialysis were returned to the folded structure. As a supporting result, the peak at 695 nm, due to the Fe-His18/Met80 ligand,26,35 was measured and is shown in Figure 2c. The Fe-His18/Met80 ligand peak was not observed in the Fe-free form (apo-form).35,36 Although the peak at 695 nm in aqueous IL solutions decreased before dialysis, this peak was observed after dialysis. The liberation of heme Fe after dialysis did not occur, and the Fe-His18/Met80 ligand of the heme state also returned to the native-like state. On the basis of these results, although cytochrome c denatures in aqueous solutions with the two ILs at X > 10, concentrations where a glassy state forms, > 90% of its activity and structure in aqueous IL solutions were recovered after the removal of ILs by dialysis. Thus, we succeeded to cryopreserve and refold proteins in concentrated aqueous IL solutions. Here we discuss the difference between the dialysis and dilution methods for the removal of ILs from aqueous protein solutions. Fujita et al.29 reported the cytochrome c activity of aqueous solutions with several ILs, which were imidazolium-, pyrrolidinium-, and choline-based ILs, containing 20 wt% water using dilution. They found that the cytochrome c activity after dilution varies considerably 8 ACS Paragon Plus Environment


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depending on ILs. On the contrary, the present results showed that >90% of the activity and structure of cytochrome c was recovered after the removal of EAN and [bmim][SCN] by dialysis. This indicates that dialysis resulted in the reduction of the influence of IL species on cytochrome c activity. Therefore, the dialysis method might be suitable for removal of ILs from proteins solutions rather than the dilution method.

3.2. Comparison with Cryoprotectant Ability of ILs and DMSO. In the previous section, the potential of cryopreservation/refolding solvents for proteins using concentrated aqueous IL solutions above X > 10 have been shown using cytochrome c. It is known that cryoprotectant ability is related to cryoprotectant-protein interactions and hydration via glass formation, influencing protein stability and inhibiting ice nucleation.37,38 In this section, we describe the potential cryoprotectant ability of both ILs combined with DMSO, another typical cryoprotectant, with respect to the cooling effect on cytochrome c activity and secondary structure, by the addition of ILs and DMSO before and after cooling. As a representative result, Figure 3a shows the time dependence of Abs550nm due to the heme Fe of cytochrome c in aqueous EAN solutions with XA and XOD at several concentrations after cooling. Addition of EAN causes a decrease in Abs550nm. Figure 3b shows the changes in the cytochrome c activity in aqueous solutions with both ILs and DMSO as a function of X before and after cooling. On the whole, cytochrome c activity decreases with increasing X before and after cooling. Although deactivation by the addition of DMSO also occurs before and after cooling, cytochrome c activity in aqueous DMSO solutions is higher than that in aqueous IL solutions. For further study of activity before and after cooling, we determined the midpoint concentration of deactivation ([X]1/2activity). The experimental data were fitted, as shown in Fig. 3b, by a sigmoidal curve under the assumption of a two-state mechanism for the change in the determined activity:39,40 a

activity  activity 0 

1  exp

 X X0   b 

  


(1)

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where activity and activity0 are the measured and initial activity values, and X and X0 are the measured and initial additive concentrations X, respectively. The values of [X]1/2activity before and after cooling are 3.87 ± 0.51 mol%IL and 4.86 ± 0.42 mol%IL for EAN, 1.52 ± 0.21 mol%IL and 1.36 ± 0.16 mol%IL for [bmim][SCN], 35.1 ± 2.24 mol%DMSO and 30.2 ± 3.16 mol%DMSO for DMSO, respectively (the values of [X]1/2activity are presented as mean ± standard deviation (n = 3)). The rank order of [X]1/2activity is DMSO > EAN > [bmim][SCN], before and after cooling. Similar results were also obtained for changes in secondary structure. Figure 4a shows a representative FTIR spectra in the amide I’ region of cytochrome c in aqueous EAN solutions at several concentrations after cooling. FTIR spectra in the amide I’ region indicate a drastic change in the secondary structure with increased EAN concentration. Figure 4b shows the changes in absorbance at 1655 cm-1 (Abs1655 cm-1) of cytochrome c in aqueous solutions with ILs and DMSO as a function of concentration X. On the whole, the decrease in Abs1655cm-1 indicates that disruption of the secondary structure was observed with increasing concentration X. Here, we determined the midpoint concentration ([X]1/2structure) of the secondary structure in the same manner as Eq.(1). The values of [X]1/2structure before and after cooling are 10.3 ± 1.62 mol%IL and 12.4 ± 0.87 mol%IL for EAN and 3.21 ± 0.35 mol%IL and 2.36 ± 0.95 mol%IL for [bmim][SCN], respectively (the values of [X]1/2structure are presented as mean ± standard deviation (n = 3)). The values of [X]1/2structure in aqueous DMSO solutions could not be determined because cytochrome c in aqueous DMSO solutions did not completely unfold. However, we can mention that the values of [X]1/2structure of cytochrome c in aqueous DMSO solution is higher than those in aqueous IL solutions. The rank order of cytochrome c unfolding is DMSO > EAN > [bmim][SCN] before and after cooling and is consistent with [X]1/2activity. Combining the results of activity and secondary structure, the cytochrome c stability in aqueous EAN solutions is higher than that in aqueous [bmim][SCN]. Many reviews showed that the protein stability in aqueous solutions with ammonium-based ILs is higher than that in aqueous solutions with imidazolium-based ILs along with the Hofmeister series.1,3 The present results are consistent with the 10 ACS Paragon Plus Environment


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previous studies. Moreover, DMSO-induced deactivation and unfolding of cytochrome c are weaker than those in aqueous solutions with both ILs throughout the studied concentration range. Here, as an intriguing result, the decrease of activity and unfolding of cytochrome c in aqueous EAN solutions after cooling was less than those before cooling. On the contrary, aqueous solutions with [bmim][SCN] and DMSO have the reverse situation. Recently, we demonstrated that [bmim][Cl] induced the decrease of lysozyme stability (activity and structure) after cooling, and EAN promoted the increase of lysozyme stability after cooling.5 The present results for ILs are in good agreement with the results from lysozyme.5 In addition, we mentioned that lysozyme aggregation in the heating process was observed in aqueous DMSO solutions, different from ILs. Related to this, cytochrome c activities in aqueous DMSO solutions by the removal of DMSO after cryopreservation were 86.0 ± 2.5% for X = 10, 79.4 ± 3.2% for X = 20, and 94.1 ± 2.9% for X = 30, respectively. DMSO showed relatively lower activity recovery rather than both ILs (> 90%) after their removal (see Fig. 1c). These indicate that the aqueous DMSO solution caused protein damage while heating from the cryogenic temperature, though DMSO-induced cytochrome c denaturation is weaker than both ILs. On the contrary, EAN had reduced ability for protein damage resulting from heating, since the activity and secondary structure of cytochrome c after cooling are higher than before cooling. Finally, we discuss the cryoprotectant ability of ILs and DMSO. It has been mentioned that cryoprotectant ability of DMSO for proteins is related to DMSO-protein interaction and hydration.38,4143

Huang et al.42 mentioned that DMSO molecules, with both hydrophobic and hydrophilic

characteristics, are expected to destabilize protein structures by weakening hydrophobic interactions between non-polar amino acid residues and by perturbing the stricture of water around protein structures. Related to this, Arakawa et al.38 proposed a mechanism for decreased protein stability in concentrated DMSO solutions. When the proteins unfold at higher DMSO concentrations, non-polar side chains (Ala, Leu, and Trp) contact with the solvent and the unfolded structure binds more DMSO. Such binding should decrease the free energy of the unfolded state in DMSO solutions. Since extensive 11 ACS Paragon Plus Environment

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DMSO binding can bring the unfolded state to a lower free energy state relative to the native state, this leads to DMSO-induced protein unfolding. Recently, Giugiarelli et al.44 reported DMSO-induced protein aggregation in blood cells using ATR-FTIR spectroscopy, and they suggest that very low water content and high DMSO concentration caused protein aggregation in the cells. On the other hand, hydration of proteins in aqueous IL solutions has been discussed.3,45,46 According to the free energy transfer (ΔGt) analysis of cyclic dipeptides from water to aqueous IL solutions performed by Attri and Venkatesu,45 ILs unfavorably interact with protein surfaces that assist in the formation of hydration layers around proteins. Jaganathan et al.46 demonstrated the organization of EAN around hydrated cytochrome c in concentrated EAN solutions using MD simulations. It has been observed that hydration of proteins in aqueous IL solutions is related to protein conformation. Moreover, our previous FTIR studies showed that EAN and [bmim][SCN] in concentrated IL solutions interacts with specific amino acid residue in proteins, Ala and Val residues for EAN23 and Lys and Arg residues for [bmim][SCN].39 Compared with the results for DMSO and ILs, DMSO or ILs interact with proteins in concentrated solutions, and the difference in the strength between IL-protein and DMSO-protein interactions might reflect the difference in the cryoprotectant ability between DMSO and ILs. In addition to this, we can speculate that the difference in the damage to proteins during cooling between DMSO and ILs might be related to the changes in hydration structure in concentrated solutions. The balance between DMSOor IL-protein interactions and hydration causes protein unfolding, aggregation, and precipitation in cryoprotectants. Unfortunately, it is difficult to know the changes in the hydration structure in the heating process from 77 K. On the basis of these results, although the cryoprotectant ability of ILs is lower than that of DMSO due to the balance between DMSO- or IL-protein interactions and hydration, both ILs showed high functional recovery (> 90%) of cytochrome c after the removal of ILs. In particular, EAN has the ability for reduced protein damage by cooling; this ability is an advantage for protein preservation 12 ACS Paragon Plus Environment


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compared to [bmim][SCN]. Related to this, we point out that other protic ILs, such as alkylammonium formates, which structurally mimic proteins more closely, might show higher cryoprotectant ability than EAN. The anion effect on the cryoprotectant ability of protic ILs of proteins will provide a better understanding of the IL-type cryoprotectant solvent. In addition to this, ILs with low vapor pressure can be removed from aqueous IL solutions under vacuum and re-condensed. Thus, concentrated IL solutions might be available to use as a renewable cryoprotectant solvent for aqueous protein solutions, as well as DMSO.

4. Conclusion We have studied the recovery of activity and structural stability after cryopreservation of bovine heart cytochrome c in aqueous IL solutions, for both EAN and [bmim][SCN], at glass-forming concentrations (X > 10) using UV-Vis, FTIR, and CD spectroscopy. Although addition of both ILs caused cytochrome c denaturation in concentrated IL solutions, > 90% recovery of activity and structure after cryopreservation was achieved after the removal of both ILs by dialysis. These recoveries with ILs are higher than those with DMSO, another cryoprotectant, though activity and structural stability of cytochrome c in aqueous IL solutions are lower than those in aqueous DMSO solutions. On the basis of these results, concentrated aqueous IL solutions have potential as cryopreservation and refolding solvents of recombinant proteins. In relation to this conclusion, as mentioned in the introduction, Fujita et al. demonstrated the solubilization of aggregated recombinant proteins (CcCel6A: 52.2 kDa) expressed in E.coli using [Chl][dhp].6 Combined with the present results, we propose that ILs might be available to create a novel “one-pot” solubilization/preservation/refolding solvent for insoluble protein aggregates such as amyloids, heat-aggregated proteins, and inclusion bodies; we are currently investigating these topics.

References (1) Weingärtner, H.; Cabrele, C.; Herrman, C. How Ionic Liquids can Help to Stabilize Native 13 ACS Paragon Plus Environment

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(a)

Before dialysis After dialysis Cytochrome c only EAN only

Absorbance

0.4 0.3 0.2 0.1 0 300

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Wavelength (nm) Absorbance at 550 nm

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(c)

80 60

40 20 0 10

30

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X (mol%IL) Figure 1. (a) Representative absorption spectra of cytochrome c in aqueous EAN solutions (X=50) before (black solid line) and after (red dashed line) dialysis combined with the spectra EAN only (blue dashed line) and cytochrome c only (green solid line). (b) Time dependence of absorbance at 550 nm of cytochrome c in aqueous solutions with EAN at X = 50 after cooling and before (dotted line) and after dialysis (dashed line) combined with the result of native state (solid line). (c) Recovery activity of cytochrome c in aqueous solutions with EAN (black bars) and [bmim][SCN](dark grey bars) at several X after dialysis combined the results of before dialysis (EAN: white bars, [bmim][SCN] : light grey bars). Bars represent standard deviation (n = 3). 17 ACS Paragon Plus Environment

4

(a)

0 -4 -8 -12

[θ]×102 (deg cm2 dmol-1)

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EAN

Water

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Figure 2. (a) Far-UV and (b) near-UV CD spectra of cytochrome c in aqueous solutions with EAN at X = 50 (red) and [bmim][SCN] at X= 30 (blue) combined with the results of folded state (black) and unfolded state (dotted) after cryopreservation by the removal of ILs. (c) Absorption spectra between 600 and 800 nm of cytochrome c in aqueous solutions with EAN at X = 50 (red) and [bmim][SCN] at X = 30 (blue) combined with the result of folded state (black). The dotted spectra in EAN and [bmim][SCN] represent the before dialysis.



Absorbance at 550 nm

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80 60 40 20 0 0

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Figure 3. (a) Time dependence of absorbance at 550 nm of cytochrome c in aqueous solutions with EAN after cooling at several X. (b) Changes in the cytochrome c activity in aqueous solutions with EAN (circles), [bmim][SCN] (triangles) and DMSO (squares) before and after cooling. Closed and open symbols represent before and after cooling. All lines are the results of the sigmoidal fitting. Bars represent standard deviation (n = 3).


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(a)

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0.25 0.24 0.23 0.22 0.21 0

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Figure 4. (a) FTIR spectra in the amide I’ band of cytochrome c in in aqueous solutions with EAN at several X after cooling. (b) Changes in the absorbance at 1655 cm-1 indicating α-helical structure of cytochrome c in aqueous solutions with EAN (circles), [bmim][SCN] (triangles) and DMSO (squares) before and after cooling. Closed and open symbols represent before and after cooling, respectively.


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