Does 1-Allyl-3-methylimidazolium chloride Act as a Biocompatible

Jun 7, 2016 - ... chain alcohol through binary mixtures containing thermophysical properties. M. Swetha Sandhya , V. Govinda , I. Bahadur , P. Venkate...
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Does 1‑Allyl-3-methylimidazolium chloride Act as a Biocompatible Solvent for Stem Bromelain? Indrani Jha, Meena Bisht, and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi, 110 007, India ABSTRACT: The broader scope of ILs in chemical sciences particularly in pharmaceutical, bioanalytical and many more applications is increasing day by day. Hitherto, a very less amount of research is available in the depiction of conformational stability, activity, and thermal stability of enzymes in the presence of ILs. In the present study, the perturbation in the structure, stability, and activity of stem bromelain (BM) has been observed in the presence of 1-allyl-3methylimidazolium chloride ([Amim][Cl]) using various techniques. This is the first report in which the influence of [Amim][Cl] has been studied on the enzyme BM. Fluorescence spectroscopy has been utilized to map out the changes in the environment around tryptophan (Trp) residues of BM and also to discuss the variations in the thermal stability of BM as an outcome of its interaction with the IL at different concentrations. Further, the work delineates the denaturing effect of high concentration of IL on enzyme structure and activity. It dictates the fact that low concentrations (0.01−0.10 M) of [Amim][Cl] are only changing the structural arrangement of the protein without having harsh consequences on its activity and stability. However, high concentrations of IL proved to be totally devastating for both activity and stability of BM. The observed decrease in the stability of BM at high concentration may be due to the combined effect of cation and anion interactions with the protein residues. The present work is successful in dictating the probable mechanism of interaction between BM and [Amim][Cl]. These results can prove to be fruitful in the studies of enzymes in aqueous IL systems since the used IL is thermally stable and nonvolatile in nature thereby providing a pathway of alteration in the activity of enzymes in potentially green systems.

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INTRODUCTION Ionic liquids (ILs) are composed of a combination of cation and anion in which the cation is an organic group while the anion can be either organic or inorganic species. By a combination of different cations and anions there is a considerable collection of ILs that can be synthesized.1 Nowadays, they are being considered as a potential replacement for volatile organic compounds due to some of their important properties such as chemical as well as thermal stability, insignificant vapor pressure, nonflammability, and many more. Over the past two decades the studies related to the use of ILs have been growing rapidly particularly as solvents for proteins.2,3 The three-dimensional arrangement of amino acid side chains of the protein is greatly influenced by its surrounding environment. Therefore, it has been observed that each IL can have a different impact on the structure and stability as well as on the activity profile of different enzymes. The mechanistic consideration of interaction between different ILs and different enzymes is very important from the biocatalysis point of view.4−7 There are experimental studies reported in the literature considering the effect of alkyl chain length of cation as well as anions of ILs on proteins.8 Noritomi et al.9 studied the thermal stability of lysozyme in the presence of IL consisting of 1-ethyl3-methylimidazolium cation with various anions. Another experimental work by Fei et al.10 showed the interaction of imidazolium IL 1-tetradecyl-3-methylimidazolium bromide © 2016 American Chemical Society

(C14mimBr) with bovine serum albumin (BSA). In the presence of same protein Shu et al.11 used 1-butyl-3methylimidazolium chloride ([Bmim][Cl]) and 1-butyl-3methylimidazolium nitrate ([Bmim][NO3]) to study the spectral behavior of aqueous systems of IL-BSA. There are several other theoretical studies also which have highlighted the interaction between protein and ILs.2,3 There are many studies that have focused on the influence of ILs on the conformational stability, aggregation, and activity of enzymes/proteins.12 Ammonium-based ILs were found to decrease the thermal stability of hemoglobin (Hb) therefore resulting in unfolding of Hb at lower temperatures as compared to Hb in buffer.13 This is an unexpected result since ammonium family ILs are generally considered to be a stabilizer for the protein structure.12,14 At the same time Hb was found to be destabilized in the presence of the imidazolium family of ILs where the destabilization tendency increased with increase in the chain length of the cation.15 Therefore, from our study as well as from various research by other groups it can be suggested that in most of the cases the proteins are destabilized in the presence of imidazolium-based ILs.12 Nevertheless, the same protein’s stability was very much improved in the presence of imidazolium-based IL, [Amim][Cl].16 This was a Received: April 18, 2016 Revised: June 7, 2016 Published: June 7, 2016 5625

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on the tertiary structure of BM and its hydrodynamic diameter (dH) has been described using near-UV CD spectroscopy and dynamic light scattering (DLS) measurements, respectively. Also, the changes in the functionality (protease activity) have been traced by performing activity measurements in buffer and in the presence of different concentrations of [Amim][Cl]. Further, to reveal the favorable binding sites of the IL in the BM, we have performed molecular docking simulations of the cation of the IL and BM using Molegro Virtual Docker (MVD).

very encouraging result in which the imidazolium-based IL stabilized the protein structure. These studies conclude that protein stability in the presence of ILs is highly dependent on the ILs used, irrespective of the protein, since the same protein Hb showed different behavior in the presence of different ILs. Intense attention has been captured by the study of interactions of biomolecules with various ILs particularly in aqueous medium which provides physiological conditions instead of neat ILs.11,17−19 The research carried out by various groups have shown [Amim][Cl] to be a potential green solvent.20−22 Therefore, these studies give a clear idea about the potential of this IL as a promising solvent for cellulose. Further, to extend the study of this IL, in protein folding studies we have recently shown the stabilizing role of this IL for Hb protein.16 Therefore, to confirm whether this IL can serve as green solvent for other proteins too, we have carried out the stability and activity of stem bromelain (BM) in the presence of this IL. 1,3-dialkylimidazolium chlorides can be used as possible solvents for a range of biomacromolecules; however, it has been reported that at room temperature the maximum number of 1,3-dialkylimidazolium chlorides salts are solid and those which are liquid have high viscosity. These pose limitations on their use as a solvent for biomacromolecules.23 It was found that the imidazolium cation consisting of an allyl group has moderate viscosity as well as a low melting point, which suggested that the presence of the allyl group has major role in lowering the melting point.24 The same conclusion was also reported by Zhang et al.21 who showed that in [Amim][Cl] the substitution in the cation with the allyl group has resulted in low melting and high thermal stability. Therefore, our previous study on the interaction of [Amim][Cl] with Hb proved that this IL stabilized the heme protein which gave additional encouragement to carry further research on the interaction of this IL with other proteins as well. Therefore, in a continuation of our work in this paper we are studying the interaction of BM in the presence of [Amim][Cl]. Obtaining a rule of thumb in the study of protein stability in the presence of ILs is almost essential to study different classes of protein in different ILs. The literature available has mostly focused on heme proteins,14−16 lysozyme,9,25 or Candida antarctica lipase B (CALB),26 indicating that there are many other classes of proteins/enzymes which are still either not or very sparsely researched as far as a study of their stability in the presence of ILs. According to the best of our knowledge no studies on the effect of ILs on the stability of BM which is a proteolytic cysteinyl protease from Ananas comosus have been carried out until now. It is a very important enzyme from a therapeutic point of view since it possesses antidematous, antiflammatory, antithrombotic, fibrinolytic, antitumoral, immunomodulator, antimetastic, and antiinvasive, etc. properties.27 BM is an α+β protein, and also its amino acid sequence is highly identical to that of papain.28 It also contains one asparagines oligosaccharide chain. BM consists of 212 amino acid residues and has a molecular weight of 23.8 kDa. It consists of three disulfide bonds. In addition, it also has one free cysteine residue (Cys). The aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) have 5, 14, and 6, respectively.27,28 In the present work a comprehensive investigation of the structural and thermal stability and activity of BM have been carried out in the presence of imidazolium-based IL [Amim][Cl]. The interaction of BM in [Amim][Cl] has been studied using various biophysical techniques. The effect of interaction

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MATERIALS AND METHODS Materials. Bromelain (E.C. 3.4.22.32) lot no. B4882 from Ananas cosmosus and [Amim][Cl] (≥97.0%) were purchased from Sigma−Aldrich, USA. Anhydrous sodium phosphate monobasic and sodium phosphate dibasic dihydrate of highest purity and analytical grade were purchased from Sisco Research Lab (SRL), India. Sodium phosphate buffer solution (10 mM) at pH 7.0 was prepared using distilled deionized water with a resistivity of 18.3 Ωcm. All the samples were prepared gravimetrically using a Mettler Toledo balance with a precision of ±0.0001 g. The protein solution was filtered with a 0.45 μm disposal filter (Millipore, Millex-GS) through a syringe prior to measurements. The concentration of the protein was fixed to be 0.5 mg/mL. We have used 0.01, 0.05, 0.10, 0.50, 1.0, and 1.0 M of [Amim][Cl]. Throughout the experimental procedure 0.5 mg/mL protein solution in buffer was used apart from DLS where we have used 1 mg/mL of BM in water.

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METHODS Steady-State Fluorescence Experiments. The measurement of the steady state fluorescence emission spectra were carried out in a Cary Eclipse fluorescence spectrofluorimeter (Varian optical spectroscopy instruments, Mulgrave, Victoria, Australia) equipped with an intense Xenon flash lamp as light source. We fixed the excitation wavelength at 295 nm to obtain the contribution only of the Trp residues. The temperature control of the Peltier thermostat cell holder is extremely stable over time, with a precision of ±0.05 °C. Thermal denaturation measurements of the sample were taken at a heating rate of 1 °C min−1. Slit width of excitation and emissions were set at 5 and 10 nm, respectively. Thermal Fluorescence Analysis. The thermal stability of BM in the presence of buffer and in the presence of different concentrations of [Amim][Cl] were analyzed by two-state equilibrium between the folded state (N) and the unfolded state (U) using fluorescence spectra of BM at different temperatures: N⇌U

(1)

The sigmoidal intensity curves for BM were obtained which were further analyzed to obtain the Tm values. Further details of the thermal analysis have been described elsewhere.13 Circular Dichroism Spectroscopy. Circular dichroism (CD) spectroscopic studies were performed using a PiStar-180 spectrophotometer (Applied Photophysics, U.K.) equipped with a Peltier system for temperature control. The protein’s concentration 0.5 mg/mL for near UV and each spectrum was collected by averaging six spectra. CD spectra of BM were measured in the absence as well as presence of [Amim][Cl]. Activity Measurements of BM in the Presence of MCNTCOOH. BM activity has been assayed using UV−vis spectrophotometer (UV-1800 Shimadzu spectrophotometer). The BM activity was assayed using azocasein as substrate by 5626

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especially around the Trp residue. Thus, the changes in the fluorescence properties of the protein in its native form can be used as potential possibility for questioning of interaction with other external molecules.30 As already mentioned BM consists of five Trp residues; however, the sequence homology with papain indicates that all the Trp are not present in the same environment. Three among the five are buried inside the hydrophobic core while the other two are located near the surface of the BM.31,32 The changes occurring in the conformation of BM as a result of addition of the IL was followed using steady-state fluorescence spectroscopy. The fluorescence spectra of BM in buffer as well as in the presence of [Amim][Cl] with excitation at 295 nm have been presented in Figure 1.

observing the rate of release of TCA soluble-azo-coupled peptides which are released from azocasein at 366 nm. The reaction mixture contained 0.5 mL of 1% w/v azocasein and 0.02 mL of enzyme which were diluted in 50 mM Tris-HCl buffer at pH 7.0. The addition of the enzyme aliquot started the reaction. The reaction was arrested by adding 0.5 mL of 20% w/v TCA after incubating for 10 min at 25 °C. After that the test mixture was centrifuged at 14 000g for 5 min, and the supernatant was collected which contained the TCA-soluble azo-coupled peptides. The absorbance of the supernatant was measured at 366 nm against the each of the simultaneously run blanks that did not contain the enzyme. One protease unit (U) is defined as the amount of enzyme required to hydrolyze (and TCA solubilize) one micromole of tyrosine equivalents per minute from soluble casein under standard assay conditions. Dynamic Light Scattering Measurements. The hydrodynamic diameter (dH) of the BM in the absence as well as presence of different concentrations of ILs have been measured by using Zetasizer Nano ZS90 dynamic light scattering (DLS) (Malvern Instruments Ltd., UK). Fixed scattering angle of 90°was used for the size measurement. The instrument is equipped with 4 mW He−Ne laser with a fixed wavelength, λ = 633 nm as a light source and utilized to characterize the obtained aggregate sizes. We have carried out all the DLS experiments at constant temperature of 25 °C. A filtered bubble free sample of around 1.5 mL was transferred into a quartz sample cell, which was sealed with a Teflon-coated screw cap to protect from dust. Then the airtight sample was introduced into the sample holder of the sample chamber of the DLS instrument. The Brownian motion of particles was detected by the DLS and it was correlated to the particle size. The relationship between the size of a particle and its speed due to Brownian motion is defined by the Stokes−Einstein equation. All data obtained were analyzed by Malvern Zetasizer Software version 7.01. Molecular Docking. Molecular docking simulations of the BM and the cation of [Amim][Cl] were performed using MVD trail version (v6.0) downloaded from Molegro with the default parameter settings.29 For the docking purpose we utilized the crystal structure of BM from the PDB bank (PDB code: 1W0Q). The cation [Amim]+, was transformed to the ligand in the MVD. The cations of [Amim]+ were created online using Marvin Sketch, and the geometry of the ligands was optimized using Hyper Chem 8.0 with the default settings. The structures were saved in PDB format for further docking studies. Both the structure of the protein (which was free from water molecules) and the optimized structure of the cation were imported into the docking program. The protein structure which was imported into the docking was free from water molecules. The docking software automatically detected the binding site, and it was manually restricted within spheres with radius of 15 Å. The other required parameters were grid resolution (0.3 Å), maximum iteration (1500 Å), maximum population size (50), and energy threshold (100) and the number of runs was fixed to be 100 for each cavity with 5 docking poses. Moldock and rerank score were used to evaluate the docked results, and the electrostatic interactions were evaluated on the basis of H-bond score.

Figure 1. Fluorescence spectra of BM in buffer (black) and in [Amim][Cl]: (red) 0.01 M, (green) 0.05 M, (blue) 0.10 M, (cyan) 0.50 M, (magenta) 1.0 M), (yellow) 1.5 M at 25 °C.

The emission maximum λmax of BM in neutral pH is around 347 nm. With the addition of increasing concentrations of IL there was a regular decrease in the intensity of fluorescence which means that there was quenching of the Trp fluorescence in the presence of [Amim][Cl]. In the presence of [Amim][Cl] up to 0.05 M concentration there was decrease in the intensity of fluorescence without any shift in the λmax. The spectrum shows quenching of fluorescence along with slight blue shift in the presence of 0.10 and 0.50 M IL concentration. At 1.0 and 1.5 M there was almost negligible fluorescence as compared to BM in buffer. The interaction of IL with the protein molecule appears to be the reason for quenching. The reason for the blue shift can be due to increase in the hydrophobicity around the Trp residues which are exposed. It can be possible that the addition of IL must have resulted in removal of molecules of water around the residues of the BM. Whenever water molecules around Trp move away from their surrounding environment, this results in blue shift of the spectrum.33,34 The λmax of BM at 347 nm indicates that few Trp residues are comparatively more exposed to the solvent. The blue shift in the BM spectra might be due to conformational changes around the Trp residues which are present near to the exposed surface. The above observation points to the existence of interaction between BM and IL which has resulted in the perturbed conformational stability of native BM. The interaction of [Amim][Cl] with BM was characterized with quenching of the Trp fluorescence. There is a clear and sharp decrease in the fluorescence intensity which is continuous with

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RESULTS Fluorescence Measurements and Quenching Analysis. The intrinsic fluorescence of protein has been recognized to be significantly responsive to the environmental conditions 5627

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The Journal of Physical Chemistry B the increasing concentration of the IL. This behavior can be represented as modifications in the microenvironment of the protein intrinsic fluorophores as a result of interactions with IL which can be considered as substantial change in the structure of BM. The graph between maximum fluorescence intensity (Imax) and concentration of IL used gives us clear idea of the region of concentration around which major perturbation of the BM structure has been carried out by the IL. It is quite obvious from Figure 2 that after 0.10 M concentration of IL there is

Figure 3. Stern−Volmer plot of [Amim][Cl] induced fluorescence quenching for BM at 25 °C.

Figure 2. Variation in the intensity of fluorescence of BM in the presence of increasing concentrations of [Amim][Cl].

some foremost transformation in the structure of BM. There is quite a possibility that after 0.10 M IL there are some cooperative interactions which ultimately result in unfolding of the BM which can be motivated by hydrophobic and electrostatic interactions. There is another possibility of growth in the observed BM−IL complex with further increase in the IL concentration. Further, there is quenching of fluorescence of BM in the presence of [Amim][Cl] which can be conveniently described in terms of the Stern−Volmer equation.35 I0 = 1 + KSV[Q ] (2) I where I0 and I are the fluorescence intensities in the absence and presence of ILs. [Q] is the concentration of the IL, and KSV is the Stern−Volmer constant. The Stern−Volmer plot as presented in Figure 3 shows deviation from linearity. In the presence of the IL the plot shows an upward curvature concave toward the y-axis. There is no absolute linear correlation between I0/I and concentration of IL which is indicative of a different quenching mechanism. In this case there is possibility of quenching of fluorescence of the BM in the presence of ILs by collisions and by complex formation with the same quencher. Thermal Stability of BM in the Presence of [Amim][Cl]. To analyze the thermal stability of BM in the presence of [Amim][Cl] we furthermore performed thermal fluorescence spectroscopy. The native BM showed a Tm of 66.9 °C in the presence of buffer (Figure 4). In the presence of [Amim][Cl] except at 0.01 M at all other concentrations there was a decrease in the thermal stability of BM as compared to control. There was a regular decrease in the thermal stability of protein with increasing concentration of ILs (except 0.01 M [Amim]-

Figure 4. Variation in Tm values of BM in buffer and obtained from thermal fluorescence analysis with 0.01 M, 0.05 M, 0.10 M [Amim][Cl]. (We did not obtain fluorescence transitions for 0.50, 1.0, and 1.5 M of [Amim][Cl]).

[Cl]). Therefore, our thermal stability results are consistent with our fluorescence results of denaturation action of the IL upon its binding with BM. In [Amim][Cl] we were unable to obtain the Tm values after 0.10 M due to excessive quenching of the fluorescence spectra at higher concentration of [Amim][Cl]. Circular Dichroism (CD) Study of BM in the Presence of [Amim][Cl]. Near-UV CD spectra were further monitored to observe the changes in tertiary structure of BM in the presence of increasing concentration of IL, and the obtained spectra have been displayed in Figure 5. We observed a perturbed tertiary structure of BM due to its interaction with IL up to 0.50 M. However, we did not observe a tertiary structure of BM at 1.0 and 1.5 M concentration of the IL. These results are again in accordance with our fluorescence results in which we assumed that at 1.0 and 1.5 M IL there is a possibility that the BM-IL has resulted in complete unfolding of the protein. For this reason only here and in the near-UV CD of BM we are not able to observe any tertiary structure of BM at 1.0 and 1.5 M IL concentrations. Changes in the Hydrodynamic Radius of BM in the Presence of [Amim][Cl]. In support of our results of 5628

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435.2, and 425.2 nm in 0.50, 1.0, and 1.5 M [Amim][Cl], respectively. These DLS results are again according to our other results confirming the complete denaturation of BM at high a concentration of [Amim][Cl]. Activity. There are possibilities of loss in enzyme activity as a result of conformational changes in the active site of protein as a result of their interaction with the ILs. On the other hand, such conformational changes can increase the enzyme activity by increasing the accessibility of substrate for the enzyme active site. Also, from the above results it is quite clear that protein structure was distorted although not to a great extent still it was significant in the low concentrations of the IL. To ascertain whether there is any alteration in the activity of BM as a result of its interaction with the ILs, we carry out the proteolytic activity of BM. The proteolytic activities of BM is considerably affected by both pH and temperature and it is found to be more active at a temperature range of 40−60 °C in neutral or basic pH. The enzyme activity for the BM in buffer and in the presence of ILs was obtained by the procedure mentioned under the Materials and Methods section, and results are shown in Figure 7. The activity of BM mainly depends on the thiol

Figure 5. Influence of [Amim][Cl] on the structure of Hb in buffer (black), from near-UV CD analysis with red (0.01 M), green (0.05 M), blue (0.10 M), cyan (0.50 M), magenta (1.0 M), yellow (1.5 M).

fluorescence and CD results we have further performed dynamic light scattering (DLS) measurements of the BM in buffer as well as BM in the various concentrations of the IL (Figure 6). The hydrodynamic diameter (dH) of BM in water

Figure 6. Volume percent distribution of BM in a buffer and in [Amim][Cl] with (red) 0.01 M, (green) 0.05 M, (blue) 0.10 M, (cyan) 0.50 M, (magenta) 1.0 M, (yellow) 1.5 M.

Figure 7. Proteolytic activity measurements of BM in absence and presence of [Amim][Cl]: the variation in relative percentage activity of BM in buffer (black) and in varying concentrations of red (0.01 M), green (0.05 M), blue (0.10 M), cyan (0.50 M), magenta (1.0 M), yellow (1.5 M).

and in the presence of 0.05−1.5 M [Amim][Cl] have been presented in Table 1. In water, the dH of BM was centered around 4.8 nm and it was 5.7, 5.4, and 6.4 in 0.01, 0.05, and 0.10 M [Amim][Cl], respectively. Such a nominal increase in the size of the protein in the presence of ILs can be viewed as a consequence of structural perturbation of BM in the presence of ILs. When there is a further increase of the concentration, there is an enormous increase in the size of BM being 360.3,

group which is present in the Cys-26 N-terminus residue along with the imidazole group present on the C-terminus His-158 residue.36 Therefore, any perturbation in the structure of the protein which can result in the change in the distance between thiol and imidazolium groups can affect the activity of the BM too. The activity of BM is due to concerted action of both imidazole group of Histidine (His) residue and the active site thiol group since there is evidence for the existence of the imidazole group within a 5 Å range from the thiol group.37 Therefore, from the changes in the activity profile of BM in the presence of [Amim][Cl] it can be predicted whether the perturbation caused by the different concentrations of [Amim][Cl] has affected the environment around these residues. From the results of BM activity it is quite apparent that [Amim][Cl] up to 0.10 M does not greatly affect the activity also. That there was decrease in the activity at 0.01 M followed by an increase at

Table 1. Hydrodynamic Diameter (dH) of BM in Different Concentrations of [Amim][Cl] BM in

hydrodynamic diameter dH/nm

buffer 0.01 M [Amim][Cl] 0.05 M [Amim][Cl] 0.10 M [Amim][Cl] 0.50 M [Amim][Cl] 1.0 M [Amim][Cl] 1.5 M [Amim][Cl]

4.8 5.7 5.4 6.4 360.3 435.2 425.2 5629

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Figure 8. Position of the four predicted cavities (interaction site) in BM using Moldock.

Figure 9. Docking images: Predicted cavities 1, 2, 3, and 4 in BM and the interaction of cation of IL [Amim][Cl].

0.05 and 0.10 M [Amim][Cl] suggests that as a result of interaction between [Amim][Cl] and BM slow changes in the structure of BM are taking place which leads to changes in the activity profile. The activity results are also in accordance with our conformational stability results. It is pertinent to observe the changes in the proteolytic activity of BM following its interaction with [Amim][Cl]. The activity profile of BM which

is the percent relative activity of BM as a function of different concentrations of [Amim][Cl] depicts trivial variation in its proteolytic activity at 0.01−0.10 M. The observed activity of BM as a function of the concentrations of ILs used dictates a marked decrease in the proteolytic activity of BM after 0.10 M [Amim][Cl] which is consistent with the denaturation of BM in the presence of ILs up to 0.10 M. 5630

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The Journal of Physical Chemistry B Molecular Docking. Mol Dock was used to prepare the protein structure and four possible interaction sites which are termed as cavities were predicted in the BM structure. The water molecules which are associated with the BM structure were not considered for the docking purpose for better prediction of the interaction sites. The dimensions of the four cavities are 346.624, 50.688, 11.264, and 10.24 Å, respectively. The cavities are termed as cavity 1, 2, 3, and 4, respectively, which has been shown in Figure 8. Cavity 1 consists of residues such as Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val. Cavity 2 composed of Asn, Gly, Trp, Leu, Ala, Glu, His amino acid residues. Ser, Pro, Glu, Ala, Asn, Asp, Cys, Gln, Glu, and Gly were the residues present in cavity 3 while cavity 4 consisted of Ala, Gly, Ile, Leu, Tyr, and Val. Our docking result analysis suggest that [Amim]+ interacts with the residues of BM through hydrogen bonding interactions between N of imidazole group and Met 188 in cavity 1 while in cavity N interacts with Cys 26. In cavity 3 we found hydrogen bonding interactions between N and Gln 53 amino acid residue while in cavity 4 we could not find any such interactions (Figure 9). Therefore, from these results it is clear that hydrogen bonding is playing a major role in the interaction between cation of IL and the protein.

almost an insignificant amount at a high concentration of IL. Because of the denaturation of BM, the required distance of the imidazole group within a 5 Å range from the thiol group must have drastically been affected due to protein unfolding, hence it had the lowest observed activity. These changes in the wavelength, the intensity of fluorescence (0.50 to 1.5 M) with decreased enzymatic activity and increased dH, and disrupted tertiary structure all together signify the unfolding of BM at high concentrations of [Amim][Cl]. According to Haq et al.31 BM when it is completely unfolded in the presence of 6 M GdnHCl showed red shift in the spectra with decreased fluorescence intensity. Therefore, from the fluorescence results it is quite clear that the conformations of BM in buffer and in the presence of the IL are different from each other and also from the completely unfolded state in 6 M GdnHCl. Also, the IL has resulted in the unfolding of the BM thereby decreasing its stability. Our results clearly indicate that the low concentration of [Amim][Cl] is not disrupting the protein stability and activity in a devastating manner, which is consistent with our previous results in which the low concentration of this IL stabilized the Hb protein.16 The experimental results suggest that up to a certain concentration of the IL (0.10 M), the IL is only nominally affecting the structure of protein while at high concentrations it has resulted in a completely disturbed BM structure ultimately resulting in protein unfolding with simultaneous loss in its biological activity. In the lower concentrations of IL, the interaction of IL with residues of protein may have resulted in the decrease in the distance between Trp and the quencher with synchronized variation (either trivial increase or decrease) in its biological activity. At high IL concentration there is change in the solvent environment of surface-exposed Trp where the hydrophobicity around the surface-exposed Trp is increased due to removal of water around the surface-exposed Trp. This is consistent with the results of Micaelo and Soares3 who suggested that imidazolium cation accumulates around the surface of the protein and removes water from its surface, while some fraction of molecules of water are still retained in the protein−water hydration layer. This might be happening in the presence of low concentrations of [Amim][Cl]. However, at higher concentrations water stripping tendency of IL must have increased to such a level which has resulted in complete hydrophobic environment around surface exposed Trp (reason for the blue shift of the λmax) and also removal of essential water molecules around the surface of protein which can be the reason for the loss in the enzymatic activity. Further, Shao38 in his simulation study of α-helix bundle in the presence of 1butyl-3-methylimidazolium chloride [Bmim][Cl] has shown that Bmim+ cation interacted with the residues present on the protein surface. This is again consistent with our docking results in which the interaction between the imidazolium group and residues of the protein through electrostatic interaction has been predicted. However, the groups present in the interior of the protein are protected from the hydrophobic cluster and inaccessible for the cation for the interaction. At high concentrations of [Amim][Cl], due to unfolding of the BM, the carbonyl groups present in the interior of the protein are now available for interaction with the cation as well as the anion. The studies of Kaar and co-workers39 have demonstrated that the stability of the α-chymotrypsin (CT) and lipase (CRL) was due to preferential exclusion of the anion from the surface of the protein which prevents the strong denaturing interaction of protein with the anion. This might be the case with our

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DISCUSSION The various spectroscopic and activity results presents interaction between IL and BM in the concentration dependent manner. Up to 0.05 M there is no shift in the fluorescence spectra while on further increasing the concentration of IL there is observable blue shift in the Trp fluorescence spectra. All these suggest that up to 0.05 M the interaction of IL has caused no change in the solvent environment of Trp residues with a decrease in proximity of the Trp and the quenchers resulting in decrease in Imax. On further increase in the concentration of IL up to 0.10 M, the interaction has resulted in slight movement of Trp toward the hydrophobic environment resulting in the blue shift of the spectra with simultaneous close approach of the quenchers to the Trp having the consequence of decreased Imax. Therefore, up to 0.10 M of the IL is interacting with BM which ultimately results in perturbation of its structure although the effect is only nominal not harsh in nature. This is supported by our other results in which we observed decrease in the thermal stability of BM from 66.9 °C in buffer to 62.2 °C in 0.10 M [Amim][Cl]. Further, near-UV CD also supported our assumption of a perturbed structure. DLS studies have shown nominal increase in the dH values. The activity profile is also consistent with our results. On further increase in the concentration the IL, the Trp fluorescence spectrum shows both fluorescence quenching as well as blue shift in the spectra. The shift toward lower wavelength shows an increased hydrophobicity in the environment surrounding the exposed Trp residues. There is a possibility that upon addition of IL some of the water molecules around the surface of the protein which interact with the the surface of the residues present have been removed. Therefore, the displacement of the water molecules by the IL through consequent interaction with amino acid residues has led to the denaturation of protein at high concentrations of the IL. This is supported from all our other results. In the near-UV CD spectra, we were unable to find the tertiary structure of protein as a result of complete protein denaturation. In DLS also, we observed larger aggregates with sizes in the range of 300−450 nm. The relative percentage activity decreased to 5631

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The Journal of Physical Chemistry B

Scheme 1. Schematic Depiction of Difference in the Mechanism of Interaction in the Low and High Concentrations of [Amim][Cl] and BM

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ACKNOWLEDGMENTS We are grateful for the support from the Department of Biotechnology (DBT), New Delhi, through the Grant/File No. BT/PR5287/BRB/10/1068/2012 for financial support. I.J. is grateful to CSIR, New Delhi, for the Senior Research Fellowship (SRF) award.

system in the low concentrations of [Amim][Cl]. It can be assumed that in the low concentrations there is compensation of the denaturing effect of anion by the cation. At lower concentration of IL the amount of water present in the system more likely results in heavy hydration of the anion and hence it is less probable to interact with the positively charged residues in the protein. While at higher concentrations due to less availability of water molecules Cl− is less hydrated and hence more available to interact with the protein. Therefore, from our results and observations from various simulation studies it can be proposed that in the low concentrations of IL the cation is mainly interacting with the residues present on the surface of the protein, and the anion being heavily hydrated participates almost negligibly in the interaction with the protein. This mechanism of interaction between ions of [Amim][Cl] and BM protein is schematically represented in Scheme 1. However, at high concentrations of IL due to less availability of water molecules, the combined effect of cation and anion interacting with the protein ultimately result in BM unfolding.

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CONCLUSION The effect of imidazolium-based IL [Amim][Cl] on BM stability and activity was investigated in this work. At low concentrations (0.01−0.10 M) of [Amim][Cl], there is ostensible only change in the stability and activity of BM, while the activity and stability of BM were severely affected at higher concentrations (0.50−1.5 M) of [Amim][Cl]. Some possible mechanisms regarding the effect of [Amim][Cl] on BM stability that can be proposed: in low concentrations of [Amim][Cl] cation interaction with BM is dominant while at high concentrations of the IL the combined effect of cation and anion interaction with BM may result in loss in BM activity as well as stability. Therefore, from both of our results it can be concluded that [Amim][Cl] can prove to be a better and greener alternative for the stability of different proteins at its low concentrations.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Tel:+91-11-27666646-142. Fax: +91-11-2766 6605. Notes

The authors declare no competing financial interest. 5632

DOI: 10.1021/acs.jpcb.6b03912 J. Phys. Chem. B 2016, 120, 5625−5633

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