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Urea and guanidine salts as novel components for deep eutectic solvents
MOLLIQ-04241; No of Pages 4 Journal of Molecular Liquids xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Urea and guanidine salts as novel components for deep eutectic solvents
2Q1
Jozef Parnica a, Marian Antalik a,b,⁎
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Article history: Received 7 December 2013 Received in revised form 7 April 2014 Accepted 18 April 2014 Available online xxxx
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Keywords: Deep eutectic solvents Guanidine salts Ionic liquids Protein stability Urea
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1. Introduction
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Room temperature ionic liquids (RTILs) are low melting-temperature salts (below 373 K) formed by bulky and asymmetric ions that hinder crystallization [1]. Properties of RTILs, such as viscosity, density, melting point or solubility in water can be tuned by changing either the anion or cation [2]. Deep eutectic solvents (DES) were reported by Abbott and co-workers [3] as extending model of ionic liquids. DES are generally defined as salts with a melting point less than 100 °C exhibiting unique characteristics: very low vapor pressures, non-flammability, high thermal and chemical stability, possibility of recycling, good solubility for a wide spectrum of compounds and high suitability for modifications [1,4–6]. DES have large depression of the freezing point of mixtures of salts with hydrogen donors (e.g. amines, amides, alcohols and carboxylic acids) [7]. The interaction of this hydrogen bond donor with the salt reduces the anion–cation electrostatic force, and thus, the freezing point of the mixture decreases. The enormously large number of salts and hydrogen bond donors that can be used to prepare DES compounds caused that there is no limit of combination of cations with anions. A classical example is the combination of choline chloride (m.p. 302 °C) with urea (m.p. 132 °C), forming a DES with a melting point of 12 °C [8]. Urea is a powerful protein denaturant as it disrupts the noncovalent bonds in the proteins. This group of chaotropic agents includes also ionic guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuSCN). These guanidine-based compounds are well known denaturing reagents and are used extensively in protein chemistry. Urea
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We present guanidinium salts in combination with urea, used to prepare a new combination of deep eutectic solvents (DES). They have recently attracted much attention as “green” alternatives to conventional organic solvents in various fields including biophysical chemistry. These were subsequently used to test the stability and solubility of proteins in a water-free DES. We performed CD analyses in order to monitor global secondary and tertiary structural changes of cytochrome c in the presence of Guanidine HCl–urea. Formation of the melt form of DES at a room temperature was an effective way to store soluble protein in the denatured form for a long time. Consequent dialysis allows one to recover proteins into their native form. © 2014 Published by Elsevier B.V.
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Department of Biochemistry, Faculty of Science, P.J. Safarik University, 04011 Kosice, Slovak Republic Institute of Experimental Physics, Slovak Republic
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⁎ Corresponding author at: Department of Biochemistry, Faculty of Science, P.J. Safarik University, 04011 Kosice, Slovak Republic. E-mail address: [email protected] (M. Antalik).
and guanidine salt denaturation curves are generally used to obtain knowledge of conformational stability of proteins [9–11]. The eutectic system GuHCl–GuSCN (eutectic point lies at 87 °C, 60 mol% of GuSCN) has been studied recently as well as several other combinations of guanidine salts such as GuFormate–GuSCN (eutecticum lies at 45 °C, 30 mol% of GuSCN) [12]. Eutectic points were not in a molar ratio as in the case of choline chloride and urea. The paper presents the results of some characteristic DES formed between urea and GuHCl as well as urea and GuSCN and its effect on the structure of ferricytochrome c detected by CD spectroscopy. These ionic liquids allow the high solubility of cytochrome c and bovine serum albumin in these non-aqueous medium and we discussed the possibility of using this type of DES for long-term storage of proteins at room temperature.
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2. Materials and methods
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Chemical denaturants, urea, GuHCl and GuSCN were raw materials of deep eutectic solvents and were obtained from Sigma-Aldrich without further purification. Stock solutions of GuHCl (5 M), GuSCN (5 M) and urea (9 M) were prepared by weighing commercial compounds and adding distilled water in the desired proportion to pH 7.0. A buffer solution, consisting of 10 mM sodium phosphate at pH 7.0 and pH 2.0, was used in some experiments and pH values of solutions were measured by pH meter HI 9017 by Hanna Instruments Company by Sensorex glass electrode. The eutectic mixtures were formed from the powders by heating and stirring two components GuHCl–urea at 70 °C and GuSCN–urea at 60 °C until homogenous liquid was formed. The molar ratios of eutectic compositions for CD measurements were found to be 1:2 (20 mM: 40 mM). The liquids were cooled at a rate of 1 °C/min and the freezing point was taken as the temperature at which the first solid
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http://dx.doi.org/10.1016/j.molliq.2014.04.016 0167-7322/© 2014 Published by Elsevier B.V.
Please cite this article as: J. Parnica, M. Antalik, Journal of Molecular Liquids (2014), http://dx.doi.org/10.1016/j.molliq.2014.04.016
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We were able to prepare a new eutectic mixture with a lower eutectic point by replacing one of the guanidine salts in GuHCl–GuSCN for urea. Isobaric phase diagram (atmospheric pressure 101.325 kPa) of these binary systems (Fig. 1) shows a two-dimensional space, where the x-axis corresponds to the composition (in molar fractions) in accordance with the lever rule and the y-axis indicates the melting point temperature of DES (°C). Experimental determination of the phase diagram is usually carried out by thermal analysis, in which the temperature of the studied sample during its cooling at a constant speed of 2–5 °C/min is registered. When the system is cooled to the eutectic temperature it will preclude the solid phase. Temperature measurements of phase diagram were made on the basis of optical observations of crystals, using a Heidolph (Germany) heater with thermometer. These values were confirmed under a light microscope (Optika Microscope) with heating and cooling of samples. Guanidine salts mixed with urea in a 1:2 molar ratio produce colorless liquid that freezes at 58 °C for GuHCl-urea and 47 °C for GuSCN-urea. For GuHClurea eutecticum is formed at a composition of 67 mol% urea which is the same as the eutectic recently reported for the choline chloride-urea system [8]. The lower melting point (42 °C) relates to GuSCN-urea mixture and occurs when the ratio of salt to hydrogen bond donor is 1:1.5 (60 mol% urea). The melting points of the mixtures are considerably lower than the melting points of either of the constituents (GuHCl melts at 185 °C, GuSCN at 120 °C and urea at 134 °C). Concentrations of the individual components at 1:2 molar ratio in the mixture GuHCl (5.7 M)–urea (11.4 M) and GuSCN (5 M)–urea (10 M) are sufficient for the complete denaturing of proteins (as in aqueous solutions). In this report we demonstrate the ability of GuSCN–urea and GuHCl– urea (1:2) to dissolve higher amounts of two different proteins—bovine serum albumin (BSA) and cytochrome c (cyt c). BSA is a globular water soluble protein with molecular weight ~ 66 kDa and isoelectric point 4.7. BSA dissolves at least 260 mg per 1 ml in both DES. Higher concentrations could not be prepared in the form of real solution because of gel forming. The second studied biomacromolecule i.e. horse hearth cyt c is a heme protein with molecular weight ~ 12 kDa and isoelectric point 10.5. Cyt c was shown to dissolve at minimum 18 mg per 1 ml in both
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DES. Detection of higher concentrations of cytochrome c caused to observe solubility difficult because the solution was very colorful. We performed CD analyses in order to monitor global secondary and tertiary structural changes of cyt c in the presence of GuHCl–urea. Although GuSCN has a stronger effect on protein denaturation as GuHCl (GuSCN–urea has a lower eutectic formation temperature), the disadvantage of its use in the far UV region is its stronger absorption than GuHCl. Therefore we used the GuHCl-urea system in the next measurements (the impact of DES on the CD spectrum of cytochrome c). However, usage of cuvettes with path length of 0.2 mm (two separate slides forming cuvette) was necessary for this DES. Cuvette constructed from two parts provides an advantage when measuring viscous samples. There is no problem loading the sample into the second enclosed cavity portion by spreading the sample evenly throughout the entire cuvette volume. Decreasing for CD signal of protein itself was offset by the high concentration of protein that dissolved as well in water as in DES. Cyt c allows deeper analysis of conformational changes by CD polarimetry as compared to BSA because heme is the internal probe characterizing the changes in the tertiary structure of the protein. Fig. 2 shows representative far- and near-UV CD spectra of cyt c in different conditions. Fig. 2 (left) shows changes in the secondary structure of cytochrome c. Native cyt c in 10 mM phosphate buffer (pH 7.0 at 25 °C) shows classical dichroic minima at 208 nm and 222 nm, specific for the proteins with high amount of α-helicity. Water solutions of GuHCl (5 M) and urea (9 M) markedly replaced these minima by a large negative minimum below 210 nm suggesting an occurrence of a typical helix to random coil transition. The CD spectra of the cyt c in 10 mM phosphate buffer pH 2.0 at 25 °C indicate that the secondary structure is qualitatively similar with spectra of the cyt c in GuHCl–urea mixture at a temperature of 70 °C. The spectra suggest randomly coiled conformations in both cases, as well as in the individual components of a mixture. Difference in the spectra may be due to expanding the unfolding of cytochrome c. Fig. 2 (right) shows changes around the heme of cytochrome c. Negative peak at 416 nm and positive peak at 402 nm has been assigned for the tertiary structure corresponding to heme-polypeptide interaction close to the heme crevice and indicates native cyt c. In the presence of GuHCl–urea we observed the absence of negative Cotton effect at 416 nm and strong positive band with a maximum at 410 nm indicating a decoupling Met80 from binding to heme iron, like this observe for in all individual components of DES in water as well as for acidic pH 2.0 buffer. The questions whether these denatured proteins in the melt form of DES can renaturate again is another problem arising here. Refolding of the solubilized proteins is initiated by the removal of the denaturant or reducing its concentration and allowing the protein to refold into the native conformation. The most often used approach here is the removal of the solubilizing agent (DES) by dialysis (as mentioned by other authors). Long-term storage of cytochrome c in GuHCl–urea DES (6 months) at room temperature, and after removal of DES in 10 mM phosphate buffer solution at pH 7 by means of dialysis, CD spectra (Fig. 2 red lines) demonstrated folding of cytochrome c into the native structure. During dialysis the concentration of the solubilizing agent decreases slowly what allows to refold the protein optimally. Dialyzed cyt c from GuHCl-urea solution was carried out in 10 mM phosphate buffer pH 7.0 at 25 °C and subsequently measured secondary (tertiary) structure and conformational stability. CD spectra of the refolded cyt c closely resemble those of the native cyt c, showing classical dichroic minima at 208 nm and 222 nm for secondary structure and negative peak at 416 nm and positive peak at 402 nm for tertiary structure (Fig. 2 — red line). The protein was in DES in melt form at RT for 6 month. Therefore, assuming the formation of solid melt in the vicinity of the protein stabilizes the denatured state and after removal of the denaturant lets his return to native form. These observations indicate that the protein could be stored at RT in the denatured state and consequently renaturate anytime becoming ready for further measurements.
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began to form. The cyt c (99%) from horse heart and BSA (98%) was obtained from Sigma Aldrich. CD spectra were recorded with a Jasco J-815 spectropolarimeter equipped with a Julabo Peltier temperature control system (CDF-426S/15 model), using a cell of 0.2 mm path length over the wavelength range from 195–250 nm, 350–500 nm. Protein concentration was 5 mg/ml. CD signal-averaged over at least three scans, and baseline was corrected by subtracting the reference spectrum.
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mol % urea Fig. 1. Binary phase diagrams of guanidinium salts GuHCl (blue), GuSCN (red) and urea in different molar ratios. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
Please cite this article as: J. Parnica, M. Antalik, Journal of Molecular Liquids (2014), http://dx.doi.org/10.1016/j.molliq.2014.04.016
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Fig. 2. (left) Far-UV CD and (right) Near-UV CD spectra of cyt c (5 mg/ml) in: 10 mM phosphate buffer pH 7.0 at 25 °C in native (solid blue) and denatured state at pH 2.0, 10 mM phosphate (violet), 5 M GuHCl (brown), 9 M urea (black), measured in 0.2 mm optical length cuvette. Eutectic mixture of GuHCl–urea with 18 mg/ml cyt c at 70 °C (green) in cuvette with two quartz plates separated by 50 μm spacer and dialyzed from DES to 10 mM phosphate buffer pH 7.0 at 25 °C (after dialyzing, cytochrome was c diluted by phosphate buffer to cca 5 mg/ml concentration and measured in 0.2 mm optical length cuvette, red spectrum). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
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Substituting in GuSCN–GuHCl by urea for the two components, there is a reduction of the melting temperature from 87 °C to 67 °C for GuHCl–urea and even at 42 °C in the GuSCN–urea, a value of more than 3 °C less for GuFormate–GuSCN. On the other hand, in the case of substituting GuHCl in GuHCl–urea for choline chloride there is still a reduction of eutectic temperature up to 15 °C. Choline chloride and other salts of choline unlike GuHCl and GuSCN rather act as stabilizing agents to denaturation of proteins, mainly driven by increasing the ionic strength environment, which, in some cases lead to the stabilization of proteins. An interesting aspect is, however, a more complex of DES than expected in terms of molar ratios with the lowest melting point DES. While in the case of GuHCl–urea, this ratio is close to 1:2 in the case of GuSCN–urea it is 1:1.15. All findings about effect of GuHCl–urea on structure of cytochrome c were completed at a higher temperature than the eutectic point. Cyt c in studied DES forms a red melt after cooling to room temperature (RT). DES may help to characterize denatured proteins with minimal presence of water. Esquembre R. et al. [13] studied the stability of hen's egg white lysozyme in different choline chloride-based (with urea and glycerol) DES (thermal unfolding and refolding od lysozyme) using circular dichroism and fluorescence techniques. They observed the secondary and tertiary structures of lysozyme in DES (below 75 wt.%) show minimal differences from the protein in buffer solutions at room temperature (partial refolding was observed even in non-diluted choline chloride–glycerol). Destabilizing character is more evident in eutectic mixture choline chloride/urea than DES with containing glycerol (might help cation desorption from the hydrophobic core of the protein on renaturation). However, the thermal stability of the protein and the reversibility of its thermal unfolding are appreciably decreased in the presence of the solvents, particularly choline chloride–urea. First report of the effect of choline chloride based deep eutectic solvents on the accumulation of compact folding intermediates of a protein [13]. Their assumptions were based on previous work of Mann J.P. et al. focused on the unfolding–refolding processes of lysozyme in aqueous dilutions of alkylammonium-based ILs have described how, upon thermal treatment, the hydrophobic core of the unfolded protein becomes available to interact with alkylammonium cations via their alkyl tail. Under these circumstances, refolding required alkylammonium desorption from the hydrophobic core. The same authors found that the use of pseudoconcentrated hydroxy derivates of alkylammonium formate salts (e.g. ethanolammonium versus ethylammonium)
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provided increased protein thermal stability through the extra hydrogen bonding donor–acceptor site, which markedly reduces the strength of hydrophobic interactions with the protein [14]. Effects of DES components acting as individual entities should begin to be observed at concentrations below 50 wt.% [13]. A more complex situation is found in pseudo-concentrated DES (e.g. DES content 86 wt.%) because of the strong interactions between the DES components. One could also consider the effect of proton activity on lysozyme stability (unfolding of lysozyme in protic ionic liquids (PILs) can proceed through reversible intermediates or cooperative pathways by selecting the proton activity of the media) [15]. It can be concluded that the composition of DES to temperature stability of proteins has the largest impact. In our conditions the denaturation of proteins is caused by chaotropic agents. Temperature of the sample should not be higher than Tm (higher temperature causes irreversible change in the structure of proteins). However, much work remains to be done on the study of novel DES properties. There is no known work which describes the behavior of the proteins in the vicinity DES in melt form. The dependence of physicochemical properties against anion nature and water content should be examined in the future and explained according to cation–anion interactions. Nardecchia S. et al. using DSC and DLS techniques demonstrated that the preferred conformation of ELbR (Elastin-like polymers– synthetic polypeptides) in DES (choline chloride–urea) is in the collapsed state. Confirmed that DES provides a nonhydrated solvent of a relatively high ionic strength that favors the stabilization of ELbR in the collapsed state. They proved a subsequent formation of aggregates—upon the loss of the structural water molecules involved in hydrophobic hydration and this collapsed state remains stable even after partial hydration [16]. Presumption for a similar condition in the case of our DES. The essential difference between the prior attempts to DES (choline chloride–urea) is the fluidity of the sample at room temperature. However, proteins are only the beginning: the examination of complex networks to DES were subsequently investigated in the area of living cells. Gutiérrez M.C. et al. [17] observed with liposomes (large/200 nm/unilamellar vesicles or LUV) as models of living cells incorporate into DES (choline chloride–urea/thiourea) full preservation of their self-assembled structure. Moreover, the incorporation of LUV in DES also opens the path for the unexplored study of transmembrane proteins in DES. Also shown how to prepare another DES method (not prepared directly in pure state). It is an alternative and easy procedure for DES preparation from aqueous solutions of their individual
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Please cite this article as: J. Parnica, M. Antalik, Journal of Molecular Liquids (2014), http://dx.doi.org/10.1016/j.molliq.2014.04.016
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The main result of the work presented here is that DES were shown to be composed of unique combination of molecules urea and GuHCl and urea–GuSCN. They exhibit high potential for preparation of a new generation of effective solvents for studies of denatured states of proteins, as well as for studies of the effect of low concentration of water on the denatured state of proteins and influence on protein stabilization or crystallization. We also report a family of biocompatible ionic liquids that are able to dissolve significant amounts of proteins such as cyt c and BSA. The eutectic mixture is suggested to exhibit good stability in melt form at RT. Knowledge of the impact of different factors on behavior of the IL is useful for developing proprietary designed solvents. Owing to their anhydrous character these molecular systems are superior for a wide range of applications in protein chemistry.
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[1] T. Welton, Chem. Rev. 99 (1999) 2071–2083. [2] M. Deetlefs, K.R. Seddon, M. Shara, New J. Chem. 30 (2006) 317–326. [3] A.P. Abbott, D. Boothby, G. Capper, D.L. Davies, R.K. Rasheed, J. Am. Chem. Soc. 126 (2004) 9142–9147. [4] P. Wasserscheid, T. Welton, Ionic liquids in synthesis, Wiley-VCH, Weinheim, 2003. [5] R.D. Rogers, K.D. Seddon, Science 302 (2003) 792–793. [6] T.G. Youngs, J.D. Holbrey, M. Deetlefs, M. Nieuwenhyuzen, C. Hardacre, ChemPhysChem 7 (2006) 2279–2281. [7] A.P. Abbott, G. Capper, D.L. Davies, H.L. Munro, R.K. Rasheed, V. Tambyrajah, Chem. Commun. (2001) 2010–2011. [8] A.P. Abbott, G. Capper, D.L. Davies, R.K. Rasheed, V. Tambyrajah, Chem. Commun. (2003) 70–71. [9] P.L. Privalov, Adv. Protein Chem. 33 (1979) 167–241. [10] C.N. Pace, Methods Enzymol. 131 (1986) 266–280. [11] D. Shortle, J. Biol. Chem. 264 (1989) 5315–5318. [12] Z. Zhao, K. Ueno, C.A. Angell, J. Phys. Chem. B 115 (2011) 13467–13472. [13] R. Esquembre, J.M. Sanz, J.G. Wall, F. del Monte, C.R. Mateo, M.L. Ferrer, Phys. Chem. Chem. Phys. 15 (2013) 11248–11256. [14] J.P. Mann, A. McCluskey, R. Atkin, Green Chem. 11 (2009) 785–792. [15] N. Byrne, C.A. Angell, J. Mol. Biol. 378 (2008) 707–714. [16] S. Nardecchia, M.C. Gutiérrez, M.L. Ferrer, M. Alonso, I.M. López, J.C. RodríguezCabello, F. del Monte, Biomacromolecules 13 (2012) 2029–2036. [17] M.C. Gutiérrez, M.L. Ferrer, C.R. Mateo, F. del Monte, Langmuir 25 (2009) 5509–5515. [18] I. Mamajanov, A.E. Engelhart, H.D. Bean, N.V. Hud, Angew. Chem. Int. Ed. Engl. 49 (2010) 6310–6314. [19] M.C. Gutiérrez, M.L. Ferrer, L. Yuste, F. Rojo, F. del Monte, Angew. Chem. Int. Ed. Engl. 49 (2010) 2158–2162.
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This research was supported by the project nos. 26220120021 and 304 Q5 26220220061 in frame of SF EU, VEGA 2/0025/12 and project APVV- 305 0171-10, APVV-0526-11. 306
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counterparts followed by freeze-drying (lyophilization) of water content. It also improves the properties of the reaction medium such as viscosity, fluidity, polarity, ionic strength, etc. [17]. This method appears as preferable alternatives for DES whose melting point is located at a higher temperature and possible incorporation of various components (protein) inside the DES would be damaged by higher temperature (denaturation temperature of proteins must be higher than the eutectic point). Similar studies were completed for DNA and RNA in anhydrous media (DES) [18] or for incorporation of bacteria into DES in its pure state with outstanding preservation of bacteria integrity and viability [19].
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Please cite this article as: J. Parnica, M. Antalik, Journal of Molecular Liquids (2014), http://dx.doi.org/10.1016/j.molliq.2014.04.016
Contents lists available at ScienceDirect
Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Urea and guanidine salts as novel components for deep eutectic solvents
2Q1
Jozef Parnica a, Marian Antalik a,b,⁎
3 4
a
5
a r t i c l e
6 7 8 9 10
Article history: Received 7 December 2013 Received in revised form 7 April 2014 Accepted 18 April 2014 Available online xxxx
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Keywords: Deep eutectic solvents Guanidine salts Ionic liquids Protein stability Urea
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1. Introduction
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Room temperature ionic liquids (RTILs) are low melting-temperature salts (below 373 K) formed by bulky and asymmetric ions that hinder crystallization [1]. Properties of RTILs, such as viscosity, density, melting point or solubility in water can be tuned by changing either the anion or cation [2]. Deep eutectic solvents (DES) were reported by Abbott and co-workers [3] as extending model of ionic liquids. DES are generally defined as salts with a melting point less than 100 °C exhibiting unique characteristics: very low vapor pressures, non-flammability, high thermal and chemical stability, possibility of recycling, good solubility for a wide spectrum of compounds and high suitability for modifications [1,4–6]. DES have large depression of the freezing point of mixtures of salts with hydrogen donors (e.g. amines, amides, alcohols and carboxylic acids) [7]. The interaction of this hydrogen bond donor with the salt reduces the anion–cation electrostatic force, and thus, the freezing point of the mixture decreases. The enormously large number of salts and hydrogen bond donors that can be used to prepare DES compounds caused that there is no limit of combination of cations with anions. A classical example is the combination of choline chloride (m.p. 302 °C) with urea (m.p. 132 °C), forming a DES with a melting point of 12 °C [8]. Urea is a powerful protein denaturant as it disrupts the noncovalent bonds in the proteins. This group of chaotropic agents includes also ionic guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuSCN). These guanidine-based compounds are well known denaturing reagents and are used extensively in protein chemistry. Urea
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We present guanidinium salts in combination with urea, used to prepare a new combination of deep eutectic solvents (DES). They have recently attracted much attention as “green” alternatives to conventional organic solvents in various fields including biophysical chemistry. These were subsequently used to test the stability and solubility of proteins in a water-free DES. We performed CD analyses in order to monitor global secondary and tertiary structural changes of cytochrome c in the presence of Guanidine HCl–urea. Formation of the melt form of DES at a room temperature was an effective way to store soluble protein in the denatured form for a long time. Consequent dialysis allows one to recover proteins into their native form. © 2014 Published by Elsevier B.V.
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Department of Biochemistry, Faculty of Science, P.J. Safarik University, 04011 Kosice, Slovak Republic Institute of Experimental Physics, Slovak Republic
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⁎ Corresponding author at: Department of Biochemistry, Faculty of Science, P.J. Safarik University, 04011 Kosice, Slovak Republic. E-mail address: [email protected] (M. Antalik).
and guanidine salt denaturation curves are generally used to obtain knowledge of conformational stability of proteins [9–11]. The eutectic system GuHCl–GuSCN (eutectic point lies at 87 °C, 60 mol% of GuSCN) has been studied recently as well as several other combinations of guanidine salts such as GuFormate–GuSCN (eutecticum lies at 45 °C, 30 mol% of GuSCN) [12]. Eutectic points were not in a molar ratio as in the case of choline chloride and urea. The paper presents the results of some characteristic DES formed between urea and GuHCl as well as urea and GuSCN and its effect on the structure of ferricytochrome c detected by CD spectroscopy. These ionic liquids allow the high solubility of cytochrome c and bovine serum albumin in these non-aqueous medium and we discussed the possibility of using this type of DES for long-term storage of proteins at room temperature.
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Chemical denaturants, urea, GuHCl and GuSCN were raw materials of deep eutectic solvents and were obtained from Sigma-Aldrich without further purification. Stock solutions of GuHCl (5 M), GuSCN (5 M) and urea (9 M) were prepared by weighing commercial compounds and adding distilled water in the desired proportion to pH 7.0. A buffer solution, consisting of 10 mM sodium phosphate at pH 7.0 and pH 2.0, was used in some experiments and pH values of solutions were measured by pH meter HI 9017 by Hanna Instruments Company by Sensorex glass electrode. The eutectic mixtures were formed from the powders by heating and stirring two components GuHCl–urea at 70 °C and GuSCN–urea at 60 °C until homogenous liquid was formed. The molar ratios of eutectic compositions for CD measurements were found to be 1:2 (20 mM: 40 mM). The liquids were cooled at a rate of 1 °C/min and the freezing point was taken as the temperature at which the first solid
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http://dx.doi.org/10.1016/j.molliq.2014.04.016 0167-7322/© 2014 Published by Elsevier B.V.
Please cite this article as: J. Parnica, M. Antalik, Journal of Molecular Liquids (2014), http://dx.doi.org/10.1016/j.molliq.2014.04.016
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We were able to prepare a new eutectic mixture with a lower eutectic point by replacing one of the guanidine salts in GuHCl–GuSCN for urea. Isobaric phase diagram (atmospheric pressure 101.325 kPa) of these binary systems (Fig. 1) shows a two-dimensional space, where the x-axis corresponds to the composition (in molar fractions) in accordance with the lever rule and the y-axis indicates the melting point temperature of DES (°C). Experimental determination of the phase diagram is usually carried out by thermal analysis, in which the temperature of the studied sample during its cooling at a constant speed of 2–5 °C/min is registered. When the system is cooled to the eutectic temperature it will preclude the solid phase. Temperature measurements of phase diagram were made on the basis of optical observations of crystals, using a Heidolph (Germany) heater with thermometer. These values were confirmed under a light microscope (Optika Microscope) with heating and cooling of samples. Guanidine salts mixed with urea in a 1:2 molar ratio produce colorless liquid that freezes at 58 °C for GuHCl-urea and 47 °C for GuSCN-urea. For GuHClurea eutecticum is formed at a composition of 67 mol% urea which is the same as the eutectic recently reported for the choline chloride-urea system [8]. The lower melting point (42 °C) relates to GuSCN-urea mixture and occurs when the ratio of salt to hydrogen bond donor is 1:1.5 (60 mol% urea). The melting points of the mixtures are considerably lower than the melting points of either of the constituents (GuHCl melts at 185 °C, GuSCN at 120 °C and urea at 134 °C). Concentrations of the individual components at 1:2 molar ratio in the mixture GuHCl (5.7 M)–urea (11.4 M) and GuSCN (5 M)–urea (10 M) are sufficient for the complete denaturing of proteins (as in aqueous solutions). In this report we demonstrate the ability of GuSCN–urea and GuHCl– urea (1:2) to dissolve higher amounts of two different proteins—bovine serum albumin (BSA) and cytochrome c (cyt c). BSA is a globular water soluble protein with molecular weight ~ 66 kDa and isoelectric point 4.7. BSA dissolves at least 260 mg per 1 ml in both DES. Higher concentrations could not be prepared in the form of real solution because of gel forming. The second studied biomacromolecule i.e. horse hearth cyt c is a heme protein with molecular weight ~ 12 kDa and isoelectric point 10.5. Cyt c was shown to dissolve at minimum 18 mg per 1 ml in both
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DES. Detection of higher concentrations of cytochrome c caused to observe solubility difficult because the solution was very colorful. We performed CD analyses in order to monitor global secondary and tertiary structural changes of cyt c in the presence of GuHCl–urea. Although GuSCN has a stronger effect on protein denaturation as GuHCl (GuSCN–urea has a lower eutectic formation temperature), the disadvantage of its use in the far UV region is its stronger absorption than GuHCl. Therefore we used the GuHCl-urea system in the next measurements (the impact of DES on the CD spectrum of cytochrome c). However, usage of cuvettes with path length of 0.2 mm (two separate slides forming cuvette) was necessary for this DES. Cuvette constructed from two parts provides an advantage when measuring viscous samples. There is no problem loading the sample into the second enclosed cavity portion by spreading the sample evenly throughout the entire cuvette volume. Decreasing for CD signal of protein itself was offset by the high concentration of protein that dissolved as well in water as in DES. Cyt c allows deeper analysis of conformational changes by CD polarimetry as compared to BSA because heme is the internal probe characterizing the changes in the tertiary structure of the protein. Fig. 2 shows representative far- and near-UV CD spectra of cyt c in different conditions. Fig. 2 (left) shows changes in the secondary structure of cytochrome c. Native cyt c in 10 mM phosphate buffer (pH 7.0 at 25 °C) shows classical dichroic minima at 208 nm and 222 nm, specific for the proteins with high amount of α-helicity. Water solutions of GuHCl (5 M) and urea (9 M) markedly replaced these minima by a large negative minimum below 210 nm suggesting an occurrence of a typical helix to random coil transition. The CD spectra of the cyt c in 10 mM phosphate buffer pH 2.0 at 25 °C indicate that the secondary structure is qualitatively similar with spectra of the cyt c in GuHCl–urea mixture at a temperature of 70 °C. The spectra suggest randomly coiled conformations in both cases, as well as in the individual components of a mixture. Difference in the spectra may be due to expanding the unfolding of cytochrome c. Fig. 2 (right) shows changes around the heme of cytochrome c. Negative peak at 416 nm and positive peak at 402 nm has been assigned for the tertiary structure corresponding to heme-polypeptide interaction close to the heme crevice and indicates native cyt c. In the presence of GuHCl–urea we observed the absence of negative Cotton effect at 416 nm and strong positive band with a maximum at 410 nm indicating a decoupling Met80 from binding to heme iron, like this observe for in all individual components of DES in water as well as for acidic pH 2.0 buffer. The questions whether these denatured proteins in the melt form of DES can renaturate again is another problem arising here. Refolding of the solubilized proteins is initiated by the removal of the denaturant or reducing its concentration and allowing the protein to refold into the native conformation. The most often used approach here is the removal of the solubilizing agent (DES) by dialysis (as mentioned by other authors). Long-term storage of cytochrome c in GuHCl–urea DES (6 months) at room temperature, and after removal of DES in 10 mM phosphate buffer solution at pH 7 by means of dialysis, CD spectra (Fig. 2 red lines) demonstrated folding of cytochrome c into the native structure. During dialysis the concentration of the solubilizing agent decreases slowly what allows to refold the protein optimally. Dialyzed cyt c from GuHCl-urea solution was carried out in 10 mM phosphate buffer pH 7.0 at 25 °C and subsequently measured secondary (tertiary) structure and conformational stability. CD spectra of the refolded cyt c closely resemble those of the native cyt c, showing classical dichroic minima at 208 nm and 222 nm for secondary structure and negative peak at 416 nm and positive peak at 402 nm for tertiary structure (Fig. 2 — red line). The protein was in DES in melt form at RT for 6 month. Therefore, assuming the formation of solid melt in the vicinity of the protein stabilizes the denatured state and after removal of the denaturant lets his return to native form. These observations indicate that the protein could be stored at RT in the denatured state and consequently renaturate anytime becoming ready for further measurements.
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began to form. The cyt c (99%) from horse heart and BSA (98%) was obtained from Sigma Aldrich. CD spectra were recorded with a Jasco J-815 spectropolarimeter equipped with a Julabo Peltier temperature control system (CDF-426S/15 model), using a cell of 0.2 mm path length over the wavelength range from 195–250 nm, 350–500 nm. Protein concentration was 5 mg/ml. CD signal-averaged over at least three scans, and baseline was corrected by subtracting the reference spectrum.
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mol % urea Fig. 1. Binary phase diagrams of guanidinium salts GuHCl (blue), GuSCN (red) and urea in different molar ratios. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
Please cite this article as: J. Parnica, M. Antalik, Journal of Molecular Liquids (2014), http://dx.doi.org/10.1016/j.molliq.2014.04.016
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Fig. 2. (left) Far-UV CD and (right) Near-UV CD spectra of cyt c (5 mg/ml) in: 10 mM phosphate buffer pH 7.0 at 25 °C in native (solid blue) and denatured state at pH 2.0, 10 mM phosphate (violet), 5 M GuHCl (brown), 9 M urea (black), measured in 0.2 mm optical length cuvette. Eutectic mixture of GuHCl–urea with 18 mg/ml cyt c at 70 °C (green) in cuvette with two quartz plates separated by 50 μm spacer and dialyzed from DES to 10 mM phosphate buffer pH 7.0 at 25 °C (after dialyzing, cytochrome was c diluted by phosphate buffer to cca 5 mg/ml concentration and measured in 0.2 mm optical length cuvette, red spectrum). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
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Substituting in GuSCN–GuHCl by urea for the two components, there is a reduction of the melting temperature from 87 °C to 67 °C for GuHCl–urea and even at 42 °C in the GuSCN–urea, a value of more than 3 °C less for GuFormate–GuSCN. On the other hand, in the case of substituting GuHCl in GuHCl–urea for choline chloride there is still a reduction of eutectic temperature up to 15 °C. Choline chloride and other salts of choline unlike GuHCl and GuSCN rather act as stabilizing agents to denaturation of proteins, mainly driven by increasing the ionic strength environment, which, in some cases lead to the stabilization of proteins. An interesting aspect is, however, a more complex of DES than expected in terms of molar ratios with the lowest melting point DES. While in the case of GuHCl–urea, this ratio is close to 1:2 in the case of GuSCN–urea it is 1:1.15. All findings about effect of GuHCl–urea on structure of cytochrome c were completed at a higher temperature than the eutectic point. Cyt c in studied DES forms a red melt after cooling to room temperature (RT). DES may help to characterize denatured proteins with minimal presence of water. Esquembre R. et al. [13] studied the stability of hen's egg white lysozyme in different choline chloride-based (with urea and glycerol) DES (thermal unfolding and refolding od lysozyme) using circular dichroism and fluorescence techniques. They observed the secondary and tertiary structures of lysozyme in DES (below 75 wt.%) show minimal differences from the protein in buffer solutions at room temperature (partial refolding was observed even in non-diluted choline chloride–glycerol). Destabilizing character is more evident in eutectic mixture choline chloride/urea than DES with containing glycerol (might help cation desorption from the hydrophobic core of the protein on renaturation). However, the thermal stability of the protein and the reversibility of its thermal unfolding are appreciably decreased in the presence of the solvents, particularly choline chloride–urea. First report of the effect of choline chloride based deep eutectic solvents on the accumulation of compact folding intermediates of a protein [13]. Their assumptions were based on previous work of Mann J.P. et al. focused on the unfolding–refolding processes of lysozyme in aqueous dilutions of alkylammonium-based ILs have described how, upon thermal treatment, the hydrophobic core of the unfolded protein becomes available to interact with alkylammonium cations via their alkyl tail. Under these circumstances, refolding required alkylammonium desorption from the hydrophobic core. The same authors found that the use of pseudoconcentrated hydroxy derivates of alkylammonium formate salts (e.g. ethanolammonium versus ethylammonium)
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provided increased protein thermal stability through the extra hydrogen bonding donor–acceptor site, which markedly reduces the strength of hydrophobic interactions with the protein [14]. Effects of DES components acting as individual entities should begin to be observed at concentrations below 50 wt.% [13]. A more complex situation is found in pseudo-concentrated DES (e.g. DES content 86 wt.%) because of the strong interactions between the DES components. One could also consider the effect of proton activity on lysozyme stability (unfolding of lysozyme in protic ionic liquids (PILs) can proceed through reversible intermediates or cooperative pathways by selecting the proton activity of the media) [15]. It can be concluded that the composition of DES to temperature stability of proteins has the largest impact. In our conditions the denaturation of proteins is caused by chaotropic agents. Temperature of the sample should not be higher than Tm (higher temperature causes irreversible change in the structure of proteins). However, much work remains to be done on the study of novel DES properties. There is no known work which describes the behavior of the proteins in the vicinity DES in melt form. The dependence of physicochemical properties against anion nature and water content should be examined in the future and explained according to cation–anion interactions. Nardecchia S. et al. using DSC and DLS techniques demonstrated that the preferred conformation of ELbR (Elastin-like polymers– synthetic polypeptides) in DES (choline chloride–urea) is in the collapsed state. Confirmed that DES provides a nonhydrated solvent of a relatively high ionic strength that favors the stabilization of ELbR in the collapsed state. They proved a subsequent formation of aggregates—upon the loss of the structural water molecules involved in hydrophobic hydration and this collapsed state remains stable even after partial hydration [16]. Presumption for a similar condition in the case of our DES. The essential difference between the prior attempts to DES (choline chloride–urea) is the fluidity of the sample at room temperature. However, proteins are only the beginning: the examination of complex networks to DES were subsequently investigated in the area of living cells. Gutiérrez M.C. et al. [17] observed with liposomes (large/200 nm/unilamellar vesicles or LUV) as models of living cells incorporate into DES (choline chloride–urea/thiourea) full preservation of their self-assembled structure. Moreover, the incorporation of LUV in DES also opens the path for the unexplored study of transmembrane proteins in DES. Also shown how to prepare another DES method (not prepared directly in pure state). It is an alternative and easy procedure for DES preparation from aqueous solutions of their individual
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The main result of the work presented here is that DES were shown to be composed of unique combination of molecules urea and GuHCl and urea–GuSCN. They exhibit high potential for preparation of a new generation of effective solvents for studies of denatured states of proteins, as well as for studies of the effect of low concentration of water on the denatured state of proteins and influence on protein stabilization or crystallization. We also report a family of biocompatible ionic liquids that are able to dissolve significant amounts of proteins such as cyt c and BSA. The eutectic mixture is suggested to exhibit good stability in melt form at RT. Knowledge of the impact of different factors on behavior of the IL is useful for developing proprietary designed solvents. Owing to their anhydrous character these molecular systems are superior for a wide range of applications in protein chemistry.
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This research was supported by the project nos. 26220120021 and 304 Q5 26220220061 in frame of SF EU, VEGA 2/0025/12 and project APVV- 305 0171-10, APVV-0526-11. 306
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counterparts followed by freeze-drying (lyophilization) of water content. It also improves the properties of the reaction medium such as viscosity, fluidity, polarity, ionic strength, etc. [17]. This method appears as preferable alternatives for DES whose melting point is located at a higher temperature and possible incorporation of various components (protein) inside the DES would be damaged by higher temperature (denaturation temperature of proteins must be higher than the eutectic point). Similar studies were completed for DNA and RNA in anhydrous media (DES) [18] or for incorporation of bacteria into DES in its pure state with outstanding preservation of bacteria integrity and viability [19].
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Please cite this article as: J. Parnica, M. Antalik, Journal of Molecular Liquids (2014), http://dx.doi.org/10.1016/j.molliq.2014.04.016