Effect of Imidazolium-Based Ionic Liquids on the ... - ACS Publications

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

The Effect of Imidazolium-Based Ionic Liquids on Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect Indrani Jha, Meena Bisht, Navin Kumar Mogha, and Pannuru Venkatesu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04661 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

The Effect of Imidazolium-Based Ionic Liquids on Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect Indrani Jha, Meena Bisht, Navin Kumar Mogha, P. Venkatesu* Department of Chemistry, University of Delhi, Delhi, India

Abstract In the present work, changes in the structure and stability of stem bromelain (BM) are observed in the presence of a set of four imidazolium-based ionic liquids (ILs) such as 1-ethyl-3methylimidazolium chloride ([Emim][Cl]), 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]) and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]) utilizing various biophysical techniques. Fluorescence spectroscopy is utilized to observe changes taking place in microenvironment around tryptophan (Trp) residues of BM and its thermal stability due to interactions with the ILs at different concentrations. Near UV CD results showed that BM native structure remained preserved at only lower concentrations of ILs. In agreement to these results, DLS revealed the formation of large aggregates of BM at higher concentrations of ILs indicating unfolding of BM. In addition to this, results also show that higher alkyl chain length imidazolium-based ILs have a more denaturing effect on BM structure as compared to the lower alkyl chain length ILs due to the increased hydrophobic interaction between ILs and BM structure. Interestingly, it is noted that that low concentrations (0.01-0.10 M) of short alkyl chain ILs are only altering the structural arrangement of the protein without significant effect on its stability. However, high concentrations of all five ILs are found to be disrupting the structural stability of BM.

1

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 23

1. Introduction Ionic liquids (ILs) are organic salts with an organic moiety is a cation, whereas an organic or inorganic group can act as an anion. A numerous type of ILs have already been synthesized using different combinations of anions and cations.1 ILs have some excellent properties such as very low vapor pressure, non-flammability, very high thermal and chemical stability. As a result of these excellent properties, ILs are now considered as the green solvents for different types of chemical and biochemical processes. A lot of attention is garnered over the use of ILs as a solvent for various proteins from past two decades.2,3 It is well known that arrangement of amino acid side chains in three-dimensional space is greatly influenced by the surroundings in which it present. Subsequently, different enzymes also get affected by the presence of various ILs in their vicinity, in terms of their structure, stability, and activity. Additionally, it is also important to consider mechanistic interactions of ILs with enzymes as it is very necessary from the point of biocatalytic activity of enzyme.4–7 Some of the experimental studies are available citing the effects of alkyl chain length of cation and anion of ILs on proteins stability.8 In some reports, the thermal stability of lysozyme (Lyz) has studied in presence of 1-ethyl-3-methylimidazolium cation with various anions9. Similarly, bovine serum albumin (BSA) has also been used for its interaction studies with 1tetradecyl-3-methylimidazolium bromide ([C14mim][Br])10 and its spectral behavior of its aqueous system in presence of 1-butyl-3-methylimidazolium chloride ([Bmim] [Cl]) and 1butyl-3-methylimidazolium nitrate ([Bmim][NO3])11. Many theoretical studies have also been conducted highlighting the interactions between ILs and proteins.2,3 Subsequently, many different studies have engrossed upon the influence of ILs on the aggregation, conformational stability, and activity of different enzymes/proteins.12 Different studies indicated that in most of the cases imidazolium-based ILs destabilizes proteins12 but in contrary to this an imidazoliumbased IL i.e. 1-allyl-3-methylimidazolium chloride [Amim][Cl] improves the stability of proteins significantly.13,14 In a similar way, Yamaguchi et al.15 studied the effects of imidazolium-based ILs on the structure of Lyz where ILs acted as a refolding agent, and the results obtained are encouraging where protein structure has been stabilized by imidazolium-based ILs. Tamura et al.16 investigated the effect of imidazolium-based ILs on thermal stability and activity of cytochrome c. In their work, a series of imidazolium-based ILs is examined and resulted in a 2


Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dramatical increase in thermal stability and retention of excellent activity even at 140 ºC, of cytochrome c, which loses its activity at 50 ºC. Interaction studies of a various aqueous solution of ILs with biomolecules have attracted a lot of attention recently as they provide better physiological conditions compared to pure ILs. 11,17–19

Our group has investigated the stability study of BM in presence of 1-butyl-3-

methylimidazolium as cation with different anions of ILs in aqueous solution.20 It is very important to study the effect of different cations and variation in alkyl chain length with fixed anion in different ILs on a different class of proteins while studying about their stability behavior. The available research suggests that research workers have focused mainly on heme proteins,14,21,22 Lyz9,23, and Candida Antarctica lipase B (CALB)24. Stem bromelain (BM) is one of the proteins on which very few reports are available for stability studies in presence of ILs.13,20 BM belongs to proteolytic cysteinyl protease class obtained from Ananas comosus. It is a very significant enzyme for its therapeutic value because of its anti-inflammatory, antiedematous, antithrombotic, antitumoral, fibrinolytic, immunomodulator, anti-invasive, antimetastatic, etc. properties.25 BM is a 23.8 kDa, α+β protein consisting of 212 amino acid residues having an identical amino acid sequence to papain.26 It also has three disulfide bonds and one asparagine oligosaccharide chain. Additionally, it contains one free cysteine (Cys) residue, five tryptophan (Trp), fourteen tyrosine (Tyr) and six phenylalanine (Phe) residues.25,26 In the present work, the structure and thermal stability of BM are studied in the presence of a series of imidazolium-based ILs with different alkyl chain length in cation viz. 1-ethyl-3methylimidazolium chloride ([Emim][Cl]), 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]) and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]). The interaction of BM in ILs is studied utilizing different biophysical techniques to understand the effect of alkyl chain length and concentration of ILs. The interaction of series of ILs on the tertiary structure of BM and its hydrodynamic diameter (dH) is described using fluorescence, near-UV CD spectroscopy, and dynamic light scattering (DLS) measurements. Also, results are compared with our previous work13 in which 1-allyl-3-methylimidazolium chloride ([Amim][Cl]) was used as IL for structural stability studies of BM. 2. Materials and Methods 2.1 Materials 3



Page 4 of 23

Bromelain (E.C. 3.4.22.32) from Ananas cosmosus and all imidazolium-based ILs (≥97.0%) were purchased from Sigma–Aldrich, USA. Anhydrous sodium phosphate monobasic and sodium phosphate dibasic dihydrate (analytical grade) were procured from Sisco Research Lab (SRL), India. Sodium phosphate buffer solution (10 mM) at pH 7.0 was prepared in distilled deionized water having a resistivity of 18.3 Ωcm. Every protein sample (0.5 mg/mL) was filtered with 0.45 µm disposal filter (Millipore, Millex-GS) using a syringe before taking measurements. ILs were used in 0.01, 0.05, 0.10, 0.50, 1.0, and 1.0 M concentrations. Only in DLS, protein samples are used in 1 mg/mL concentration instead of 0.5 mg/mL. 2.2 Methods The detailed experimental methods used for different techniques has been described our previous articles13,22,27 and also, briefly, in supporting information. 3. Results and Discussions 3.1 Steady-state fluorescence analysis of BM in the presence of imidazolium-based ILs Proteins show some intrinsic fluorescence, which is recognized as highly responsive to the change in its environmental conditions, particularly around the Trp residues. Maximum intensity and an emission wavelength of fluorescence spectra are highly sensitive to the change in microenvironment around fluorophore of the protein. Consequently, any change in the fluorescence properties of the native protein indicates possible interaction with some external molecule leading to change in microenvironmental conditions.28 As aforementioned, BM comprises of five Trp residues and all are not surrounded by the same microenvironment. Three Trp residues are buried in the hydrophobic core of the protein, however, remaining two are present near the surface of BM.29,30 Steady-state fluorescence spectroscopy is used to monitor the changes occurred in the conformation of Trp in BM with the addition of ILs. Figure 1. shows the fluorescence spectra of BM in the presence of different concentrations of ILs for example [Emim][Cl], [Bmim][Cl], [Hmim][Cl] and [Dmim][Cl] as well as in buffer. Along with these ILs results are compared with [Amim][Cl] whose results were already reported by our group previously.13 For neutral pH excitation wavelength for the BM kept at 295 nm while its emission maximum (λmax) of BM is around 347 nm. It is clearly evident 4


Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from Figure 1 that different ILs are influencing the microenvironment of Trp in BM. A regular decrement in intensity is noted with the increasing concentrations of IL, indicating effective quenching of Trp fluorescence except in some concentrations. The decrement in fluorescence intensity for BM in the presence of [Amim][Cl] up to 0.05 M. Followed by quenching of fluorescence with the minor blue shift from 0.10 to 0.50 M of IL concentration13. While a complete quenching of fluorescence is noted for the concentration of 1.0 to 1.5 M. In case of [Emim][Cl] as illustrated in Figure 1(a) a notable decrease in fluorescence intensity is observed with the addition of 0.01 to 0.1 M of IL. Whereas, at 0.5 M, a complete quenching of fluorescence intensity is observed, moreover, on further increase in the concentration of IL (1.0-1.5 M) a comprehensive red shift is noted with respect to emission observed in the pure buffer for BM. The observed red shift can be attributed to the possible conformational changes in Trp microenvironment of BM. Upon the addition of [Bmim][Cl] (Figure 1(b)), decreased intensity of BM is observed at lower concentrations (0.0 to 0.5 M) which accompanied by the blue shift at higher concentrations (1.0-1.5 M). Figure 1 (c) and 1 (d) depicts the BM fluorescence properties in presence of [Hmim][Cl] and [Dmim][Cl], respectively. At low concentration 0.01 M, fluorescence intensity increases in both cases. With further increase in the concentration, there is a decrease in intensity in both the cases. However, at higher concentration (0.5 to 1.5 M), a complete red shift in case of [Dmim][Cl] is observed, while no such observation is noted in case of [Hmim][Cl]. This remarkable decrease in fluorescence intensity and red shift with the increasing concentration of [Dmim][Cl] indicates the conformational changes occurring due to exposure of Trp residues of BM towards the more polar environment. From these results, it is clear that [Hmim][Cl] and [Dmim][Cl] are behaving in a similar fashion only at a lower concentration. However, conformational changes induced by [Hmim][Cl] and [Dmim][Cl] at higher concentration vary at large extent. Effect of alkyl chain length of imidazolium-based IL i.e from [Emim][Cl] to [Dmim][Cl], contributing in the modification of both Imax and λmax are more pronounced as compared to lower alkyl chain ILs. Therefore, fluorescence studies indicate that interaction between ILs and BM depends on concentration and alkyl chain length of the ILs.

5



Page 6 of 23

Figure 1. Fluorescence spectra analysis of BM in buffer (black), (a) [Emim][Cl], (b) [Bmim][Cl], (c) [Hmim][Cl] and (d) [Dmim][Cl] with red (0.01 M), green (0.05 M), blue (0.1 M), cyan (0.5 M), magenta (1.0 M) and yellow (1.5 M) at 25 0C. Spectra in (c) and (d) were taken at lower working voltage and reduced slit width of emission.

Observed quenching in fluorescence might result from the interaction between protein and ILs, while blue shift is attributed to the increase in hydrophobicity around the exposed Trp residues of BM. As a result of the addition of ILs, there is a possibility of water molecules to be removed from the vicinity of BM residues, in particular, Trp residues. Consequently, resulted in a blue shift of fluorescence spectra.31,32 The λmax of BM at 347 nm suggests that some Trp residues are relatively more exposed to the solvent, and the noted blue shift in BM spectra might be due to conformational changes occurred around exposed Trp residues. As a result, the interaction of BM and ILs contribute to an alteration in microenvironment protein intrinsic fluorophores which result in a considerable change in the structure of BM. Hence, the

6


Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fluorescence analysis results compelled us to observe the thermal stability of BM in the presence of ILs. 3.2 Thermal fluorescence analysis of BM in the presence of imidazolium-based ILs Thermal fluorescence spectroscopy for BM in imidazolium-based ILs is performed to analyze protein’s thermal stability. Transition temperature (Tm) of protein gives a clear evidence for its thermal stability. Therefore, Tm measurement is used as a tool for examining the thermal stability of BM in presence of ILs. Figure 2 displays obtained Tm values of BM in the buffer and in ILs, results are also provided in Table S1, offering significant information into how ILs affects BM structure under similar conditions. The results in Figure 2 show that Tm of native BM is 66.9 °C in the presence of a buffer. Figures 2 (a) and 2 (b) represent the effect of [Emim][Cl] and [Bmim][Cl] on the Tm of BM, respectively. At lower concentrations (0.01-0.1 M) of both ILs, there is negligible change in Tm values as compared to buffer. At higher concentration (1 M) of [Emim][Cl], Tm value is decreased from 66.90 to 60.01°C and no transition plot was obtained for 1.5 M. However, in case of [Bmim][Cl], there is insignificant change in Tm values. The results in Figures 2(c) and 2(d) shows that Tm values are significantly decreasing for higher concentrations of [Hmim][Cl] and [Dmim][Cl] which show the decrease in the stability of BM during thermal unfolding. Thermal stability order of ILs: [Bmim][Cl] > [Emim][Cl] > [Amim][Cl] > [Hmim][Cl] > [Dmim][Cl] as is compared by Tm values provided in Table S1. From our previous work13, where [Amim][Cl] is used as IL a regular decrease in Tm value is observed except at 0.01 M hence decrement in thermal stability of BM was observed. On the other hand, the results indicate the denaturing conditions imposed by [Cnmim][Cl] (n=2,4,6 and10) ILs which follows order: [Dmim][Cl] > [Hmim][Cl] > [Emim][Cl] > [Bmim][Cl] as can be clearly observed from Tm values given in Table S1. Consequently, from lower to higher concentration and [Emim][Cl] to [Dmim][Cl], Tm values are decreasing. Therefore, it can be concluded that higher concentration and higher alky chain ILs are imposing denaturing condition which caused decrease in Tm values.

7



Page 8 of 23

Figure 2. The variation in Tm values of BM in buffer (black), (a) [Emim][Cl], (b) [Bmim][Cl], (c) [Hmim][Cl] and (d) [Dmim][Cl] with red (0.01 M), green (0.05 M), blue (0.1 M), cyan (0.5 M), magenta (1.0 M) and yellow (1.5 M).

3.3 Circular dichroism study of BM in the presence of ILs The structural changes of BM in the presence of imidazolium-based ILs are further observed by using CD spectroscopy. Unfortunately, due to high absorbance of the ILs, spectra in the far-UV region is not observed, only near -UV CD spectrum (260–350 nm) of BM could be investigated, which can provide further insights into protein-ILs interactions. The near UV CD band in the wavelength range from 260 to 350 gives information about the tertiary structure of the protein. Therefore, near-UV CD spectra are monitored to observe the changes in the tertiary structure of BM in the presence of increasing concentration of ILs and obtained spectra are illustrated in Figure 3. The CD spectra of BM show positive bands around 270, 280 nm and a negative band at 299 nm. Figure 3(a) shows the near-UV CD spectra of BM in [Emim][Cl] 8


Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


respectively. Results suggest that tertiary structure of BM is almost preserved up to 0.5 M in [Emim][Cl], after this concentration, there is a complete change in tertiary structures. Figure 3(b) and 3(c) shows that tertiary structure of BM is maintained up to 0.01-0.05 M of [Bmim][Cl] and [Hmim][Cl], respectively. Figure 3(d) revealed that the tertiary structures completely vanished after 0.05 M in the case of [Dmim][Cl]. Moreover, the tertiary structure of BM is found to be more destabilized in the presence of more hydrophobic ILs. This destabilization tendency increased with increase in the chain length of the cation. Consequently, no tertiary structure of a protein is observed in case of [Dmim][Cl] except 0.01 M due to complete denaturation of BM. The perturbed tertiary structure of BM observed is due to its interaction with ILs at high concentration of all ILs, also with the interaction of hydrophobic core with longer alkyl chains of ILs. However, we don’t observe the tertiary structure of BM at 1.0 and 1.5 M concentration of all ILs. These results are in agreement with fluorescence results discussed in previous sections and supports the complete unfolding of the BM in 1.0 and 1.5 M of ILs.

9



Page 10 of 23

Figure 3. near-UV CD analysis of imidazolium family ILs on the structure of BM in buffer (black) from (a) [Emim][Cl], (b) [Bmim][Cl], (c) [Hmim][Cl] and (d) [Dmim][Cl] with red (0.01 M), green (0.05 M), blue (0.1 M), cyan (0.5 M), magenta (1.0 M) and yellow (1.5 M).

3.4 Influence of ILs on the hydrodynamic diameter of BM by dynamic light scattering measurements Furthermore, DLS measurements of the BM in the buffer as well as in various concentrations of the ILs are performed to support the results of fluorescence and CD. The hydrodynamic diameter (dH) of BM in the buffer and in the presence of ILs is presented in Figure 4 and also reported in Table S2. In pure water, the dH of BM is found to be 4.8 nm. DLS data for all the ILs is represented in Figure 4, including [Amim][Cl]13 for comparison. It is very interesting to note that a slight increment in size of BM up to a concentration of 0.10 M for all ILs is observed. This insignificant increase in the size of protein can be attributed to structural perturbation of BM in the presence of ILs. However, a further rise in the concentration of ILs resulted in a vast increase in the size of BM. To sum up the DLS results, larger aggregates with sizes in the range of 300-700 nm are observed at higher concentrations and higher alkyl chain ILs. Furthermore, these DLS results are according to our other results, thus, confirming complete denaturation of BM at high concentration of all ILs.

10


Page 11 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Influence of imidazolium-based ILs on the hydrodynamic diameter (dH) of BM in the buffer and in ILs

The changes observed in wavelength and the intensity in fluorescence spectra with a significant increment in dH and disordered tertiary structure suggest unfolding of BM at high concentrations of ILs. In a completely unfolded state BM shows red shift accompanied by decreased fluorescence intensity in presence of 6 M Guanidinium chloride (GdnHCl).29 Consequently, Fluorescence results suggest that there is a significant difference in conformation of BM in the buffer and in presence of ILs, additionally, BM is not in the completely unfolded state. Accordingly, ILs at lower concentration does not disrupt protein stability significantly but high concentrations of ILs have completely disrupted BM structure causing it to unfold, which is consistent with previous studies performed by our group.14 Lesser disruption in BM structure at lower concentrations of ILs may be due to the fact that protein and ILs interaction might have decreased the distance between the quencher and Trp residues. However, higher concentrations of ILs induces a modification in the solvent microenvironment of surface exposed Trp. Consequently, increasing the hydrophobicity around 11



Page 12 of 23

surface exposed Trp as a result of water molecules removal from their vicinity. It is in accordance with earlier report2 suggesting that imidazolium cation can accumulate around the surface of a protein and tends to eradicate water molecules from its surface. However, some fraction of water molecules can still remain in the protein-water hydration layer. Moreover, in one simulation study33 of the α-helix bundle in the presence of [Bmim][Cl] has revealed that Bmim+ cation can interact with residues on the protein surface. Though, the interior of the protein is inaccessible to cation for interaction due to the presence of hydrophobic clusters for protection. Still, at higher concentration of ILs unfolding of protein occurs leading to the availability of protein interior for interaction to cation as well as anions of ILs. Haq et.al.34 revealed the role of inorganic salts in inducing conformational changes to partially folded BM at pH 2. The authors reported that salts reduce only electrostatic repulsion and do not have a significant effect on the hydrogen bond and hydrophobic interaction at low concentration34. Though, Nordwald and Kaar35 confirmed that preferential exclusion of anion from the surface of protein results in high stability of the α-chymotrypsin (CT) and lipase (CRL). This preferential exclusion avoids strong denaturing interaction of between protein and anion. Similarly, in this present work, the cation is compensating the denaturing effects of anion at lower concentrations of ILs. Additionally, due to the presence of a large amount of water in the vicinity of a protein results in heavy hydration of anion and prevent its direct interaction with positively charged residues of the protein. While, at higher concentrations, vice versa happens due to a lesser amount of water molecules anion is free to interact with the protein. Interestingly, it is also observed that the stability of BM in imidazolium-based ILs decreases significantly as the alkyl chain of the ILs in increased from ethyl to butyl to hexyl and decyl. This conclusion is also supported by various studies.36–39 Huang et al.36 worked with three imidazolium-based ILs such as [Bmim][Cl], [Hmim][Cl] and octyl-3-methylimidazolium chloride ([Omim][Cl]), and observed that the binding strength of ILs with BSA followed, [Bmim][Cl] < [Hmim][Cl] < [Omim][Cl] trend. Here, [Omim][Cl] tends to bind more strongly as compared to other ILs, confirming predomination of hydrophobic forces in ILs-protein interactions. Similarly, in another report by Lange et al.38 mentioned that hydrophobic imidazolium cations carrying longer alkyl chains of ILs are increasingly destabilizing the structure of lysozyme. Similarly, Yamaguchi et al.40 studied several imidazolium-based 12


Page 13 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrophobic cations coupled with a Cl anion and reported that refolding was best realized with short alkyl chain cations as compared to longer alkyl chain cations. According to Silva et al.,41 the longer alkyl groups from imidazolium-based ILs compete with hydrophobic, non-polar patches of the protein driving to its inherent destabilization. This may due to the fact that from lower to higher alkyl chain length, the hydrophobicity of the IL increases, as a result, these ILs can form strong hydrophobic interactions with the polypeptide backbone or binding sites of the enzyme. These hydrophobic interactions may dissociate the Hbonds that maintain the structural integrity of the α-helices and β-sheets, causing the protein to unfold partially or completely.5,42,43 The electrostatic interaction between cationic hydrocarbon chain of IL and hydrophobic amino acid residues in the core of BM is also possible. These electrostatic favorable interactions decrease the free energy of the unfolded state to a large extent, thus, decreased the Tm values. This can be due to stronger interaction between the hydrophobic cation and protein surface which exposes the hydrophobic cores of the protein. 4. Hofmeister series effect on the structure and stability of BM. According to the Hofmeister series, protein shows higher activity and stability with a decrease in the kosmotropicity of the cations. Additionally, the kosmotropic behavior of imidazolium cations increases as with increment in alkyl chain length. In the present work, kosmotropicity increases from [Emim]+ to [Dmim]+. The Hofmeister ion effects become important when the electrostatic forces are screened by higher ions interaction. At very low ILs concentrations (up to ∼0.01M), ions affect enzyme’s performance primarily via electrostatic interactions.44 The experiments also show that both concentration and chain length of the cation of the ILs has a very deep influence on destabilization of the proteins. Results are also suggesting that destabilization tendency of the protein is increased with increase in the kosmotropicity of the cation and concentration. Therefore, it is reasonable to conclude that the imidazolium-based cations have followed the Hofmeister series at higher concentration of ILs in present work (only after 0.01M) in the following order Dmin > Hmim > Bmim > Emim. Thus, it can be depicted that the hydrophobic forces are the main interaction forces in binding the imidazolium-cations of the ILs to BM at higher concentration. Overall, literature concludes that the impact of ILs on enzyme stability is the result of a combination of numerous complex interactions between the enzyme and ILs 13



Page 14 of 23

system. Therefore, kosmotropicity/chaotropicity of the ions is not only the factor affecting enzyme’s stability. Thus, at low concentrations of IL, the cation is mainly interacting with the residues present on the surface of the protein and anion being heavily hydrated participate almost negligibly in the interaction with the protein. However, at high concentrations of ILs, due to less availability of water molecules both the combined effect of cation and anion interacting with the protein, ultimately, resulting in BM unfolding. Similarly, lower alkyl chain length to higher alkyl chain length hydrophobicity in ILs increase so is there interaction with protein hydrophobic core, which ultimately results in a decrement of protein stability. For clarity and representation, the mechanism of interaction between ions of [Bmim][Cl] and BM protein has schematically represented in Scheme 1.

Scheme 1. A plausible mechanism of interaction between ILs and BM

5. Conclusions Results in the present article, illustrate that interactions of ions of ILs with protein are important for understanding the stability of proteins. It is found that the cationic moieties and 14


Page 15 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentration of the imidazolium family ILs play a very crucial role in the interactions of ILs with BM. Our results conclude that stability of BM is IL-concentration dependent where stability of the protein decreases with the increasing the concentration of the imidazolium-based ILs. At low concentrations (0.01-0.5 M) of all ILs, there is an only apparent change in the BM stability, however, the stability of BM is severely affected at higher concentrations depending on the nature of ILs. A possible mechanism concerning the effect of alkyl chain length and concentration of imidazolium-based ILs on the stability of BM stability is also proposed in present work. At low concentration of ILs cation interaction with BM plays a major role, however, at high concentration of ILs, combined effect of anions and cations has resulted in disruption of BM stability. In addition to this, higher alkyl chain length imidazolium-based ILs are more denaturing as compared to the lower alkyl chain length ILs. Stability of BM significantly decreases as an increase in the alkyl chain cation of the ILs from ethyl to butyl and hexyl to decyl. This can be due to strong hydrophobic interaction with the enzyme polypeptide backbone that may dissociate structural integrity of the protein. Therefore, from these results, it can be concluded that at lower concentrations, imidazolium-based ILs can substitute other solvents as better and potentially greener co-solvents for stability studies of different types of proteins. Supporting Information Detailed experimental methods, Transition temperatures, Tm (0C), which are determined by thermal fluorescence analysis of BM in the presence of imidazolium-based ILs (Table S1) and Hydrodynamic diameter (dH) of BM in different concentrations of imidazolium- based ILs (Table S2).

AUTHOR INFORMATION Corresponding Author: [email protected]; [email protected]; Tel: +9111-27666646-142; Fax: +91-11-2766 6605. Acknowledgments

15



Page 16 of 23

We gratefully acknowledge the Council of Scientific & Industrial Research (CSIR), New Delhi, Delhi, India through the Grant No. 01/2871/17/EMR-II for their financial support. ORCID Pannuru Venkatesu: 0000-0002-2066-9807 Navin Kumar Mogha: 0000-0002-2483-1170 References (1)

Zhang, S.; Sun, N.; He, X.; Lu, X.; Zhang, X. Physical Properties of Ionic Liquids: Database and Evaluation. J. Phys. Chem. Ref. Data 2006, 35 (4), 1475–1517.

(2)

Micaêlo, N. M.; Soares, C. M. Protein Structure and Dynamics in Ionic Liquids. Insights from Molecular Dynamics Simulation Studies. J. Phys. Chem. B 2008, 112 (9), 2566– 2572.

(3)

Summers, C. A.; Flowers, R. A. Protein Renaturation by the Liquid Organic Salt Ethylammonium Nitrate. Protein Sci. 2000, 9 (10), 2001–2008.

(4)

Patel, D. D.; Lee, J. Applications of Ionic Liquids. Chem. Rec. 2012, 12 (3), 329–355.

(5)

van Rantwijk, F.; Sheldon, R. A. Biocatalysis in Ionic Liquids. Chem. Rev. 2007, 107 (6), 2757–2785.

(6)

Moniruzzaman, M.; Kamiya, N.; Goto, M. Activation and Stabilization of Enzymes in Ionic Liquids. Org. Biomol. Chem. 2010, 8 (13), 2887–2899.

(7)

Yang, Z. Hofmeister Effects: An Explanation for the Impact of Ionic Liquids on Biocatalysis. J. Biotechnol. 2009, 144 (1), 12–22.

(8)

Patinha, D. J. S.; Alves, F.; Rebelo, L. P. N.; Marrucho, I. M. Ionic Liquids Based Aqueous Biphasic Systems: Effect of the Alkyl Chains in the Cation versus in the Anion. J. Chem. Thermodyn. 2013, 65, 106–112.

(9)

Noritomi, H.; Minamisawa, K.; Kamiya, R.; Kato, S. Thermal Stability of Proteins in the Presence of Aprotic Ionic Liquids. J. Biomed. Sci. Eng. 2011, 4 (2), 94–99.

(10)

Geng, F.; Zheng, L.; Liu, J.; Yu, L.; Tung, C. Interactions between a Surface Active 16


Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Imidazolium Ionic Liquid and BSA. Colloid Polym. Sci. 2009, 287 (11), 1253–1259. (11)

Shu, Y.; Liu, M.; Chen, S.; Chen, X.; Wang, J. New Insight into Molecular Interactions of Imidazolium Ionic Liquids with Bovine Serum Albumin. J. Phys. Chem. B 2011, 115 (42), 12306–12314.

(12)

Jha, I.; Venkatesu, P. Endeavour to Simplify the Frustrated Concept of ProteinAmmonium Family Ionic Liquid Interactions. Phys. Chem. Chem. Phys. 2015, 17 (32), 20466–20484.

(13)

Jha, I.; Bisht, M.; Venkatesu, P. Does 1-Allyl-3-Methylimidazolium Chloride Act as a Biocompatible Solvent for Stem Bromelain? J. Phys. Chem. B 2016, 120 (25), 5625–5633.

(14)

Jha, I.; Venkatesu, P. Unprecedented Improvement in the Stability of Hemoglobin in the Presence of Promising Green Solvent 1-Allyl-3-Methylimidazolium Chloride. ACS Sustain. Chem. Eng. 2016, 4 (2), 413–421.

(15)

Yamaguchi, S.; Yamamoto, E.; Tsukiji, S.; Nagamune, T. Successful Control of Aggregation and Folding Rates during Refolding of Denatured Lysozyme by Adding NMethylimidazolium Cations with Various N’-Substituents. Biotechnol. Prog. 2008, 24 (2), 402–408.

(16)

Tamura, K.; Nakamura, N.; Ohno, H. Cytochrome c Dissolved in 1-Allyl-3Methylimidazolium Chloride Type Ionic Liquid Undergoes a Quasi-Reversible Redox Reaction up to 140°C. Biotechnol. Bioeng. 2012, 109 (3), 729–735.

(17)

Singh, T.; Bharmoria, P.; Morikawa, M.; Kimizuka, N.; Kumar, A. Ionic Liquids Induced Structural Changes of Bovine Serum Albumin in Aqueous Media: A Detailed Physicochemical and Spectroscopic Study. J. Phys. Chem. B 2012, 116 (39), 11924– 11935.

(18)

Wang, X.; Liu, J.; Sun, L.; Yu, L.; Jiao, J.; Wang, R. Interaction of Bovine Serum Albumin with Ester-Functionalized Anionic Surface-Active Ionic Liquids in Aqueous Solution: A Detailed Physicochemical and Conformational Study. J. Phys. Chem. B 2012, 116 (41), 12479–12488.

(19)

Kumar, A.; Venkatesu, P. Overview of the Stability of α-Chymotrypsin in Different 17



Page 18 of 23

Solvent Media. Chem. Rev. 2012, 112 (7), 4283–4307. (20)

Kumar, P. K.; Jha, I.; Venkatesu, P.; Bahadur, I.; Ebenso, E. E. A Comparative Study of the Stability of Stem Bromelain Based on the Variation of Anions of Imidazolium-Based Ionic Liquids. J. Mol. Liq. 2017, 246, 178–186.

(21)

Attri, P.; Jha, I.; Choi, E. H.; Venkatesu, P. Variation in the Structural Changes of Myoglobin in the Presence of Several Protic Ionic Liquid. Int. J. Biol. Macromol. 2014, 69, 114–123.

(22)

Jha, I.; Kumar, A.; Venkatesu, P. The Overriding Roles of Concentration and Hydrophobic Effect on Structure and Stability of Heme Protein Induced by ImidazoliumBased Ionic Liquids. J. Phys. Chem. B 2015, 119 (26), 8357–8368.

(23)

Bisht, M.; Kumar, A.; Venkatesu, P. Analysis of the Driving Force That Rule the Stability of Lysozyme in Alkylammonium-Based Ionic Liquids. Int. J. Biol. Macromol. 2015, 81, 1074–1081.

(24)

Klähn, M.; Lim, G. S.; Seduraman, A.; Wu, P. On the Different Roles of Anions and Cations in the Solvation of Enzymes in Ionic Liquids. Phys. Chem. Chem. Phys. 2011, 13 (4), 1649–1662.

(25)

Rani, A.; Venkatesu, P. Insights into the Interactions between Enzyme and Co-Solvents: Stability and Activity of Stem Bromelain. Int. J. Biol. Macromol. 2015, 73, 189–201.

(26)

Cohen, L. W.; Coghlan, V. M.; Dihel, L. C. Cloning and Sequencing of Papain-Encoding CDNA. Gene 1986, 48 (2–3), 219–227.

(27)

Jha, I.; Attri, P.; Venkatesu, P. Unexpected Effects of the Alteration of Structure and Stability of Myoglobin and Hemoglobin in Ammonium-Based Ionic Liquids. Phys. Chem. Chem. Phys. 2014, 16 (12), 5514–5526.

(28)

Lakowicz, J. R. Principles of Fluorescence Spectroscopy Principles of Fluorescence Spectroscopy; Springer Science+Business Media New York,1999.

(29)

Haq, S. K.; Rasheedi, S.; Khan, R. H. Characterization of a Partially Folded Intermediate of Stem Bromelain at Low PH. Eur. J. Biochem. 2002, 269 (1), 47–52.

(30)

Gupta, P.; Khan, R. H.; Saleemuddin, M. Trifluoroethanol-Induced “Molten Globule” 18


Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


State in Stem Bromelain. Arch. Biochem. Biophys. 2003, 413 (2), 199–206. (31)

Toptygin, D.; Brand, L. Spectrally- and Time-Resolved Fluorescence Emission of Indole during Solvent Relaxation: A Quantitative Model. Chem. Phys. Lett. 2000, 322 (6), 496– 502.

(32)

Wright, W. W.; Guffanti, G. T.; Vanderkooi, J. M. Protein in Sugar Films and in Glycerol/Water as Examined by Infrared Spectroscopy and by the Fluorescence and Phosphorescence of Tryptophan. Biophys. J. 2003, 85 (3), 1980–1995.

(33)

Shao, Q. On the Influence of Hydrated Imidazolium-Based Ionic Liquid on Protein Structure Stability: A Molecular Dynamics Simulation Study. J. Chem. Phys. 2013, 139 (11), 115102–115109.

(34)

Haq, S. K.; Rasheedi, S.; Sharma, P.; Ahmad, B.; Khan, R. H. Influence of Salts and Alcohols on the Conformation of Partially Folded Intermediate of Stem Bromelain at Low pH. Int. J. Biochem. Cell Biol. 2005, 37 (2), 361–374.

(35)

Nordwald, E. M.; Kaar, J. L. Mediating Electrostatic Binding of 1-Butyl-3Methylimidazolium Chloride to Enzyme Surfaces Improves Conformational Stability. J. Phys. Chem. B 2013, 117 (30), 8977–8986.

(36)

Huang, R.; Zhang, S.; Pan, L.; Li, J.; Liu, F.; Liu, H. Spectroscopic Studies on the Interactions between Imidazolium Chloride Ionic Liquids and Bovine Serum Albumin. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2013, 104, 377–382.

(37)

Pan, X.; Liu, R.; Qin, P.; Wang, L.; Zhao, X. Spectroscopic Studies on the Interaction of Acid Yellow With Bovine Serum Albumin. J. Lumin. 2010, 130 (4), 611–617.

(38)

Lange, C.; Patil, G.; Rudolph, R. Ionic Liquids as Refolding Additives: N′-Alkyl and N′(ω-Hydroxyalkyl) N-Methylimidazolium Chlorides. Protein Sci. 2005, 14 (10), 2693– 2701.

(39)

Buchfink, R.; Tischer, A.; Patil, G.; Rudolph, R.; Lange, C. Ionic Liquids as Refolding Additives: Variation of the Anion. J. Biotechnol. 2010, 150 (1), 64–72.

(40)

Yamaguchi, S.; Yamamoto, E.; Tsukiji, S.; Nagamune, T. Successful Control of Aggregation and Folding Rates during Refolding of Denatured Lysozyme by Adding N19



Page 20 of 23

Methylimidazolium Cations with Various N’-Substituents. Biotechnol. Prog. 2008, 24 (2), 402–408. (41)

Silva, M.; Figueiredo, A. M.; Cabrita, E. J. Epitope Mapping of Imidazolium Cations in Ionic Liquid–protein Interactions Unveils the Balance between Hydrophobicity and Electrostatics towards Protein Destabilisation. Phys. Chem. Chem. Phys. 2014, 16 (42), 23394–23403.

(42)

Wu, Y.; Zhang, T. Structural and Electronic Properties of Amino Acid Based Ionic Liquids: A Theoretical Study. J. Phys. Chem. A 2009, 113 (46), 12995–13003.

(43)

Herrera, C.; García, G.; Atilhan, M.; Aparicio, S. A Molecular Dynamics Study on Aminoacid-Based Ionic Liquids. J. Mol. Liq. 2016, 213, 201–212.

(44)

Kumar, A.; Venkatesu, P. Does the Stability of Proteins in Ionic Liquids Obey the Hofmeister Series? Int. J. Biol. Macromol. 2014, 63, 244–253.

20


Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphic The Effect of Imidazolium-Based Ionic Liquids on Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect Indrani Jha, Meena Bisht, Navin Kumar Mogha, P. Venkatesu* Department of Chemistry, University of Delhi, Delhi, India

21



Page 22 of 23

22


Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphic The Effect of Imidazolium-Based Ionic Liquids on Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect Indrani Jha, Meena Bisht, Navin Kumar Mogha, P. Venkatesu* Department of Chemistry, University of Delhi, Delhi, India


Effect of Imidazolium-Based Ionic Liquids on the ... - ACS Publications

Recommend Documents