Thermodynamic Contribution of Amino Acids in Ionic Liquids Towards Protein Stability

Send Orders for Reprints to [email protected]

Current Biochemical Engineering, 2014, 1, ��-��0 1��

Thermodynamic Contribution of Amino Acids in Ionic Liquids Towards Protein Stability

Awanish Kumara, Pannuru Venkatesua,*, Mohamed Tahab and Ming-Jer Leeb

aDepartment of Chemistry, University of Delhi, Delhi-110 007, India;

bDepartment of Chemical Engineering, National

Taiwan University of Science and Technology, 43 Keelung Road, Section 4, Taipei 106-07, Taiwan

Abstract: Amino acids (AAs) combine to form a three-dimensional protein structure and are of very much importance in understanding the biophysical properties of biomolecules. Basically, the nature and the arrangement of the AAs in a pro-tein backbone is only responsible for the individual characteristics of the macromolecule. The AAs in a protein backbone are influenced by the solvent molecules hence, it is very important to have a clear idea on the solubility, stability, and thermodynamic properties of these AAs in various solvents and co-solvents. A basic level of quantifying protein-solvent interactions involve the use of transfer free energies, �Gtr from water to solvents. The values of �Gtr for side chains and peptide backbone quantify the thermodynamic consequences of solvating a protein species in a co-solvent solution rela-tive to pure water. Based on the transfer model and experimental �Gtr for these AAs, it has been proposed that these co-solvents exert their effect on protein stability primarily via the protein backbone. The �Gtr of AAs from water to another solvent system will be either favorable or unfavorable. By definition, an unfavorable transfer free energy, �Gtr > 0, means that the protein becomes solvophobic on transfer to a solvent, whereas a favorable transfer free energy, �Gtr < 0, repre-sents that the protein becomes solvophilic on transfer to a solvent. The sign and magnitude of the measured �Gtr quantifies the protein response to changes in solvent quality. Therefore, this review will provide the basis of a universal mechanism for co-solvent-mediated (that includes the new novel biocompatible ionic liquids (ILs)) protein stabilization and destabilization as the protein backbone is shared by all proteins, regardless of side chain sequence.

Keywords: Amino acids, free energy, ionic liquids, molecular dynamics, protein stability, thermodynamics.

1. INTRODUCTION

The co-valent structure of a natural protein molecule is primarily because of the 20 different amino acids (AAs). These AAs are the building blocks that differ in size, shape, charge, hydrogen-bonding capacity, hydrophobicity and chemical reactivity. The polar or charged AAs participate in hydrogen bonding and electrostatic interactions, with co-solvents or solvent itself. On the other hand non-polar AAs have unfavorable interactions with the solvent molecules (especially water). Clustering of these hydrophobic AAs in the interior of the protein provides a thermodynamic driving force for protein folding (the hydrophobic effect). Both these interactions of the AA sequence spontaneously and selec-tively give rise to a unique protein structure that is an attrac-tive aspect of the structural features in a biomolecule [1]. Basically, the nature and the arrangement of the AA side-chain in a protein backbone is responsible for the biophysical properties of the macromolecule and it has been recognized in the open literature that all of the information pertaining to protein can be obtained through investigating the properties of the AA sequence [1, 2]. Moreover, proteins differ from

*Address correspondence to this author at the Department of Chemistry, University of Delhi, Delhi-110 007, India; Tel: +91-11-27666646-142; Fax: +91-11-2766 6605; E-mails: [email protected]; [email protected]

each other (in physical properties/functions) due to the varia-tions in AAs sequence. Basically, the disulphide bridges are influenced by the aqueous solutions in the protein backbone hence, it is crucial to have an initiative in understanding the solubility, stability, and bio-thermodynamic properties of AAs in aqueous solutions [3, 4]. Theoretical and computer simulation studies on the thermodynamic properties of AAs and the role of electrostatics in particular, in this context, become very important in developing a molecular view of how different residues interact with each other and with sol-vent and an ion atmosphere [5-7].

Most of the proteins found in nature have to adopt a spe-cific three dimensional conformation, called folded or native state for proper functioning. Considering the vital impor-tance of proteins in living organisms, the investigation of the structural and functional properties of proteins has been al-ways a priority with biochemists. A challenging and rapidly emerging field of biotechnology is the tailoring of proteins to carry out unique functions at different physiological and process conditions. Protein folding is of particular concern in the production of industrial biocatalysts as well as for storage purpose, where the enzymes are often inactive due to mis-folding. Protein folding is a reversible transition state of a protein composed of AA residues that is in rapid equilibrium between its ordered and disordered states. This equilibrium between the folded and the unfolded states of the protein can

2212-7127/14 $58.00+.00 © 2014 Bentham Science Publishers

�2� Current Biochemical Engineering, 2014, Vol. 1, No. 2 Kumar et al.

be perturbed by changing the thermodynamic state of the system (temperature, pressure, pH) or by changing the com-position with the addition of co-solvents to the solution. In other words, the process of shifting the conformational equi-librium toward the unfolded state is termed as denaturation [8-13]. We may infer that an environmental stress causes a protein to lose its native structure and hence denature it to a state that is much less compact, somewhat more flexible, and highly hydrated. The stability of a protein reflects the extent to which its conformation resists change when subject to stress. Understanding the protein folding process helps us to understand the behavior of the macromolecule and the ob-tained information are of remarkable in overcoming the chal-lenges in both modern biophysical and pharmaceutical con-texts [14]. The folding and unfolding of proteins under co-solvent conditions are dependent on the nature of the co-solvent. In the presence of co-solvents the protein’s struc-tural properties are changed via significant interactions with its functional groups with the co-solvent [14]. Additionally, the stability of protein is also marginally stable against tem-perature and pressure. Various experimental studies have been made in improving the protein stability in co-solvents by various research groups and it has been established and widely accepted through the experimental observations that the protein stability is balanced between intra-molecular in-teraction of protein functional groups (i.e., with AAs) and their interactions with solvents as well [15].

Several existing reviews are available on how the addi-tion of small organic molecules, known as co-solvents, af-fects the folding equilibrium of proteins [16, 17]. However, protein structures have evolved to be stable enough to func-tion effectively, but not so stable that they cannot be readily processed and metabolized in test tubes, as needed by nor-mally active cells. In this context, AAs have been used exten-sively as model compounds for specific aspects of the more complex proteins in aqueous solutions as these small solutes incorporate some of the structural features found in globular proteins [18-22].

Ionic liquids (ILs) have emerged as a new class of green solvents that have considerably replaced the volatile and toxic organic solvents [14, 23-26]. In support for this, Wood and Stephens [27] explicitly elucidate the impact and poten-tial of ILs on the eco-system. ILs are composed of weakly coordinating ions such as organic cation and inorganic or organic anion that have designable properties [28, 29]. Moreover, the ions of ILs induce changes in the solvent structure that either promotes or hinders the binding of the hydrated ions to the protein surface, leading to the changes in protein stability [30]. Depending on the composition and the solvation environment, these ILs can be tuned to fit a spe-cific solute and process requirements, such as solubility or phase separation from an organic or an aqueous phase [31-35]. The ability of ILs to reform water structure (kosmo-tropic) or to destroy the water structure (chaotropic) which influence on protein stability is not well understood. How-ever, in a review Zhao has quantified and explained the ILs hydration properties through several thermodynamic parame-ters including B-coefficients, structural entropies, structural volumes, and ion mobility [36]. Obviously, an interesting aspect is that ILs have emerged as solvents being able to

influence not only physicochemical properties of mixtures but also influencing protein folding properties [37-45]. As solvents, ILs have proved themselves in solubilizing various biomolecules, in increasing the protein activity, high selec-tivity, as a refolding solvent system in biocatalysis, and also in preserving the enzyme’s stability [46-49]. Even though, protein stability in the presence of ILs is in its infancy. At-tention must be paid to see the influence of ILs solubilization on the higher order structure of the proteins/enzymes. Our aim is to successfully illustrate the effective interactions be-tween AAs and ions of ILs in aqueous media through investi-gating their thermodynamic properties. The data collected will surely provide adequate and fundamental understanding of the behavior and stability of AAs in ILs.

2. THE IDEA OF TRANSFER FREE ENERGY FOR

AMINO ACIDS IN AQUEOUS IONIC LIQUID SOLU-TION

The stability of a protein in IL solutions is an important and fundamental behavior of biomolecules and has been the subject of extensive study. An understanding of solvent-protein interactions is necessary in order to comprehend the profound influence of the solvent on protein structure. It is clear that water can influence protein conformations and stability by competing for hydrogen-bonding sites of the biomolecule and effectively solvating the polar groups. Therefore, to gain further insight into the role of co-solvents such as ILs, we turn our attention towards consideration of the interactions between ILs and AAs through the aqueous environment by transfer free energy (�Gtr). Since highly potential applications of ILs in combination with complex biomolecules in the field of protein stabilities, a better under-standing of the mechanism of dissolution and of the interac-tion with fundamental units, AAs, is essential [50, 51]. How-ever, before we move into our main subject, we would like to present a brief idea of��Gtr

and its importance in this section. In 1959, Kauzmann [52] elucidated the interactions contrib-uting to the protein stability that the hydrophobic effect, as manifested in the burial of non-polar groups in the native protein. Later, a more quantitative analysis was exploited by Nozaki and Tanford [53-55], who accepted the paradigm of transfer between two phases and estimated the hydrophobic contributions to the stability of biomolecules. Protein stabil-ity and associations depend on the electrostatic, hydrophobic, van der Waals, and steric interactions with itself and with solvent molecules. Apparently, evaluating �Gtr

is based on measuring the differential solubility of AAs in water-co-solvent mixture which is schematically shown in (Scheme 1). The net stability of a protein is defined as the difference in the free energy (�G) of the native (folded) and denatured (unfolded) states. We can represent the equilibrium between these two states with a simple mechanism as

Thus, the net protein stability is defined as follows:

(1)

Thermodynamic Contribution of Amino Acids Current Biochemical Engineering, 2014, Vol. 1, No. 2 ��

where [U], [F], GU, and GF denote the concentration and free energies of the unfolded and folded states, respectively. This has been attributed to the exposure of non-polar residues to the aqueous solvent during the unfolding process. However, all approaches in understanding the molecular basis of pro-tein stability ultimately depend on reliable experimental de-terminations of the thermodynamics of protein unfolding for protein structure.

Nozaki and Tanford [53-55] introduced the group trans-fer model, which states that the �Gtr of a given protein in its natural or denatured state is simply the sum of the �Gtr of the solvent exposed residues. Based on the solubility measurements of small peptides mixed in water-co-solvent, and combining the thermodynamic hypothesis with the group transfer model, the remaining problem is to accu-rately model the �Gtr of individual AAs, which are assumed to be embedded in proteins in a solvent-accessible manner. Whether this scenario allows in fully understanding the IL-induced AAs stability/destability, it poses a fundamental and important challenge on its own which we treat in this review.

�Gtr is best suited property for identifying the favorabil-

ity or unfavorability of model proteins from water to other co-solvent media, i.e., ILs. A basic level of quantifying protein-solvent interactions involve the use of �Gtr. The �Gtr of AAs from water to a co-solvent system will be ei-ther favorable or unfavorable. By definition, an unfavorable �Gtr > 0, means that the protein becomes solvophobic on transfer to a co-solvent, which accounts for an increase in protein stability, whereas a favorable transfer free energy, �Gtr < 0, represents that the protein becomes solvophilic on transfer to a kind of co-solvents, which indicates the

destabilization of proteins. The net interaction �G of a co-solvent for the protein surface, whether it is the native or unfolded form, may be considered on the sign and magni-tude of �Gtr contributions of each protein component group with the co-solvent. The sign and magnitude of the meas-ured �Gtr quantifies the protein’s response to changes in solvent quality [56].

The �Gtr of transporting a solute molecule between dif-

ferent solvent environments provides the thermodynamic framework needed to explain protein chemical denaturation, and the hydrophobic effect. Early work on obtaining the �G for AAs involves through calculating their thermodynamic properties in presence of co-solvents [56]. In this review, we explore how the hydrophobic effect for a solute can be modi-fied by the addition of solubility modifiers or “Ionic Liq-uids”. The objective of this review is to use, �Gtr data for the purpose of identifying the role of the major functional groups in stabilizing and/or destabilizing the native and unfolded forms of the protein in the presence of ILs. In this review we have systematically explored the �Gtr determinations of AAs in ILs, especially the novel protic ionic liquids (PILs). The structure of these PILs is provided in (Fig. 1).

The study of inter- and intra-molecular interactions in an aqueous environment is very complex. The energetic role of peptide hydrogen bonds (H-bonds) was studied as long ago as 1955 [57] but the subject has made slow progress since then, chiefly because of difficulty in determining how water interacts with the peptide group both in the unfolded and folded forms of a protein. In the words of Baldwin [58], the peptides H-bonds are likely to make a significant contribu-tion to the energetics of folding because about two-thirds of the residues in folded proteins make peptide H-bonds. This

(Scheme 1). Schematic representation of relationship between transfer free energies of AAs (�Gtr) and free energy of a protein (�G).

Water Phase Aqueous Ionic Liquid Phase

The sum of the transfer free energies (ΔGtr)

of individual amino acids from water to

ionic liquid

Free energy (ΔG) of a protein

in aqueous ionic liquid=

�� Current Biochemical Engineering, 2014, Vol. 1, No. 2 Kumar et al.

peptide backbone solvation can be predicted through the experimental measurements of peptide solvation that is lim-ited to amides as models for the peptide group.

The �Gtr of side chains and peptide backbone quantify the thermodynamic consequences of solvating a protein spe-cies in a co-solvent solution relative to pure water. Based on the transfer model and experimental �Gtr for these groups it has been proposed that these co-solvents exert their effect on protein stability primarily via the protein backbone. This provides the basis of a universal mechanism for co-solvent-mediated protein stabilization or destabilization by various co-solvents as the protein backbone is shared by all proteins, regardless of side chain sequence. The evaluation of group-values defined the classic Nozaki-Tanford model for urea-induced protein denaturation [53-55]. In that model, the thermodynamic interaction of the side chains with the urea solution gave rise to the long held concept of favorable urea interaction with hydrophobic groups as a driving force in urea's denaturation effect. Recent work corrected the previ-ous measurements for the activities of the model compounds in solution and showed that the strength of the hydrophobic interactions does not change substantially on transfer to urea, and that urea's favorable interaction with the protein back-bone is responsible for its denaturing ability [53-55]. Thus, the �Gtr

dependence of protein stability as a function of co-solvent concentration can be predicted if one assumes that the �Gtr of solvent exposed side chain and backbone groups on the native and denatured states are additive.

3. THE TRANSFER MODEL OF AAS FROM WATER TO ILs

3.a. The Determination of Solubility of AAs in Aqueous

ILs

The procedure of solubility measurements has been de-picted elsewhere in detail [3, 17, 40, 59, 60]. Solubility of

the AAs in aqueous ILs was measured through a solid-disappearance method. The related procedure includes each sample vial containing a fixed amount of solvent i.e. an aqueous IL solution to which a weighed amount of AA was added in order to make a series of mixtures with increasing composition (by weight) of AA. Each sample mixture was prepared by weighing pure compounds to ± 0.1 mg. The samples are sealed in a small glass vial and placed in a cold bath to crystallize the liquid sample. The solidified sample is then immersed in a visual thermostatic bath and shaken vig-orously for observation of solid disappearance at a fixed temperature. The bath temperature was elevated by a tiny increment each time, if the solid in the vial still existed after about 30-min observation. The increment must be as small as 0.1 K, when the temperature is near that of solid disappear-ance.

The solid solubility at 298.15 K was determined experi-mentally by densimetry. After shaking to 36-48 h, the pre-pared solution was left overnight so that the undissolved solid particles settle at the bottom of the sample vial. The supernatant of each solution was removed with the help of a syringe and the solution was filtered through a 0.47 �m dis-posal filter (Millipore, Millex-GS) prior to the density meas-urements. The density measurements (�) are performed with a high precision densimeter, while the temperature of the vibrating tube was maintained within ± 0.03 K. The � values of the samples were obtained at 298.15 K under ambient pressure with an uncertainty of ± 0.00005 g cm-3. Proper calibration at each temperature must be achieved with dou-bly distilled, deionized water and with air as standards after each measurement. The uncertainty of the solubility limit must be lower than ± 1.2 %.

3.b. Methodology for Obtaining��Gtr

The solubility data are used to determine the �Gtr of the AAs from water to IL. A detailed description of the evaluat-ing �Gtr has been delineated in our previous papers [3, 17,

Fig. (1). Structural representation of protic ionic liquids (PILs).


40, 59, 60]. At the solubility limit of the AAs, solid and liq-uid phases are at equilibrium, and thus the fugacities of the AAs in the solid and the liquid phases should be equal. As-suming that the solid phase is pure AAs, the fugacity equality becomes

(2)

or

(3)

where xi is the solubility of the solute i in water or in the IL

solutions, i� the activity coefficient of component i in the

liquid phase, and o

if is the standard-state fugacity to which�

i� refers. Because no liquid exists for pure solute i at the

equilibrium temperature, the standard-state may be chosen as either pure, sub-cooled liquid or the solution at infinite dilu-tion. Eq. (3) can thus be expressed, respectively, as

(4)

or

(5)

where i� is defined with reference to an ideal solution in the

sense of Raoult’s law; i.e., i� = 1 as xi = 1. *

i� is defined with

reference to an ideal dilute solution; i.e., *i� = 1 at infinite

dilution and its formulation can be derived fromi� by

(6)

where is the activity coefficient of component i at infinite dilution.

Based on eq. 5, the solid-liquid equilibrium (SLE) equa-tions for AAs in water and in the aqueous IL system are given as,

(7)

(8)

where the subscript ‘W’ refers to the aqueous system and ‘Wil’ to the aqueous ILs system. Since the left hand sides of eqs. 7 and 8 and are the same at a given temperature, these two equations are thus combined as

(9)

or

(10)

As a result, the �Gtr of the AAs from the water to the IL solutions, �Gtr, can be calculated from the following equa-tion;

(11)

(12)

(13)

(14)

(15)

Eq. 15 can also be expressed in terms of molar concentra-tion;

(16)

where the activity coefficient in terms of molar concentration and its value approaches unity at infinite dilution. Several researchers have ignored the activity coefficient term on the right hand side of eq. 16 for determining �Gtr of various solutions, because obtaining the activity coefficients of AAs in multi-component systems is extremely problematic and difficult [17, 18, 22, 40-44, 53-55, 59-65]. When the activity coefficient term was neglected, �Gtr is best denoted as ap-parent transfer free energies ( ). Nozaki and Tanford

[53-55] noted that the activity coefficient term is a self-interaction coefficient term and found that the ratio of the activity coefficient term makes only a small contribution and was not much greater than the experimental uncertainty. Since then, the activity coefficient calculating problem has been neglected in finding the total �Gtr�from water to solvent (ILs). The �G of transporting a solute molecule between different solvent environments provides the thermodynamic framework that is needed to explain the protein denaturation, and the hydrophobic effect.

4. SOLUBILITY OF AMINO ACIDS IN IL

The solubility limit of AAs was determined graphically at the point of intersection of the two fitted lines for each sys-tem as shown in (Fig. 2). The solubility limits of the AAs in the aqueous solutions are expressed as grams of AAs per 100 grams of co-solvent (ILs), and the densities of the saturated solutions (i.e., at the solubility limit) are represented by �*. In this context, several significant research papers have been documented during the last few years on the solubility and �Gtr of AAs in ILs [40-44]. These AAs range from hydropho-bic to hydrophilic in nature on the polarity scale. The AAs, including Tryptophan (Trp), Tyrosine (Tyr), Histidine (His), Valine (Val), Alanine (Ala), Leucine (Leu) have been stud-ied recently by our group (data not shown). Moreover, to determine the thermodynamic contribution of the peptide backbone unit, dipeptide units such as glycine (Gly), digly-cine (Gly2), triglycine (Gly3) and tetraglycine (Gly4), cy-clo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala), and cyclo(Val-Val) were also studied by Venkatesu and group [40, 41] and their solubilities are discussed in next section of this review.

��

�


Fig. (2). The schematic depiction of obtaining the solubility limit of amino acids (AAS) in aqueous solution. Densities of AAS in water vs. composition of AAS in water at 298.15 K under atmospheric pressure. The solubility limit of the model compound was deter-mined at the point of the intersection of the two fitted lines in each system.

The solubilities as well as the thermodynamic properties of the AAs were studied in detail by Venkatesu‘s group [40-44] in the presence of protic ionic liquids (PILs). Interest-ingly, PILs stand as a versatile subclass of ILs family. These PILs are easily produced by the combination of a Brønsted base and a Brønsted acid [66, 67]. A comparison with the aprotic ILs (APILs) reveals that PILs often have higher fluid-ities than the APILs. On the other hand, in PILs, the sizes of the ions are small; they also tend to melt at lower tempera-tures than their APILs analogues. PILs contain dissociable protons and the obvious difference between the PILs and APILs is the reversible hydrogen transfer between the acid and the base [38]. This implies that for PILs, the properties are closer to the corresponding binary liquid where the trans-fer is weak, whereas the aprotic ILs keep their ionic charac-ter until decomposition [68]. The key property that distin-guishes PILs from APILs is the proton transfer from the acid to the base, leading to the buildup of a hydrogen-bonded network, which is similar to the effects that occur in protein aqueous solutions [69, 70]. The above discussion on PILs suggests that PILs can be used as media for those enzymatic processes, where constant polarity, fluidity and stability of the solvent components are strictly needed at higher tem-peratures. However, stabilizing proteins in ILs for the long term is a multivariate problem [71]. ILs as solvents provide thermal stabilization, and enhance refoldibility, while other ILs act as denaturants and induce aggregation [72]. Our ex-perience with the protic ammonium-based ILs suggest that these ILs act as biocompatible solvent media for the globular protein and as stabilizing agents for several amino acids [37-45].

We have studied the of AAs from water to PILs such

as diethyl ammonium acetate ([Et2NH][CH3COO], DEAA), diethyl ammonium sulfate ([Et2NH][HSO4], DEAS), triethyl ammonium acetate ([Et3NH][CH3COO], TEAA), triethyl ammonium sulfate ([Et3NH][HSO4], TEAS), triethyl ammo-

nium phosphate ([Et3NH][H2PO4], TEAP) and trimethyl ammonium acetate ([Me3NH][CH3COO], TMAA) from solubility measurements. Fig. 3 and 4 represents the solubil-ity limit of AAs at 298.15 K under atmospheric pressure which is plotted as a function of the concentration of ammo-nium-based IL. The solubility of AA in water decreases in the order as Ala > Val > His > Leu > Trp > Tyr (Fig. 3). The order appears to be grouped according to the hydrophobicity with the aliphatic (Ala), whereas, the aromatic (Tyr) residues are more hydrophobic than the charged AA. The results are in correlation with the hydrophobicity scale for AAs obtained by Nozaki and Tanford [73] by comparing the �G of the AAs from water to ethanol phase. The variations in the solubility from Ala to Tyr are due to the long chain in AA. Moreover, as relevant from (Fig. 4), the solubility of glycine and gly-cine peptides (GPs) in water decreases from Gly to Gly4 and c(GG). The trend in the aqueous solubility of GPs follows the trend Gly > Gly2 > Gly3 > c(GG) > Gly4 [41].

Consequently, the aqueous solubility of cyclo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala), and cy-clo(Val-Val) is represented in (Fig. 4 and 5), that follows the order; cyclo(Ala-Gly) > cyclo(Ala-Ala) > cyclo(Gly-Gly) > cyclo(Leu-Ala) > cyclo(Val-Val) [40]. It shows that the highest solubility in water is c(AG) and substantially lower solubility in water is c(LA) or c(VV). This is due to the in-crease in the number of �CH2 groups in the side chains of Leu and Val, that varying the shape and physical properties of c(LA) or c(VV), and correspondingly increasing the hy-drophobic character.

The variation in the solubility of AAs in water is due to the formation of a cage like structure or ‘‘clathrate’’ due to the long chain residues of AAs in an aqueous medium which restricts the motion and the arrangements of the water mole-cules, resulting in the low solubility of branched AAs. Due to this, the AA is only partially surrounded by water molecules. A polarity scale based on the tendencies of AA to leave wa-ter and accumulate at the surface has been developed and �G based on the surface tension of aqueous solutions of AA has been evaluated by Maheshwari and Dhathathreyan [74] which correlates with experimental findings in (Fig. 3 and 4). As a result, the structure of the long chain AA leads to lower values of solubility in water. For these reasons, Ala and Val are very soluble, His and Leu are moderately, and Trp and Tyr are weakly soluble in water (Fig. 3). Analysis of the solubility of AA in ILs in (Figs. 3, 4 and 5), reveal that there is a monotonic decrease in the AA solubility with increasing concentrations of ILs. The decrease in the solubility of AAs reflects that the salting-out effect is dominant in the presence of ILs. The salting-out of the AA reflects an increase in the stability of the native structure of AAs in the presence of ILs. The magnitude of this salting-out effect depends on the na-ture of ILs and generally follows the physical interface of AA structure. The salting-out effect mainly indicates that ILs increase the stability of AA in the presence of ILs. To the best of our knowledge, no solubility data of AA in ammo-nium ILs have been reported in the literature.

The interpretation of the solubility behavior of AA based on the specific individual effect of cation and anion of IL is

��

�

Unsaturated state

Saturated state

Solubility limit


Fig. (3). Plot of solubility against concentration to illustrate the solubility limits (SAA) for Ala (red), Val (green), Leu (blue), His (cyan), Trp (magenta) and Tyr (yellow) in an aqueous solution of ILs: DEAA, DEAS, TEAA, TEAS, TEAP, TMAA at 298.15 K. The values of SAA for Ala and Val are collected from Ref. [43] and those for the rest of AAs are obtained from our recent work (unpublished). The SAA values of Tyr are in between the range of 0.05-0.03, hence not represented in the graph.

difficult to predict, since a balance between competitive in-teraction of the cation, anion and water exist with the AA. The combination of these complex interactions determines the preferential interaction as well as solvation of the IL with AA which is helpful in predicting the differences in the solu-bility limits of the AA in the presence of varying concentra-tions of the ILs in aqueous solution. Moreover, Tome et al. [75, 76] proposed through experimental as well as computa-tional techniques that salting-in and salting-out phenomena possess a common basis which is the competition between water-AA side chain, IL-AA side chain, and water-IL inter-actions. They concluded that the delicate balance between

these interactions is dependent on the relative affinities of the biomolecules to water or to IL cation and anion and the magnitude of the solubilities. As presented, in (Fig. 3), the water affinity of the AA decreases in the order ranging from Ala to Tyr, reflecting an increasing hydrophobicity as the side chain increases. The solubility data from (Fig. 3), pre-dicts that TMAA ILs acted as the strong stabilizer for all AA, whereas these AAs were having a lower solubility in TEAS. This trend in solubility of each of the AA is observed even at higher concentrations of the ILs too. Moreover, the solubility of GPs does not follow a trend in ILs. For the sake of comparison in with 30 % (v/v) of ILs, Gly is more soluble

0%

IL

70

%IL

50

%IL

30

%IL

TMAA

0%

IL

70

%IL

50

%IL

30

%IL

TEAS

0%

IL

70

%IL

50

%IL

30

%IL

DEAS

So

lub

ilit

yo

fa

min

oa

cid

s(g

/10

0g

of

ILs)

Concentration of ILs (V/V)

0%

IL

70

%IL

50

%IL

30

%IL

DEAA

0%

IL

70

%IL

50

%IL

30

%IL

TEAA

0%

IL

70

%IL

50

%IL

30

%IL

TEAP


Fig. (4). Plot of solubility against concentration to illustrate the solubility limits (SAA) for Gly (red), Gly2 (green), Gly3 (blue), Gly4 (cyan), c(GG) (magenta), c(AG) (yellow), c(AA) (dark yellow), c(LA) (navy) and c(VV) (purple) in an aqueous solution of ILs: DEAA, DEAS, TEAA, TEAS, and TEAP at 298.15 K. The values of SAA for selected peptides are collected from Ref. [41].

in TEAP, Gly2 in TEAS, Gly3 in DEAA, Gly4 in DEAA and c(GG) in DEAA (Fig. 4).

It therefore seems that at each IL concentration the elec-trostatic forces are dominant based on the effect of ions of ILs on their ability to screen effective charges on the AA surface. Additionally, it was found that the performance of AA extraction in IL water systems is sensitive to the hydro-phobic character and chemical structures of ions in the ILs. Experimentally, the partitioning behavior of AAs by ILs from aqueous media is mainly influenced by the hydropho-bic character of the AAs, indicating the importance of the hydrophobic effect of AAs as a driving force for their parti-

tion into a particular IL [77]. The polarity of the AA pro-vokes an increase interaction with the more polar ILs. Wang et al. [77] proposed that the electrostatic interactions be-tween the AA surface and the ILs are important for the pref-erence of AA towards various ILs. Since, the presence of water particularly disrupts the hydrogen bond association between the cation and the anion of the ILs, leading possibly to an increase in the number of free ions (cations and anions) and the ionic mobility in the IL phase. This increase would then enhance the electrostatic interactions between the charged surfaces of the AA and the ions of the ILs. Accord-ingly, the partitioning of the AAs will be preferred in an IL with higher water solubility. In support to Wang et al. [77],

0%

IL

70

%IL

50

%IL

30

%IL

TEAS

0%

IL

70

%IL

50

%IL

30

%IL

DEAS

Solu

bil

ity

of

pep

tid

esu

nit

s(g

/10

0g

of

ILs)


0%

IL

70

%IL

50

%IL

30

%IL

DEAA

0%

IL

70

%IL

50

%IL

30

%IL

TEAA

0%

IL

70

%IL

50

%IL

30

%IL

TEAP

Thermodynamic Contribution of Amino Acids Current Biochemical Engineering, 2014, Vol. 1, No. 2 �

Tome et al. [78] proposed that the partitioning behavior of L-Trp between aqueous and IL phases is ruled by a complex interplay of intermolecular forces between the solute and the IL solvent, such as electrostatic interactions between the charged form of the AA and the ions of the IL. The more polar AA, the strong interaction with the IL, consequently the magnitude of the interaction of IL with AA is expected to vary as compared with the solubility of the AA in ILs aque-ous solution.

5. APPARENT TRANSFER FREE ENERGY OF

AMINO ACIDS IN ILs

Attempts to understand the thermodynamic contribution of protein-solvent versus protein-co-solvent interactions have provided a systematic mechanism for understanding the pro-tein folding/unfolding pathways which is very important for both academic and industrial applications [41, 79]. These efforts have begun to provide us the thermodynamic details of the molecular origins of the stabilizing/ destabilizing ac-tion of various ILs towards protein structure [80, 81]. New biophysical methods, such as NMR, fluorescence or circular dichroism, has been developed for examining proteins in their natural environment, but such techniques may not be feasible or adequate for characterizing simple AAs which mimic groups that are solvent-accessible in a protein [15, 82]. The �Gtr measurements play a key role in understanding the mechanism of stabilization or destabilization of AAs in

aqueous ILs [41]. of AA from the water to the ILs at 298.15 K under atmospheric pressure is obtained from the solubility measurements as discussed in early section 3. The

dependencies of the for AA from water to IL solution

as a function of IL concentration is displayed in (Fig. 6).

Importantly, the values of the are positive that point towards the unfavorable interaction between ILs and AA. These unfavorable interactions between ILs and AAs leads to stabilize the AA structure in ILs.

The performance of PILs towards simple AAs is still not clear. In our recent findings (data no published but repre-sented in Fig. 6) we observed that AAs such as Ala, Val, His, Leu, Trp, Tyr are stabilized in the presence of six PILs; TEAS, DEAS, TEAP, TEAA, DEAA, and TMAA. The sta-bility of these AAs follows the Hofmeister series order as TEAS > DEAS > TEAP > TEAA > DEAA > TMAA. Vas-antha et al. [41] obtained the results of values obtained

for GPs from water to ammonium ILs at T = 298.15 K which are plotted in (Figs. 7 and 8). These findings depict that the

values are positive for GPs from water to ammonium IL solutions at T = 298.15 K. We have observed that the

value of GPs linearly increases with the increasing concentration of ILs. The stabilities of GPs as revealed from

the varies with the structure of the GPs. Considering each of the GPs; the for the simple Gly AAs in ILs show

that the DEAS IL is the most efficient stabilizer and the sta-bility order for Gly in ammonium ILs is as follows: DEAS > TEAA > TEAS > DEAA > TMAA > TEAP. On the other hand, the stability of the native state of Gly2 varied from IL to IL; therefore, the stabilizing effects of ILs follow the trend TEAA > TEAP > DEAA > DEAS > TMAA > TEAS. Addi-tionally, the experimental results reveal that the stabilization order for a Gly4 structure in the ILs is; TEAP > TEAA > TEAS > TMAA > DEAS > DEAA. The stability order of

Fig. (5). Plot of solubility against concentration to illustrate the solubility limits (SAA) for Gly (red), Gly2 (green), Gly3 (blue), Gly4 (cyan), c(GG) (magenta), c(AG) (yellow), c(AA) (dark yellow), c(LA) (navy) and c(VV) (purple) in an aqueous solution of ILs: TMAA and DEAP at 298.15 K. The values of SAA for selected peptides are collected from Ref. [41].

��

�

��

�

��

�

��

�

��

�

��

��

�

��

�

So

lub

ilit

yo

fp

epti

des

un

its

(g/1

00

go

fIL

s)

0%

IL

70

%IL

50

%IL

30

%IL

TMAA

0%

IL

70

%IL

50

%IL

30

%IL

DEAP


1� Current Biochemical Engineering, 2014, Vol. 1, No. 2 Kumar et al.

Fig. (6). Plot of apparent transfer energy, against concentration for (a) Ala, (b) Val, (c) Leu, (d) His, (e) Trp and (f) Tyr in 30 % (red),

50 % (green) and 70 % (blue) aqueous solution of ILs: DEAS, TEAS, DEAA, TEAA, TEAP, and TMAA at 298.15 K.

Gly4 in ILs illustrates that the protein stabilization process changes with the variation in ammonium cation from triethyl to trimethyl amine and to diethyl amine. Moreover, the ILs stabilizes c(GG) in the order as; TEAS > DEAS > TEAA > DEAA > TEAP > TMAA.

The varying stability behavior of GPs in combinations of ILs with TEA+ and DEA+ cations with acetate, sulfate and phosphate anions depends not only on the cationic proper-ties. Rather, it is the combined influence of the ionic behav-ior of each ion in the solvent system on the surfaces of GPs. Therefore, it is very interesting to compare the stability be-tween the common acetate anion with varying alkyl chain length in the cation of ammonium ILs (Figs. 7 and 8). Ac-cordingly, more unfavorable interactions were observed for TEA+ as compared to TMA+ on the surface of Gly and Gly2. Additionally, the stability of higher glycyl residues, such as Gly3, Gly4 and c (GG), is of the order; TEA+ > DEA+ [41].

Interestingly, the combination of a common cation such as TEA or DEA with acetate, sulfate and phosphate does not follow the Hofmeister series stability order for the GPs struc-ture. Gly and Gly2 tend to stabilize in the ILs in the follow-ing order of different anions for TEA cation: TEAA > TEAS > TEAP. The acetate anion is represented to be a good stabi-lizer for the Gly structure over phosphate or sulfate anion. Subsequently, in the triethylamine family, the order of stabil-ity of Gly3 structure is TEAP > TEAS > TEAA, whereby the phosphate ion is dominated over the sulfate and acetate ion. Gly4 and c(GG) stability in ILs follow the order for TEA+ with different anions as; TEAP > TEAS > TEAA and TEAS > TEAA > TEAP, respectively. Following Ref. [41], the triethylammonium (TEAA, TEAS, and TEAP) family ILs failed completely to follow the Hofmeister series, hence they are not suitable for explaining protein stability and its struc-tural behavior in hydrophilic ILs. These discrepancies are attributed to the weak hydration properties due to an increase in the hydrophobicity of the anion which has a stabilizing

0

1000

2000

3000

4000

5000

6000

DE

AA

DE

AS

TE

AA

TE

AS

TE

AP

TM

AA

Ala

ΔG' t

r(J

.m

ol-1

)

0

1000

2000

3000

4000

5000

6000

DE

AA

DE

AS

TE

AA

TE

AS

TE

AP

TM

AA

ΔG' t

r(J

.m

ol-1

)

Val

0

1000

2000

3000

4000

5000

6000

DE

AA

DE

AS

TE

AA

TE

AS

TE

AP

TM

AA

Leu

ΔG' t

r(J

.m

ol-1

)

0

500

1000

1500

2000

2500

3000

His

DE

AA

DE

AS

TE

AA

TE

AS

TE

AP

TM

AA

ΔG' t

r(J

.m

ol-1

)

0

250

500

750

1000

DE

AA

DE

AS

TE

AA

TE

AS

TE

AP

TM

AA

Trp

ΔG' t

r(J

.m

ol-1

)

0

500

1000

1500

2000

DE

AA

DE

AS

TE

AA

TE

AS

TE

AP

TM

AA

Tyr

ΔG' t

r(J

.m

ol-1

)

Thermodynamic Contribution of Amino Acids Current Biochemical Engineering, 2014, Vol. 1, No. 2 1�

Fig. (7). Plot of against concentration to illustrate the solubility limits (SAA) for Gly (red), Gly2 (green), Gly3 (blue), Gly4 (cyan), c(GG) (magenta), c(AG) (yellow), c(AA) (dark yellow), c(LA) (navy), and c(VV) (purple) in an aqueous solution of ILs: DEAS, TEAS, DEAA, TEAA, TEAP and TMAA at 298.15 K. The values of SAA for selected peptides are collected from Ref. [41].

effect on the native state of the protein. On the other hand, from (Figs. 7 and 8), the stabilities of cyclic dipeptides (CDs), such as cyclo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala), and cyclo(Val-Val), follow Hofmeister series of ILs in the following order: TEAS > DEAS > TEAA > DEAA > TEAP > DEAP. Obviously, sulfate anions are strong stabilizers, acetates are moderate stabilizers and phos-phate ions are weak stabilizers for CDs [40]. The polarity of the AA provokes an increase interaction with the more polar ILs. The more polar AA, the strong interaction with IL, con-sequently the magnitude of the interaction of IL with AA is expected to vary as compared with the solubility of the AA in ILs aqueous solution [69, 83].

The alkyl chain length in the cation of PILs can lead to the variations in the efficiency of ILs to stabilize the AAs structures. In this context, Greaves and Drummond [66] ex-tensively reviewed the properties of PILs and concluded that for the PILs with alkylammonium cations, the viscosity in-creases with increasing alkyl chain length and significantly increased with methyl substitution on the alkyl chain. It may be possible that the longer alkyl chain on the cation of IL has a screening effect or a steric hindrance effect on the electro-static attractive interactions of the cationic form of the AA with anion of the IL. For the sake of comparison, we con-sider the case of triethylammoniumcation (TEA+) and the diethylammoniumcation (DEA+); TEA+ and DEA+ behaved as stabilizers for all AA. This difference in the behavior of

��

�

TEAP70

%IL

50

%IL

30

%IL

TEAS

70

%IL

50

%IL

30

%IL

DEAS

70

%IL

50

%IL

30

%IL

Ap

pa

ren

ttr

an

sfer

free

ener

gy

(ΔG

´ tr)

,k

Jm

ol-1


DEAA

70

%IL

50

%IL

30

%IL

TEAA

70

%IL

50

%IL

30

%IL

1� Current Biochemical Engineering, 2014, Vol. 1, No. 2 Kumar et al.

water-soluble ILs is attributed to an increase in the alkyl chain length of their cations that may lead to increase in the viscosity of the ILs. Apparently, TEAA has a higher viscos-ity (24.12 mPa s) than DEAA (16.36 mPa s) thereby result-ing in higher stabilizing ability in TEAA than in DEAA for the structure of AA [43]. The possible explanation is that the high viscosity of ILs slows down the conformational changes of biomolecules, allowing a more compact structure of AA. Moreover, because the strong interaction of the ions of IL with water competes effectively for water molecules associ-ated with AA surface, thus the strongly solvated IL is ex-cluded from AA surface. This encourages AA surface to be primarily and preferentially solvated by water molecules.

The role of anion in predicting AA stability in ILs is a complex phenomenon and has not been completely explored yet. However, we observed that the HSO4� anion acted as the best stabilizer for the AA whereas, H2PO4� and CH3OO� anions acted as moderate and weak stabilizers and obeys the Hofmeister order as HSO4� > H2PO4� > CH3OO�. Moreover,

the values corresponding to each of the AA in various ILs at different concentrations follow the same order. The

trend in the values of the AA in ILs is well correlated with the results of distinguishing kosmotropic ions and cha-otropic ions based on the Gibbs free energies of hydration (-�G) [84]. It is seen that the kosmotropic ions have larger negative Gibbs free energies of hydration (-�G) than those of chaotropic ions. Thus, it can be expected that the larger -�G, the larger the strength of hydration.

Tome et al. [76] through simulation studies calculated that contrary to the hydrophobic species, AA with a more polar character do not interact with the IL cation and are less effectively bound to the hydrophobic moieties of the IL an-ion, showing thus a clear preference for polar environment.

This can be considered as one of the mechanism by which AA prefers to be highly stabilized by the ILs. According to Yang and Pan [85] the stability through anions of ILs usually follows two types of mechanism. Firstly, due to the lower hydrogen bond basicity of biocompatible anions of ILs, the tendency of the anions to interfere with the internal hydrogen bonds of the biomolecule is minimized [84, 85]. Secondly, the enzyme-compatible anions exhibit lower nucleophilicity and thus would show a lower tendency to change the enzyme conformation by interacting with the positively charged sites in the enzyme structure [85, 86]. Because of the difference in the cations and anion species, different ILs are solvated to different extent [87]. Thereby, strongly solvated ILs stabilize AA and promote salting-out. The solvated ILs interact poorly with weakly solvated AA and are excluded from the surface of AA. This indicates that AA adopts a stabilized structure with a minimum of exposed surface area to water molecules. Recently, results by Buchfink et al. [28] contributed to our understanding that preferentially excluded ILs tends to stabi-lize protein structure.

AAs are amphoteric substances and exist as anions, cations, or neutral molecules depending on their isoelectric points and solution conditions. Therefore, it is deduced that the electrostatic interaction between AA and ILs play an important role in protein stabilization. Therefore, hydropho-bic interaction between AAs and ILs is also suggested to account for the partitioning behavior of AAs observed in various ILs systems [88, 89]. However, the explanation of IL induced protein model compound stability is still not clearly understood. This discrepancy may be primarily due to an increased hydrophobicity of the anion which has an indirect stabilizing effect on the structure of the proteins [90]. Over-all, the results of the anion and cation, demonstrated that the combination of sulfate and acetate anions with higher alkyl

Fig. (8). Plot of against concentration to illustrate the solubility limits (SAA) for Gly (red), Gly2 (green), Gly3 (blue), Gly4 (cyan), c(GG) (magenta), c(AG) (yellow), c(AA) (dark yellow), c(LA) (navy) and c(VV) (purple) in an aqueous solution of ILs: TMAA and DEAP at 298.15 K. The values of SAA for selected peptides are collected from Ref. [41].

��

�

��

�

��

�


TMAA

70

%IL

50

%IL

30

%IL

DEAP

70

%IL

50

%IL

30

%IL

Ap

pare

nt

tran

sfer

free

ener

gy

(ΔG

' tr)

kJm

ol-1

Thermodynamic Contribution of Amino Acids Current Biochemical Engineering, 2014, Vol. 1, No. 2 1�

chain at the substituted ammonium cation, ILs show essen-tially effective and strong stabilizer for the structure of AA.

6. SIMULATION STUDIES OF ENZYMES IN IONIC

LIQUIDS

Molecular dynamics (MD) simulation has established as a valuable tool for studying the protein-solvent interactions at the atomistic level. MD has been used to explore how the ILs interact with enzymes and how they affect on their con-formation structure and dynamics [91-94]. Micaelo and Soare have performed MD of serine protease cutinase in aqueous solution of two ILs, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium nitrate ([BMIM][NO3]) [91]. Klahn et al. have simulated Candida antarcticalipase B (CAL-B) in eight different ILs [92, 93]. The influence of 1-ethyl-3-methylimidazolium trifluoromethanesulfonate on the con-formational dynamic of the protein ubiquitin and a zincfinger motif was also investigated by MD [94]. The simulations revealed that IL with a high charge density, anions, preferen-tially clusters in the vicinity of the oppositely charged AAs. The interactions of the anions with the protein surface are dominated by Coulomb interactions, and thus their mean residence time at protein surface is much longer as compared with those of ions having a low charge density or water. The anions are also strongly hydrated in the bulk of the solution. On the other hand, the affinity of ions with low charge den-sity, cations, for the oppositely residues is less pronounced. They aggregate in hydrophobic clusters in the bulk of the solution and can be found nearby the anions in the vicinity of AAs with like charge, as well as in the proximity of the polar and non-polar residues. The interactions of the hydrophobic parts of cations with the protein surface are dominated by van der Waals interactions. Therefore, their mean residence time is shorter and more mobile at the protein surface. In-deed, the binding of anions to protein backbone and the exis-tence of cations on the protein surface play a vital role for the protein stability.

To get insights into the polarity of the cations and anions DEAA, DEAS, TEAA, TEAS, TEAP, and TMAA, we calcu-lated their �-profiles (Fig. 9), which show the surface charge density distribution on the molecular surface. The �-profiles of the individual compounds of these ILs were calculated by the conductor-like screening model for a realistic solvation (COSMO-RS). The molecular geometries were optimized on the density functional theory (DFT) on BP-TZVP-COSMO level as implemented in TURBOMOLE quantum chemical program package [95]. The �-profile is divided into three regions; H-bond acceptor, non-polar region, and H-bond donor, with H-bond cutoff is 0.079 e�Å-2. H2PO4

2- shows three peaks at (0.024, 0.017, and 0.010) e�Å-2 arising from the strongly negative polar regions of the oxygen atoms, while a strong negative peak at 0.015 e�Å-2 appears for HSO4

-. Two peaks at (0.02 and 0.013) e�Å-2 also appear for acetate ions due to the oxygen atoms. The hydrogen atoms of H2PO4

- and HSO4- give positively peaks at -0.014 e�Å-2 and

-0.016 e�Å-2, respectively, and the acidic hydrogen of the later is much more polar than those of the former. The methyl group of the acetate ion gives two peaks at (0.004 and -0.002) e�Å-2 in the non-polar region. It is clear from these profiles that the polarity of these anions follows the

order, H2PO4- > CH3COO- > HSO4

-. Note that due to the sign inversion of the polarization charge density, �, compared with the molecular polarity, the peaks from the negatively polar oxygen atoms are located on the right side, while the peaks arising from the positively polar hydrogen atoms are located on the left side. The color code of the negative and positive polar regions can be recognized clearly as deep red and deep blue, respectively. In the profiles of the cations, we see that their surfaces are mainly non-polar, i.e., their areas under non-polar regions follow the order [TEA]+ > [DEA]+ > [TMA]+. The polar hydrogens of these cations show strongly positively polar peaks at (-0.019, -0.02, and -0.021) e�Å-2 for [TEA]+, [DEA]+, and [TMA]+, respectively. These hydro-gens are strongly polarized and appear as deep blue. The surface polarization charge densities �, can be useful for understanding the interactions of the ILs with the protein surface and water molecules.

Fig. (9). The �-profiles of HSO4- (black line), CH3COO- (red line),

H2PO4- (blue line), [TEA]+ (pink line), [DEA]+ (dark cyan line), and

[TMA]+ (olive line).

CONCLUSION AND FUTURE PERSPECTIVES

In conclusion, the degree of stabilization through the de-termination of �Gtr of AAs depends on the type of the sol-vent and the additive and is directly providing information related to the stability of a biomolecule existing in the sys-tem. We reviewed that solvents like ILs influenced the sta-bility of the AAs that depends also on the nature of the sur-rounded hydration shell, which is composed of water mole-cules attached to the AAs surface. Moreover, ILs structure and polarity also influences the solubility behavior of AAs in aqueous phase by affecting the hydration level of the en-

-0.04 -0.03 -0.02 -0.01 0.00 0.01 0.02 0.03 0.04024681012141618202224262830

non-polarregion

H-bond donorregion

P(�

)

��e/Å2]

H-bond acceptorregion

138 Current Biochemical Engineering, 2014, Vol. 1, No. 2 Kumar et al.

zyme. In this context, ammonium based PILs represent a large class of non-volatile and also biocompatible solvents for AAs. The results show the decrease in the aqueous solu-bility (salting-out) of the AAs with increasing the concentra-tion of PILs, indicating towards the unfavorable interaction of the ILs with the AA surface. The findings are informative which brings forward the influences of solvation behavior of PILs on AA. This detailed information concludes that PILs are biocompatible and can stabilize three dimensional protein structures through unfavorable interactions with the AA of the protein. Hence, the combined conclusions of the obtained results converge to the point that the PILs are very efficient co-solvent media which are capable of stabilizing the struc-ture of proteins. Moreover, to broaden the applications of PILs in biochemistry and biophysics, further work is re-quired and it is likely that PILs will be an attractive alterna-tive reaction media for enzymes and the most preferred among the biophysical chemists in the future.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

We gratefully acknowledge the Council of Scientific In-dustrial Research (CSIR), New Delhi; Department of Sci-ence and Technology (DST), New Delhi; University Grants Commission (UGC), New Delhi and DU/DST-PURSE grant, New Delhi, India, for financial support.

REFERENCES

[1] S. Doonan, Peptides and Proteins, Royal Society of Chemistry, Cambridge, U.K., 2002.

[2] C. Branden and J. Tooze, “Introduction to protein structure”, 2nd Ed. Garland Publishing, Inc. New York, 1999.

[3] P. Venkatesu, M. J. Lee, and H. M. Lin, “Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K,” Biochem. Eng. J., vol. 32, pp. 157-170, 2006.

[4] S. Damodaran and B. S. Kyung, “The role of solvent polarity in the free energy of transfer of amino acid side chains from water to or-ganic solvents,” J. Biol. Chem., vol. 261, pp. 7220-7222, 1986.

[5] D. Horinek and R. R. Netz, “Can simulations quantitatively predict peptide transfer free energies to urea solutions? Thermodynamic concepts and force field limitations,” J. Phys. Chem. A, vol. 115, pp. 6125-6136, 2011.

[6] S. B. Dixit, R. Bhasin, E. Rajasekaran and B. Jayaram, “Solvation thermodynamics of amino acids assessment of the electrostatic con-tribution and force-field dependence,” J. Chem. Soc. Faraday Trans., vol. 93, pp. 1105-1113, 1997.

[7] C. Y. Hu, H. Kokubo, G. C. Lynch, D. W. Bolen and B. M. Pettitt, “Backbone additivity in the transfer model of protein salvation,” Prot. Sci., vol. 19, pp. 1011-1022, 2010.

[8] D. R. Canchi and A. E. Garcia, “Cosolvent Effects on Protein Sta-bility,” Annu. Rev. Phys. Chem., vol. 64, pp. 273-293, 2013.

[9] G. I. Makhatadze and P. L. Privalov, “Energetics of protein struc-ture,” Adv. Protein Chem., vol. 47, pp. 307-425, 1995.

[10] P. L. Privalov, “Cold denaturation of proteins,” Crit. Rev. Biochem.

Mol. Biol., vol. 25, pp. 281-305, 1990. [11] K. Heremans, “High-pressure effects on proteins and other bio-

molecules,” Annu. Rev. Biophys. Bioeng., vol. 11, pp. 1-21, 1982. [12] S. N. Timasheff, “The control of protein stability and association

by weak interactions with water: How do solvents affect these processes? Annu. Rev. Biophys. Biomol. Struct., vol. 22, pp. 67-97, 1993.

[13] W. Kauzmann, “Protein stabilization: thermodynamics of unfold-ing,” Nature, vol. 325, pp. 763-764, 1987.

[14] A. Kumar and P. Venkatesu, “Overview of the stability of �-chymotrypsin in different solvent media”, Chem. Rev., vol. 112, pp. 4283-4307, 2012.

[15] J. L. England and G. Haran, “Role of solvation effects in protein denaturation: from thermodynamics to single molecules and back”, Annu. Rev. Phys. Chem., vol. 62, pp. 257-277, 2011.

[16] D. R. Canchi and A. E. Garcia, “Cosolvent Effects on Protein Sta-bility,” Annu. Rev. Phys. Chem., vol. 64, pp. 273-293, 2013.

[17] P. Venkatesu , M. J. Lee and H. M. Lin, “Thermodynamic Charac-terization of the Osmolyte Effect on Protein Stability and the Effect of GdnHCl on the Protein Denatured State,” J. Phys. Chem. B, vol. 111, pp. 9045-9056, 2007.

[18] P. Venkatesu , M. J. Lee and H. M. Lin, “Trimethylamine N-oxide counteracts the denaturing effects of urea or GdnHCl on protein denatured state,” Arch. Biochem. Biophys. vol. 466, pp. 106-115, 2007.

[19] G. Barine, G. Castronuovo, P. D. Vecchio and C. Giancola, “The peptide-urea interaction”, J. Chem. Soc., Faraday Trans. I, vol. 85, pp. 2087-2097, 1989.

[20] A. Pertsemlidis, A. M. Saxena, A. K. Soper, T. Head-Gordon and R. M. Glaeser, “Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid,” Proc. Natl. Acad. Sci. U.S.A., vol. 93, pp. 10769-10774, 1996.

[21] Y. Deng and B. Roux, “Hydration of amino acid side chains: non-polar and electrostatic contributions calculated from staged mo-lecular dynamics free energy simulations with explicit water mole-cules,” J. Phys. Chem. B, vol. 108, pp. 16567-16576, 2004.

[22] M. Auton and D. W. Bolen, “Additive transfer free energies of the peptide backbone unit that are independent of the model compound and the choice of concentration scale,” Biochemistry, vol. 43, pp. 1329-1342, 2004.

[23] T. Welton, “Room-Temperature Ionic Liquids. Solvents for Syn-thesis and Catalysis”, Chem. Rev., vol. 99, pp. 2071-2084, 1999.

[24] T. de Diego, P. Lozano, S. Gmouh, M. Vaultier and J. L. Iborra, “Fluorescence and CD spectroscopic analysis of the �-chymotrypsin stabilization by the ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide”, Biotech-

nol. Bioeng., vol. 88, pp. 916-924, 2004. [25] D. Constantinescu, H. Weingärtner and C. Herrmann, “Protein

Denaturation by Ionic Liquids and the Hofmeister Series: A Case Study of Aqueous Solutions of Ribonuclease�A”, Angew. Chem.

Int. Ed., vol. 46, pp. 8887-8889, 2007. [26] N. V. Plechkova and K. R. Seddon, “Application of ionic liquids in

the chemical industry”, Chem. Soc. Rev., vol. 37, pp. 123–150, 2008.

[27] N. Wood and G. Stephens, “Accelerating the discovery of biocom-patible ionic liquids,” Phys. Chem. Chem. Phys., vol. 12, pp. 1670-1674, 2010.

[28] R. Buchfink, A. Tischer, G. Patil, R. Rudolph and C. Lange, “Ionic liquids as refolding additives: variation of the anion”, J. Biotech-nol., vol. 150, pp. 64-72, 2010.

[29] L. P. N. Rebelo, N. Visak, Z. P. Visak, M. Nunes da Ponte, J. Szydlowski, C. A. Cerderina, J. Troncoso, L. Romani, J. M. S. S. Esperanca, H. J. R. Guedes, H. C. de Sousa, “A detailed thermody-namic analysis of [C4mim][BF4] + water as a case study to model ionic liquid aqueous solutions”, Green Chem., vol. 6, pp. 369-381, 2004.

[30] Mu. Naushada, Z. A. ALOthmana, A. B. Khanb and M. Ali, “Ef-fect of ionic liquid on activity, stability, and structure of enzymes: A review,” Int. J. Biol. Macromol., vol. 51, pp. 555-560, 2012.

[31] X. Han and D. W. Armstrong, “Ionic Liquids in Separations,” Acc.

Chem. Res., vol. 40, pp. 1079-1086, 2007. [32] E. Alvarez-Guerra, A. Irabien, “Extraction of lactoferrin with hy-

drophobic ionic liquids,” Sep. Purif. Technol., vol. 98, pp. 432-440, 2012.

[33] C. F. Poole and S. K. Poole, “Extraction of organic compounds with room temperature ionic liquids,” J. Chromatography A, vol. 1217, pp. 2268-2286, 2010.

[34] J. Ranke, A. Othman, P. Fan, A. Muller, “Explaining Ionic Liquid Water Solubility in Terms of Cation and Anion Hydrophobicity”, Int. J. Mol. Sci., vol. 10, pp. 1271-1289, 2009.

[35] E. Feher, B. Major, K. Belafi-Bako and L. Gubicza, “On the back-ground of enhanced stability and reusability of enzymes in ionic liquids”, Biochem. Soc. Trans., vol. 35, pp. 1624-1627, 2007.

[36] H. Zhao, “Are ionic liquids kosmotropic or chaotropic? An evalua-tion of available thermodynamic parameters for quantifying the ion

Thermodynamic Contribution of Amino Acids Current Biochemical Engineering, 2014, Vol. 1, No. 2 139

kosmotropicity of ionic liquids,” J Chem. Technol. Biotechnol.,vol.81, pp. 877-891, 2006.

[37] A. Kumar and P. Venkatesu, “Prevention of insulin self-aggregation by protic ionic liquid”, RSC Adv. (Commun.), vol. 3, pp. 362-367, 2013.

[38] P. Attri, P. Venkatesu and A. Kumar, “Water and a protic ionic liquid acted as refolding additives for chemically denatured en-zymes”, Org. Biomol. Chem., vol. 10, pp. 7475-7478, 2012.

[39] P. Attri, P. Venkatesu and A. Kumar, “Activity and stability of �-chymotrypsin in biocompatible ionic liquids: enzyme refolding by triethyl ammonium acetate”, Phys. Chem. Chem. Phys., vol. 13, pp. 2788-2796, 2011.

[40] P. Attri and P. Venkatesu, “Thermodynamic characterization of the biocompatible ionic liquid effects on protein model compounds and their functional groups”, Phys. Chem. Chem. Phys., vol. 13, pp. 6566-6575, 2011.

[41] T. Vasantha, P. Attri, P. Venkatesu and R. S. Rama Devi, “Struc-tural basis for the enhanced Stability of protein model compounds and peptide backbone unit in ammonium ionic liquids”, J. Phys.

Chem. B, vol. 116, pp. 11968-11978, 2012. [42] T. Vasantha, P. Attri, P. Venkatesu and R. S. Rama Devi, “Ther-

modynamic contributions of peptide backbone unit from water to biocompatible ionic liquids at T = 298.15 K”, J. Chem. Thermo-

dyn., vol. 45, pp. 122-136, 2012. [43] T. Vasantha, A. Kumar, P. Attri, P. Venkatesu and R. S. Rama

Devi, “Influence of biocompatible ammonium ionic liquids on the solubility of l-alanine and l-valine in water”, Fluid Phase

Equilibria, vol. 335, pp. 39-45, 2012. [44] T. Vasantha, P. Attri and P. Venkatesu, “Ammonium based ionic

liquids act as compatible solvents for glycine peptides,” J. Chem. Thermodyn., vol. 56, pp. 21-31, 2013.

[45] P. Attri and P. Venkatesu, “Exploring the thermal stability of �-chymotrypsin in protic ionic liquids,” Process Biochemistry, vol. 48, pp. 462-470, 2013.

[46] A. Chandran , D. Ghoshdastidar , and S. Senapati, “Groove binding mechanism of ionic liquids: a key factor in long-term stability of DNA in hydrated ionic liquids,” J. Am. Chem. Soc., vol. 134, pp. 20330-20339, 2012.

[47] R. Vijayaraghavan, A. Izgorodin, V. Ganesh, M. Surianarayanan, and D. R. MacFarlane, Long-term structural and chemical stability of DNA in hydrated ionic liquids,” Angew. Chem. Int. Ed., 2010, 49, 1631-1633.

[48] Z. Yang, “Hofmeister effects: an explanation for the impact of ionic liquids on biocatalysis”, J. Biotechnology, vol. 144, pp. 12-22, 2009.

[49] H. Zhao, “Methods for stabilizing and activating enzymes in ionic liquids-a review”, J. Chem. Technol. Biotechnol., vol. 85, pp. 891-907, 2010.

[50] A.A. Tietze, P. Heimer, A. Stark, D. Imhof, “Ionic Liquid Applica-tions in Peptide Chemistry: Synthesis, Purification and Analytical Characterization Processes,” Molecules, vol. 17, pp. 4158-4185, 2010.

[51] J. C. Plaquevent, J. Levilliain, F. Guillen, C. Malhiac, A. C. Gaumont, Ionic Liquids: “New Targets and Media for �-Amino Acid and Peptide Chemistry”, Chem. Rev., vol. 108, pp. 5035-5060, 2008.

[52] W. Kauzmann, “Some factors in the interpretation of protein de-naturation”, Adv. Prot. Chem., vol. 14, pp. 1-63, 1959.

[53] Y. Nozaki and C. Tanford, “The solubility of amino acids and related compounds in aqueous urea”, J. Biol. Chem., vol. 238, pp. 4074-4081, 1963.

[54] Y. Nozaki and C. Tanford, “The solubility of amino acids and related compounds in aqueous thylene glycol solutions”, J. Biol.

Chem., vol. 240, 3568-3573, 1965. [55] Y. Nozaki and C. Tanford, “The solubility of amino acids, digly-

cine, and triglycine in aqueous guanidine hydrochloride solutions”, J. Biol. Chem., vol. 245, pp. 1648-1652, 1970.

[56] P. H. Yancey, M. E. Clark, S. C. Hand, R. D. Bowlus and G. N. Somero, “Living with water stress: evolution of osmolyte systems”,Science, vol. 217, pp. 1214-1222, 1982.

[57] J. A. Schellman, “The stability of hydrogen-bonded peptide struc-tures in aqueous solution”, C.R. Trav. Lab. Carlsberg Ser Chim,vol. 29, pp. 230-259, 1955.

[58] R. L. Baldwin, Protein Folding Handbook. Part I. Edited by J. Buchner and T. Kiefhaber Copyright 8, 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30784-2.

[59] P. Venktesu, H. M. Lin and M. J. Lee “Counteracting effects of trimethylamine N-oxide and betaine on the interactions of urea with zwitterionic glycine peptides,” Thermochim. Acta, vol. 491, pp. 20-28, 2009.

[60] P. Attri and P. Venkatesu, “Refolding of urea-induced denaturation of model proteins by trimethylamine N-oxide,” Thermochim. Acta,vol. 526, pp. 143-150, 2011.

[61] Y. Liu and D. W. Bolen, “The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes,” Bio-

chemistry, vol. 34, pp. 12884-12891, 1995. [62] A. Wang and D. W. Bolen, “A naturally occurring protective sys-

tem in urea-rich cells: mechanism of osmolyte protection of pro-teins against urea denaturation,” Biochemistry, vol. 36, pp. 9101-9108, 1996.

[63] D. R. Robinson and W. P. Jencks, “The effect of compounds of the urea-guanidinium class on the activity coefficient of acetyltetragly-cine ethyl ester and related compounds,” J. Am. Chem. Soc., vol. 87, pp. 2462-2470, 1965.

[64] H. Talukdar, S. Rudra and K. K. Kundu, “Thermodynamics of transfer of glycine, diglycine, and triglycine from water to aqueous solutions of urea, glycerol, and sodium nitrate,” Canad. J. Chem.,vol. 66, pp. 461-468, 1988.

[65] P. Ramasami, “Solubilities of amino acids in water and aqueous sodium sulfate and related apparent transfer properties,” J. Chem. Eng. Data, vol. 47, pp. 1164-1166, 2002.

[66] T. L. Greaves and C. J. Drummond, “Protic ionic liquids: proper-ties and applications”, Chem. Rev., vol. 108, pp. 206-237, 2008.

[67] P. Stange, K. Fumino, and R. Ludwig, “Ion Speciation of Protic Ionic Liquids in Water: Transition from Contact to Solvent-Separated Ion Pairs”, Angew. Chem. Int. Ed., vol. 52, pp. 2990-2994, 2013.

[68] M. Yoshizawa, W. Xu and C. A. Angell, “Ionic Liquids by Proton Transfer: Vapor Pressure, Conductivity, and the Relevance of �pKa

from Aqueous Solutions”, J. Am. Chem. Soc., vol. 125, pp. 15411, 2003.

[69] H. Weingrtner, A. Knocks, W. Schrader and U. Katze, “Dielectric Spectroscopy of the Room Temperature Molten Salt Ethylammo-nium Nitrate”, J. Phys. Chem. A, vol. 105, pp. 8646-8650, 2001.

[70] D. F. Evans, S. H. Chen, G. W. Schriver and E. M. Arnett, “Thermodynamics of solution of nonpolar gases in a fused salt. Hydrophobic bonding behavior in a nonaqueous system”, J. Am.

Chem. Soc., vol. 103, pp. 481-482, 1981. [71] C. Lange, G. Patil and R. Rudolph, “Ionic liquids as refolding

additives: N�-alkyl and N�-(�-hydroxyalkyl) N-methylimidazolium chlorides”, Protein Sci., vol. 14, pp. 2693-2701, 2005.

[72] D. Constatinescu, C. Herrmann and H. Weingartner, “Patterns of protein unfolding and protein aggregation in ionic liquids”, Phys.

Chem. Chem. Phys., vol. 12, pp. 1756-1763, 2010. [73] Y. Nozaki and C. Tanford, “The Solubility of Amino Acids and

Two Glycine Peptides in Aqueous Ethanol and Dioxane Solutions”, J. Biol. Chem., vol. 246, pp. 2211-2217, 1971.

[74] R. Maheshwari and A. Dhathathreyan, “Investigation of surface properties of amino acids: polarity scale for amino acids as a means to predict surface exposed residues in films of proteins,” J. Col. Int. Sci., vol. 277, pp. 79-83, 2004.

[75] L. I. N. Tome, M. Dominguez-Perez, A. F. M. Claudio, M. G. Freire, I. M. Marrucho, O. Cabeza and J. A. P. Coutinho, “On the interactions between amino acids and ionic liquids in aqueous me-dia”, J. Phys. Chem. B, vol. 113, pp. 13971-13979, 2009.

[76] L. I. N. Tome, M. Jorge, J. R. Gomes and J. A. Coutinho, “Molecu-lar dynamics simulation studies of the interactions between ionic liquids and amino acids in aqueous solution”, J. Phys. Chem. B,vol. 116, pp. 1831-1842, 2012.

[77] J. Wang, Y. Pei, Y. Zhao and Z. Hu, “Recovery of amino acids by imidazolium based ionic liquids from aqueous media, Green Chem-

istry, vol. 7, pp. 196-202, 2005. [78] L. I. N. Tome, V. R. Catambas, A. R. R. Teles, M. G. Freire, I. M.

Marrucho, J. A. Coutinho, “Tryptophan extraction using hydropho-bic ionic liquids”, Sep. Purif. Technol., vol. 72, pp. 167-173, 2010.

[79] C. Wu, J. Wang, H. Wang, Y. Pei, “Effect of anionic structure on the phase formation and hydrophobicity of amino acid ionic liquids aqueous two-phase systems”, J. Chromatography A, vol. 1218, pp. 8587-8593, 2011.

[80] L. Jackson, Z. Song and O. Olubajo, “Using ionic liquid [EMIM][CH3COO] as an enzyme-‘friendly’ co-solvent for resolu-

140 Current Biochemical Engineering, 2014, Vol. 1, No. 2 Kumar et al.

tion of amino acids”, Tetrahedron: Asymmetry, vol. 17, pp. 2491-2498, 2006.

[81] J. L. Kaar, A. M. Jesionowski, J. A. Berberich, R. Moulton and A. J. Russell, “Impact of ionic liquid physical properties on lipase ac-tivity and stability,” J. Am. Chem. Soc., vol. 125, pp. 4125-4131, 2003.

[82] Y. L. Shek and T. V. Chalikian, “Volumetric characterization of interactions of glycine betaine with protein groups,” J. Phys. Chem. B, vol. 115, pp. 11481-11489, 2011.

[83] G. Absalan, M. Akhond and L. Sheikhian, “Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liq-uids”, Amino Acid, vol. 39, pp. 167-174, 2010.

[84] T. Takeuchi, B. Jiang and L. W. Lim, “Separation of inorganic anions on hydrophobic stationary phases in ion chromatography”, Anal. Bioanal. Chem., vol. 402, pp. 551-555, 2012.

[85] Z. Yang and W. Pan, “Ionic liquids: green solvents for non-aqueous biocatalysis”, Enzy. Microb. Technol., vol. 37, pp. 19-28, 2005.

[86] S. Park and R. Kazlauskas, “Biocatalysis in ionic liquids-advantages beyond green technology”, Curr. Opin. Biotechnol.,vol. 14, 432-437, 2003.

[87] Z. Yang, Y. J. Yue, W. C. Huang, X. M. Zhuang, Z. T. Chen and M. Xing, “Importance of the ionic nature of ionic liquids in effect-ing enzyme performance,” J. Biochem., vol. 145, pp. 355-364, 2009.

[88] Y. Pei, Z. Li, J. Wang, “Partitioning behavior of amino acids in aqueous two-phase systems formed by imidazolium ionic liquid and dipotassium hydrogen phosphate”, J. Chromatography, vol. 1231, pp. 2-7, 2012.

[89] Y. C. Pei, J. J. Wang, X. P. Xuan, X. J. Lu, “Ionic liquid-based aqueous two-phase extraction of selected proteins”, Sep. Purif. Technol., vol. 64, pp. 288-295, 2009.

[90] S. Chun, S. V. Dzyuba, “Influence of structural variation in room-temperature ionic liquids on the selectivity and efficiency of com-petitive alkali metal salt extraction by a crown ether”, Anal. Chem.,vol. 73, pp. 3737-3741, 2001.

[91] N. M. Micaelo, C. M. Soares, “Protein structure and dynamics in ionic liquids. Insights from molecular dynamics simulation stud-ies”, J. Phys. Chem. B, vol. 112, pp. 2566–2572 , 2008.

[92] M. Klahn, G. S. Lim, A. Seduraman, P. Wu, “On the different roles of anions and cations in the solvation of enzymes in ionic liquids” Phys. Chem. Chem. Phys. Vol. 13, pp. 1649–1662, 2011.

[93] M. Klahn, G. S. Lim, P. Wu, “How ion properties determine the stability of a lipase enzyme in ionic liquids: A molecular dynamics study”, Phys. Chem. Chem. Phys., vol. 13, pp. 18647–18660, 2011.

[94] M. Haberler, C. Schro �der, O. Steinhauser, “Hydrated ionic liquids with and without solute: The influence of water content and protein solutes”, J. Chem. Theory Comput., vol. 8, pp. 3911�3928, 2012.

[95] a.d.o.U.o.K.a.F.K.G. TURBOMOLE V6.1 2009, 1989–2007, 25 GmbH, since 2007; available from http://www.turbomole.com.

Received: November 07, 2013 Revised: December 16, 2013 Accepted: December 20, 2013

Thermodynamic Contribution of Amino Acids in Ionic Liquids Towards Protein Stability

Documents