Impact of imidazolium-based ionic liquids on the structure and stability of lysozyme

Article abstract of DOI:10.1080/00387010.2016.1167089

Various types of water-miscible aprotic ionic liquids (ILs) with different cations (1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, 1-octyl-3-methylimidazolium) and anions (ethylsulfate and chloride) were used as co-solvents to investigate the s

Full text of DOI:10.1080/00387010.2016.1167089

Spectroscopy Letters
An International Journal for Rapid Communication
ISSN: 0038-7010 (Print) 1532-2289 (Online) Journal homepage: http://www.tandfonline.com/loi/lstl20
Impact of imidazolium based ionic liquids on the
structure and stability of lysozyme
Lakkoji Satish, Shubhasmin Rana, Manoranjan Arakha, Lipeeka Rout,
Basanti Ekka, Suman Jha, Priyabrat Dash & Harekrushna Sahoo
To cite this article: Lakkoji Satish, Shubhasmin Rana, Manoranjan Arakha, Lipeeka Rout,
Basanti Ekka, Suman Jha, Priyabrat Dash & Harekrushna Sahoo (2016): Impact of imidazolium
based ionic liquids on the structure and stability of lysozyme, Spectroscopy Letters, DOI:
10.1080/00387010.2016.1167089
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Date: 01 April 2016, At: 05:25

Impact of Imidazolium based Ionic Liquids on the Structure and Stability of
Lysozyme
Lakkoji Satish1, Shubhasmin Rana1, Manoranjan Arakha2, Lipeeka Rout1, Basanti Ekka1,
Suman Jha2, Priyabrat Dash1, Harekrushna Sahoo1
1Department of Chemistry, NIT Rourkela, Rourkela, Sundergarh, Odisha, 2Department of
Life Sciences, NIT Rourkela, Rourkela, Sundergarh, Odisha
Corresponding Address: Dr. Harekrushna Sahoo, Department of Chemistry, National
Institute of Technology, Rourkela-769008, Sundergarh, Odisha, Email:
sahooh@nitrkl.ac.in
Abstract
Various types of water-miscible aprotic ionic liquids with different cations (1-ethyl-3-
methylimidazolium, 1-butyl-3-methylimidazolium, 1-octyl-3-methylimidazolium) and
anions ( ethylsulfate and chloride) were used as co-solvents to investigate the stability of
Lysozyme. Different techniques such as fluorescence, thermal absorption and circular
dichroism spectroscopy have been used for the study. Fluorescence results reveal that the
addition of ionic liquids (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-
methylimidazolium) increases the hydrophobicity around the tryptophan environment in
Lysozyme. circular dichroism analysis and temperature dependant studies were done to
investigate the stability of the protein. From the circular dichroism analysis, it was
observed that the ionic liquids keep the native structure of protein intact. Thermal
denaturation studies depicted that the melting temperature of the protein increased in the
presence of ionic liquids (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-
methylimidazolium), which indicates the stabilization of the protein.
1

1. INTRODUCTION
Biological processes such as protein kinetics, stability, and activity are largely influenced
by the physical and chemical properties of solvent/co-solvent, environmental condition,
additives, etc.[1-4] Generally, the biological processes are governed by the three-
dimensional structure of the protein at its folded or native state.[2,3] Chemically, the three-
dimensional native states of proteins are stable due to the existence of several weak
interactions such as hydrogen bonds, ionic and hydrophobic interactions with solvents as
well as other entities present in a medium. The signal of these weak interactions is
dependent on the local/global chemical environment. Keeping these in mind, it is very
much interesting to study the stability (local and global) of protein in different co-solvent
conditions.[5,6] In this regard, ionic liquids (ILs) can act as a potential solvent as they have
already shown promising applications in various pharmaceutical, biomedical and
biotechnological research fields because of their unique properties such as negligible
vapour pressure, good thermal stability, tunable viscosity, water miscibility, favourable
solvating properties, and non-ionizing nature.[7-11] Due to the tunable properties as
discussed above, ILs are used as designer solvents.
Various studies carried out by many research groups have shown that most of the ILs
acted as stabilizers for biomolecules.[12-17] Lau, R M et al. observed that the enzyme,
CALB retains its native structure in the [Et3NMe][MeSO4] by maintaining its acitivity.[18]
Water miscible ammonium and imdazlium based ILs was found to improve the activity
and stability of CALB.[17] In another work, it was investigated that lysozyme effictivley
refolds in ammonium based ILs.[19] Byrne et al. reported that ethylammonium nitrate
2

increases the thermal stability of lysozyme and provides long-term stabilization against
aggregation.[15] It was observed that [chol]Cl markedly increases the thermal stability of
the two enzymes, hen egg white lysozyme and α-lactalbumin.[20] Further, a work by Wei
and Danielson explains the structural stability of cyt c in methyl ammonium and ethyla
ammonium formate.[21] It was reported that ammonium, phosphonium and imidazolium
ILs are able to stabilize the native structure and activity of a-chymotrypsin (CT).[12]
Recently, Kumar, A et al. explored that imidazolium-based ILs act as stabilizers for the
insulin structure.[22]
There were few reports demonstrating that some proteins are highly denaturated by the
ILs. Baker and Heller observed that human serum albumin and equine heart cytochrome c
were destabilized by 1-butyl-3-methylimidazolium chloride.[13] Furthermore,
Constantinescu et al. pointed out that the native structure of protein is also perturbed by
some ILs.[16] Recent work by Mandal et al. reveals the destabilizing effect of 1-butyl-3-
methylimidazolium octylsulfate on lysozyme.[23] Thus, it is very desirable to know the
effect (stabilizing or destabilizing) of ILs on the protein’s structure and stability and a
molecular level understanding is needed to gain a deeper insight into the nature of
interactions between ILs and protein. To gain a mechanistic insight, we explore the effect
of imidazolium based ILs as a function of various concentrations on the structure and
stability of lysozyme with the help of UV-Vis, Fluoroscence and Circular-dichroism
spectrophotometric methods. For the comparison among different types of ILs, we have
used four different types varying the cations and anions. (Table 1) The chemical
structures of ILs are given in Figure 1a.
3

We employed the protein, Lysozyme because of its broad and well-characterized
applications.[5,6] Lysozyme, N-acetyl-muramic-hydrolase, is a bacteriolytic enzyme
commonly found as a single peptide chain consisting of 129 amino acids (~14 kD). In
Lysozyme polypeptide sequence, the nature of amino acid side chain and its arrangement
play a significant role in generating hydrophobicities and polarities in the molecular
structure. Although, Lysozyme is found as a monomer in nature (Hen egg white is an
easy and rich source for Lysozyme), it is more active in dimeric or polymeric form.
Dimeric form exhibits therapeutic, antiviral and anti-inflammatory properties.[24,25]
Therefore, because of their wide applications we used Lysozyme to study its stability as a
function of different types of above mentioned ILs with varying concentrations. Very
limited number of studies in the literature on the effect of ILs on the stability of
Lysozyme make this study very intresting not only from academic view but also from the
industrial aspect. The effect of employed ILs in this study on the thermal stability of
Lysozyme as a function of concentration was not studied earlier as per our knowledge.
2. MATERIALS AND METHODS
2.1 Materials
Chicken egg white Lysozyme (Product No- MB098) was purchased from Himedia. The
protein was used as such received from Himedia without any further purification.
EmimESO4 (Product No-51682), EmimCl (Product No-30764) and BmimCl (Product
No-94128) were purchased from Sigma-Aldrich and used as it is. Whereas, OmimCl was
synthesized as per the procedure given in Figure 1b.[26] 18 MΩ.cm Milli-Q water
4

(Millipore, Bedford, MA) was used as the solvent to prepare 0.01 M phosphate buffer
(pH -7.4), which was used to prepare samples for all the experiments.
2.2 Synthesis Of 1-Octyl-3-Methylimidazolium Chloride
To a 100 ml round bottom flask equipped with reflux condenser and magnetic stirring bar
connected with a nitrogen cylinder, 10 ml (125 mmol) of 1-methyl imidazole and 12.6 ml
toluene was added to an ice bath. After 5 min of stirring 23.4 ml (138 mmol), 1-chloro
octane was added. The solution was heated to reflux at 125 °C for 72 h, yielding a two-
phase mixture of [OmimCl] and toluene. Then the toluene was decanted and the product
was recrystallized with acetone three times. The final product was then dried in a schlenk
line at 80 °C for 7 h before use.
1H NMR (CdCl3, 400 MHz), δ(ppm) : 0.69 (t), 1.00 (m), 1.70 (m), 3.98 (s), 4.17 (t), 4.68
(s), 5.27 (s), 7.38 (d) and 8.44 (d)
2.3 Methods
2.3.1 UV-Visible Spectroscopy
The ultraviolet spectra and thermal profiling of Lysozyme were studied by using UV-Vis
spectrophotometer (Agilent, Cary 100) equipped with a peltier system for maintaining the
temperature of the sample. For measurements, 10 µM protein solution, containing ILs of
varying concentrations, 0, 0.2, 0.6 and 1 M, were employed. The ILs absorption was
subtracted from the respective protein sample containing IL to remove the effect of ILs.
For thermal denaturation studies, absorbance at 280 nm was observed at different
5

temperatures. Thermal profiling of Lysozyme in the absence of IL was taken as control.
Temperature range for the study was maintained at 30 to 90 oC.
2.3.2 Steady-State Fluorescence Spectroscopy
The fluorescence measurements were done using Horiba Jvon Spectrometer (Fluoromax-
4P). In order to perform the signal-based fluorescence measurements, we took the
advantage of fluorescent properties of Trp residue as an intrinsic fluorophore. Trp was
excited at 295 nm to avoid the interference from Tyr (tyrosine) and Phe (Phenylalanine)
and the fluorescence was collected as a function of wavelength (or steady-state) in the
range of 325 to 450 nm. The effect of ILs on the Trp-emission of Lysozyme was
analyzed in terms of the fluorescence signal. For measurements, 10 µM protein solution
containing ILs of varying concentrations (0 - 1 M) were used. The width of the excitation
and emission slit was set to 5 and 5 nm, respectively. Fluorescence signals are corrected
by subtracting blank solutions containing only ILs. This kind of correction was validated
in earlier reports.[27,28]
2.3.3 Circular Dichroism Spectroscopy
Tertiary structures of the protein was studied by a circular dichroism (CD)
spectrophotometer (JASCO-1500) at room temperature under constant nitrogen flush.
The scan speed (100 nm/min) was fixed with a response time of 1 s and 1 nm bandwidth.
Each spectrum was collected by averaging three spectra. The tertiary structure of
Lysozyme was monitored by using near-UV (250–300 nm) (0.1 cm path length cuvette)
spectra. The protein’s concentration was maintained at 100µM and IL concentration was
6

kept at 0.2, 0.6 & 1.0 M. Each sample spectrum was obtained by subtracting the
appropriate background (without protein) from the experimental protein spectrum.
3. RESULTS AND DISCUSSION
3.1. Absorption Measurements
Absorption spectra of Lysozyme in the presence of different ILs are presented in Figure 3
(a-d). The spectra classified into two regions; a) 200-250nm (refers to the backbone
polypeptide chain and corresponds to π–π* transition) and b) 260-290nm (refers to
aromatic amino acid residues, i.e., Trp, Tyr, and Phe and corresponds to n–π*
transition).[29] From Figure 3, it was observed that the absorbance at 280nm was
decreased in the presence of ILs. In case of EmimESO4, the decrease is more profound
with a blue shift in absorption maxima as a function of concentration. The observed
changes may be as a result of the changes in the local environment around aromatic
amino acid residues.[30] But in case of other ILs, there is a marginal decrease in
absorbance was noticed with the increase in the hydrophobic moiety (i.e., from ethyl to
butyl to octyl) in the imidazolium ring of the ILs.
From the absorption results, it is concluded that the hydrophobic groups have more
impact on the local environment of aromatic residues which cause those perturbations.
When considering the peak at lower wavelength range, it can be noticed that, the signal
decreases in the presence of ILs, and a red shift was observed in all the ILs as a function
of concentration. It is known that when amide moieties are exposed to aqueous medium
they undergo low energy π–π*. In presence of ILs, the water in contact with peptide
7

backbone was replaced by ILs. It means the ILs change the polarity of the solvent
medium, which lowers the π–π* transition energy causing a bathochromic shift. These
observations may be attributed to disturbances in the microenvironment of the peptide
backbone structure.[31] This kind of results was earlier reported in protein-IL interaction
studies.[31,32] From the above observations, it is proved that the protein forms a complex
with the ILs.[33]
3.2. Steady-State Fluorescence Study
Fluorescence spectroscopy has been widely applied to study interactions between protein
and ligand and simultaneously, also to explore the quenching mechanism, binding
constants, and binding sites. We used the intrinsic fluorescence of Lysozyme to explore
the interaction between ILs and Lysozyme. From the crystal structure and amino acid
sequence, it is evident that Lysozyme has several solvent exposed and unexposed
tryptophan(s).[34] As shown in Figure 2, Lysozyme contains six Trp residues at positions
28, 62, 63, 108, 111, and 123. The Trp residues at positions 28, 108, 111, and 123
present in the α-domain, and the residues at positions 62, 63 and 108 are in the substrate
binding sites. Trp 62 & 63 are situated close to the hinge region between the α and β
domains and they are found to be highly susceptible to chemical modifications as they are
more exposed to the solvent medium whereas Trp 108 is in hydrophobic core, away from
the aqueous medium.[35,36] According to the detailed studies reported earlier, Trp 62 and
108 are found to have more impact on the fluorescence emission of Lysozyme, whereas
other four residues have small contributions.[37,38] Therefore, the exposed Trp 62 and 63
residues are supposed to be the active site of binding and the fluorescence emission study
8

by exciting at 295 nm could provide the information of local environment around these
Trp residues.
The emission spectra of Lysozyme in ILs were shown in Figure 4 (a-d). In the presence
of ILs, EmimESO4 & EmimCl, a sharp blue shift of the emission maxima was observed
(345 nm to 336 nm). This is assigned to the change in local environment around
fluorophore residues upon interaction with the IL indicating the movement of
fluorophores toward a more hydrophobic environment, which is possible during
stabilization of Lysozyme. The stabilization effect is evidenced by thermal denaturation
studies discussed later. In case of BmimCl & OmimCl, a small blue shift of emission
maxima observed (345 nm to 342 nm) which may be possible either by stabilization of
Lysozyme where the fluorophores move towards hydrophobic core or by the interaction
between hydrophobic groups of IL and the fluorophores during unfolding process. In
these ILs, the unfolding process occurs considering the results obtained from thermal
denaturation studies and circular dichroism studies discussed later.
Fluorescence quenching mechanisms: Quenching of Lysozyme fluorescence by ILs
depend differently on the type of IL as evident from the Figure 4. It indicates different
mechanism of interaction with Lysozyme. Quenching of the fluorescence can be static
(ground state complex formation between fluorophore and quencher) or dynamic (excited
state collision of fluorophore and quencher). These mechanisms can be distinguished by
the Stern−Volmer plot, which give the idea about quenching efficiency of ILs after
9

binding with Lysozyme in the vicinity of fluorophore and protein backbone. The
quenching process can be described by the Stern–Volmer equation (Eq.1) [39,32,40-42]
F
0
1 KSV Q 1 k  Q
  0  
(1)
q
F
where F0 and F are the fluorescence signal of BSA in the absence and presence of
quencher IL, [Q] is the quencher concentration, and KSV = Kqτ0 is the Stern- Volmer
quenching constant (Kq is the bimolecular quenching constant, and τ0 is the lifetime of
fluorescence in the absence of quencher). Ksv values were obtained from the slope of the
Stern-Volmer plot and Kq values were calculated by considering τ0 = 10-8. Kq values
were found to be less than the largest dynamic bimolecular quenching constant in
aqueous medium, which is 2 × 1010 L mol−1s−1, indicating that the quenching of
Lysozyme fluorescence by ILs is dynamic. It reveals that the quenching of fluorescence
is mainly by the interaction of IL with Lysozyme and the unfolding of Lysozyme in the
present case doest not contribute much to the decreased fluorescence signal.
When small molecules bind independently to a set of equivalent sites on a biomolecule,
the equilibrium between free and bound molecules is given by the following equation
(Eq.2).[43]
F  F
0
log
 logK  nlog Q
 
(2)
b
F
Where, n is the number of binding sites and Kb is the binding constant. The values of n
and Kb were obtained from the slope and intercept of the plot of log(F0 - F)/F0 versus
log[Q] respectively.
10

The values of n and Kb were shown in table 2. Gibbs energy for the binding of the IL to
the protein can be calculated from the following equation (Eq.3).[44]
Gb  2.303RT log Kb
(3)
The “n” value is helpful to know the number of binding sites and to locate the binding
site in Lysozyme. The calculated “n” value is closer to unity indicates that there was one
independent class of binding sites on Lysozyme with IL. Kb values reveal that the
reactivity of ILs with Lysozyme follows the trend OmimCl > EmimESO4 > BmimCl >
EmimCl which are in agreement with UV difference spectra where the strongest effect
observed for OmimCl and EmimESO4. The negative ∆Gb values indicate the binding
interaction between the ILs and Lysozyme is a spontaneous process.
The low values of Kb suggest very weak interactions between ILs and Lysozyme. In
general, ligand - protein complexes have the binding affinity in the range of 105-108
Lmol-1.[45] However, lower binding constants (102-104 Lmol-1) have been reported for
several ligand-protein complexes.[44,39,41,42] Recently, imidazolium ILs have been
reported to possess very weak interactions (binding constants in the order of 102) with
BSA having ∆Gb values in the range of -10 to -15 kJ mol-1.[32] The low free energy values
are attributed to the use of higher concentration of ILs, by which the IL-IL interactions
dominate the IL-lysozyme interactions in the formation of micelle.[46] Hence the driving
force for IL binding with lysozyme gets reduced.
11

3.3 Thermal Denaturation Studies
Figure 5a shows the thermal denaturation curve of Lysozyme in the buffer solution, as
monitored by absorption measurements. Thermal denaturation of Lysozyme in absence
and presence of ILs was found to follow a single step, two state transition. These results
are in accordance with previous reports where single step, two state transition unfolding
was observed.[47-49]
Experimentally, the fraction of unfolded molecules (Melting temperature, Tm) can be
measured by taking the first derivative of the sigmoidal curve of the absorbance vs
temperature graph.[50,51]
The effect of different ILs (EmimESO4, EmimCl, BmimCl, and OmimCl) on thermal
stability of Lysozyme were illustrated in Figure 5b. (The complete analysis included in
supplementary information).
Figure 5b shows that some of the ILs (EmimSO4 & EmimCl) rapidly increase the Tm of
the Lysozyme and some (BmimCl & OmimCl) follow the reverse order, which also
depend on the concentration of the IL. These observations reveal that the native structure
of Lysozyme keeps intact in the presence of some ILs. The stability of the proteins in the
presence of ILs follows the trend EmimESO4 > EmimCl > BmimCl > OmimCl and the
stability is found to be higher at low concentrations of EmimCl. The increased thermal
stability may be due to the interaction of ILs (cationic moiety) with the negatively
charged residues on the surface of Lysozyme (via electrostatic, hydrophobic forces /
12

hydrogen bonding through amine protons). It is assumed that in this case these ILs do not
interact with the functional groups of Lysozyme. The extra stabilization of EmimESO4
-
can be predicted by the interaction of both cation, Emim+ as well as anion, ESO4 with
oppositely charged sites of Lysozyme via electrostatic and hydrophobic forces. In case of
BmimCl & OmimCl, with increasing concentration, the disruption is found to be more
and follows the trend of 0.2 M < 0.6 M < 1.0 M. It means when the size of the
hydrophobic group in the cation moiety increases, the protein is more prone to disruption.
The unfolding of protein in these ILs may be due to the interactions between ions of the
ILs and the functional groups of protein (specifically hydrophobic interactions between
the hydrophobic core of protein and hydrophobic groups of the ILs) and the repulsive
intramolecular interactions between the protein functional groups which may disrupt the
protein’s native structure.[52,53]
Further, to have a better understanding on the structural changes in ILs we performed CD
analysis.
3.4 CD Spectral Analysis
CD spectroscopy can be used to observe the secondary and tertiary structural changes
occurred in the presence of ILs. The secondary structure of proteins can be calculated
from the far-UV CD spectrum, but the working concentrations of ILs produce high values
of HT voltage which leads to high noise in the spectrum. Therefore, we confined our
study to near-UV CD spectral region (250–350 nm), which is sensitive to the
chromophore’s local environment and, consequently, the protein’s tertiary structure.
13

The near-UV CD spectra of 100 µM Lysozyme in different ILs are presented in Figure 6
(a-d). The tertiary structure of Lysozyme is confirmed by a positive triplet-like signal in
the range of 280–300 nm and which indicates the active conformation of Lysozyme.[54,19]
The net positive CD bands are associated with the Trp and Tyr Lb transitions and net
negative CD bands are associated with Trp La and disulfide transitions.[55] The positive
triplet feature (signals at 283, 289 and 295 nm) of Lysozyme is considered to be obtained
from the transitions of the Trp residues.[56] Therefore, any changes in CD spectra in the
presence of ILs reveals the alteration of the environment of these aromatic amino acid
residues indicating the unfolding of Lysozyme.
In the presence of EmimESO4, the spectra were not changed much except the signal of
the peak at 289 nm which decreases as a function of concentration. But, the width of the
peak is lesser as compared to the native Lysozyme. This observation is attributed to the
proteins compact structure in the IL. But in case of EmimCl, the signal of the triplet peak
increases at lower concentration (0.2 M) and at higher concentration the triplet feature
diminished, which means the protein is unfolded at higher concentration. Therefore,
comparing these two ILs, we can conclude that the anion is playing a crucial role in
stabilizing and destabilizing protein structure. It is also supported by the melting
temperature of Lysozyme in the presence of these ILs.
When comparing the effects of the hydrophobic moiety present in ILs on protein
structure, it was observed that, with increasing alkyl chain length in the imidazolium
group of the ILs, the protein gets destabilized. In case of BmimCl at higher concentration
14

(0.6 M & 1.0 M), the signal of triplet signal was found to be higher, but those peaks are
broader. This observation suggests, the presence of IL in the vicinity of Trp residues
changes the local environment and consequently the tertiary structure altered.[19]
Considering the effect of OmimCl, it was noticed that the triplet feature keeps intact at
low concentration but with increasing concentration the triplet signal reduced and finally
at 1.0 M the tertiary structure disappeared. These results are attributed to the increased
hydrophobic interactions between protein hydrophobic core and the hydrophobic group
of the ILs during the unfolding process. Recent simulation studies of protein
conformation reveal that direct interaction between protein core - IL leads to
destabilization of the protein.[57,58] During the process, ILs induce conformational
changes in protein enabling ILs to directly bind to the protein surface, thereby unfolding
of the biomolecule. Klahn et al. elucidate that the van der Waals interactions placed a
dominant role in the interaction of the protein with the cation of the ILs, which leads to
the disruption of the protein structure in the ILs.[57] These processes reduce the free
energy evidenced by the decrease of Tm value and, therefore, destabilize the native
structure of the protein.
4. CONCLUSION
In conclusion, among the ILs used in this study, EmimESO4 acted as most promising and
biocompatible IL for the native structure of Lysozyme at all studied concentrations
followed by EmimCl at low concentration. From the results, it can be concluded that not
only the IL type but also concentration of the ILs play a major role in
stabilizing/destabilizing a protein. It was found that anion ESO4 has a great influence on
15

increasing the thermal stability of Lysozyme compared to Cl ion and hydrophobicity of
the cation influences the thermal stability profoundly in destabilizing protein. The present
wotk explains the mechanism of lysozyme-IL interaciotns and it was observed that very
weak interactions exist between lysozyme and ILs elucidated by spectrophotometric
studies. We hope that the results obtained here will be useful in the selection and
designing of ILs with varying combination of different cations and anions, which can
provide green, biocompatible co-solvents for the stabilization of proteins in biochemical
processes.
ACKNOWLEDGEMENT
Authors are highly acknowledging MHRD (New Delhi, India) for financial support and
thankful to Department of Chemistry as well as Life Science (NIT Rourkela) for
extending their support in terms of the facility to carry out the experiments.
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24

Table 1. Briefing of employed Ionic liquids for protein stability investigation.
Name of the Ionic liquid
Molecular
Availability*
Weight /gm
Abbreviation
EmimESO4
Chemical Name
1-Ethyl-3-Methyl-Imidazolium
EthylSulfate
236.29
146.62
174.67
230.78
Commercial
Commercial
Commercial
Synthesized
EmiImCl
BmimCl
OmimCl
1-Ethyl-3-Methyl-Imidazolium
Chloride
1-Butyl-3-Methyl-Imidazolium
Chloride
1-Octyl-3-Methyl-Imidazolium
Chloride
* Details are mentioned in “Materials and Methods” section
25

Table 2. Stern–Volmer quenching constant (KSV), bimolecular quenching constant (kq),
binding constant (Kb), binding sites (n) and Gibbs energy (∆Gb) for the binding of Ionic
iquids with Lysozyme at 298.15 K
IL
KSV/Lmol−1
Kq/Lmol-1s-1
Kb/Lmol-1
n
∆Gb/kJ mol-1
EmimESO4
EmimCl
1.71
1.26
1.44
2.31
1.7*108
1.2*108
1.4*108
2.3*108
1.61
1.18
1.37
2.29
0.79
0.78
0.80
0.92
-1.19
-0.42
-0.78
-2.06
BmimCl
OmimCl
* EmimESO4: 1-Ethyl-3-Methyl-Imidazolium EthylSulfate, EmimCl: 1-Ethyl-3-Methyl-
Imidazolium Chloride, BmimCl: 1-Butyl-3-Methyl-Imidazolium Chloride, OmimCl: 1-
Octyl-3-Methyl-Imidazolium Chloride
26

Figure 1
27

Figure 2
28

Figure 3
29