Mechanism and Dynamics of Long-Term Stability of Cytochrome c

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Mechanism and Dynamics of Long Term Stability of Cytochrome c Conferred by Long Chain Imidazolium Ionic Liquids at Low Concentration Upendra Kumar Singh, Meena Kumari, Sabab Hasan Khan, Himadari Bihari Bohidar, and Rajan Patel ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03168 • Publication Date (Web): 03 Nov 2017 Downloaded from http://pubs.acs.org on November 4, 2017

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ACS Sustainable Chemistry & Engineering

Mechanism and Dynamics of Long Term Stability of Cytochrome c Conferred by Long Chain Imidazolium Ionic Liquids at Low Concentration Upendra K. Singha, Meena Kumaria, Sabab H. Khana, Himadri B. Bohidarb and Rajan Patela* a

Biophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia (A Central University), New Delhi-110025, India. b

Polymer and Biophysics Laboratory, School of Physical Sciences, Jawaharlal Nehru

University, New Delhi 110067, India.

*Corresponding author. Tel.: +91-8860634100; fax: +91-11-26983409. Email address: [email protected], [email protected] (Dr. R. Patel), (orcid.org/0000-0002-3811-2898)

1 ACS Paragon Plus Environment


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Abstract Protein stability have been a concern for researchers from long time as they are sensitive towards their environment. Mostly proteins during experiments require medium that keep them stable at room temperature (RT). Recent research utilizing ionic liquids (ILs) to stabilize protein has gained much importance. Although a few ILs have been claimed to suit the requirement, reported studies employ IL concentrations that remains an issue which might produce irreversible denaturation and aggregation. . This study demonstrates first report for long term stabilization of horse heart cytochrome c (h-cyt c) by long chain imidazolium ILs at far low concentration (1mM) of IL when stored at RT. Long chain imidazolium ILs until now were less familiar for their stabilizing nature towards protein. A significant increase in the helical content of h-cyt c (dissolved state) were observed with prolonged structural stability (secondary and tertiary) for about 6 months in the aqueous solution of 1-methyl-3-octyl imidazolium chloride [C8mim][Cl] and 1-decyl-3-methylimidazolium chloride [C10mim][Cl]. The in-depth mechanism discussed suggests interaction of ILs with amino acid residues of h-cyt c, rigidifies the loop regions with reduced mobility and hence prolonged the stability is achieved. The study firmly advocates the use of long chain imidazolium ILs as the potent inhibitor against denaturation during storage of h-cyt c at RT.

KEYWORDS: Cytochrome c, Ionic liquid, Long-term stability, Time resolved fluorescence spectroscopy, Peroxidase activity.


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Introduction Protein stability have been a muse for over years and controlling factors that influence stability and folding have been a key challenge. Any altered environment around proteins affect their stability and hence their function. Therefore, ineffective preservation can lead to instability of proteins and severely hit the protein-based pharmaceuticals. Hence, there exists strong need for solvents that confine these molecules and confer stability by influencing their physical and chemical properties of the proteins at room temperature (RT). Use of ionic liquids (ILs) as solvents for protein stabilization have been reported at much higher concentration (>1M)

1-3

,

even not all of the stabilizing ILs support the long term stability 4. There exists the need for preservation of proteins in native like environment to resist aggregation and denaturation for maintaining stability at very low concentration (micromolar to millimolar) of ILs. The most important factor is the reusability of protein after storage for different purposes also remains an issue. Hence, it is quite fascinating to study the long term stability and interaction of the proteins with co-solvents, as stability and activity of proteins are largely dependent on the nature of cosolvents in which they are stored as they are influenced by their physical and chemical properties. Also, understanding about the mechanism of their bimolecular interactions on structural basis causing stabilization/destabilization is fundamental to our accumulating knowledge. The dissolution of proteins in a stable medium is important for advances in liquid formulation for protein pharmaceuticals and various other applications 5. Several challenges confront scientists involved in the development of proteins based biotechnological medicine. First, the stability of proteinss (physical and chemical) is confronted if they have to deal with external factors (pH, impurities, thermal conditions etc.) during handling and processing at RT as many of them are dramatically prone to aggregation and denaturation upon exposure to RT 3 ACS Paragon Plus Environment


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which raises seriuos doubts about the long-term stabilization (protein based drugs like Aldesleukin, Insulin, Enbrel, Refacto etc.)

5-6

. The development of protein based drugs is a

challenge due to need of maintaining proteins in its native state, from processing till the end point of use. Second, mitigate the expense of liquid formulations, it's tough to develop protein drugs that can ease the complexity of administration through desired approach 5. Additionally the use of long chain ILs is a leapfrog in advancements for stabilizing protein and other biomolecules in various sectors of material research and other biotechnological applications. Hence, long term stability at processing temperature of biomolecules which is preferably the RT, makes the IL a convenient and sustainable medium to preserve protein for applications. These ILs provide an extra advantage that extends shelf-life of protein based drugs without any issues of aggregation and attain easy reusability. A quest for liquids that possess such fascinating properties of molecular liquids and provide a unique possibility to modulate protein stability are ionic liquids (ILs), a reason for this burst of interest include their wide tunable properties

3-4, 6-11

. The different interaction behavior of ILs

with proteins has long been reported in neat IL and ILs in aqueous medium

4, 10

. Significant

advances in the ability of ILs to impart stability to protein and ultimately conserve their structure have been made 7, 9-10, 12-16. Although, a few ILs have been reported having outstanding ability to stabilize proteins over time and high temperature

3, 6, 10, 17

. A study by Bihari et al. showed that

the presence of neat IL alters the tertiary structure of h-cyt c, however it was not accompanied by aggregation of the protein 4. Recently, a fascinating study of newly synthesized proteinpolymer surfactant nanoconstructs reported by Alex et al. imparts high thermal stability to protein

18

. However, in existing literatures use of high concentrations of ILs have been shown

that raise question about reusability of protein for other purposes and even it is quite not


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necessary. The impact of these chemicals on environment raises concern about environmental safety. A constant use of neat ILs as reaction media for heme proteins have been seen to monitor activity and stability of IL at high temperatures 1, 10, but such high concentration of ILs are quite not necessary, as they perturb the tertiary structure of the heme proteins by interfering with charge transfer band

4, 19

. So, it is desirable to sprung up with ILs which at low concentration

(micromolar to millimolar) possess the ability to sustain the stability and activity of proteins in the dissolved state for long term storage periods and that can be easily removed by dialysis before use 1. Thus, such biomolecular interactions causing stabilization-destabilization have been an active subject of considerable interest. Horse heart cytochrome c (h-cyt c) is the most thoroughly studied model metalloprotein, commonly used for folding/unfolding experiments

20

. It contains heme prosthetic group which

has been exploited as probe to monitor structural changes during folding. The h-cyt c possess separable nature of cooperative folding/unfolding units (foldons) 21. We have used the difference in alkyl chain length of the imidazolium cation containing two ILs, and consecutively studied the effect of chain length of ILs on long term stability and interaction at RT they offer dramatically improved protein stability with respect to time. Reports for h-cyt c with ILs stability and solubility were reported that showed the enhanced stability of h-cyt c 1, 4, 18, 22-24. This encouraged us to check into the utility of long chain imidazolium ILs, 1-methyl-3-octylimidazolium chloride ([C8mim][Cl]) and 1-decyl-3-methylimidazolium chloride ([C10mim][Cl]) to preserve structure & stability of h-cyt c for long periods at RT. Fortunately, we obtained surprising results in which both ILs not only stabilized h-cyt c, but also increased their shelf life for a long period of time at RT at low concentration (1 mM) which is sufficient to meet the requirements for in vitro applications of protein.



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MATERIALS AND METHODS Equine heart cytochrome c (h-cyt c, type IV), sodium monobasic dihydrate, sodium dibasic dihydrate, 1-methyl-3-octylimidazolium chloride [C8mim][Cl], 1-decyl-3-methylimidazolium chloride [C10mim][Cl], and sodium chloride were purchased from Sigma Aldrich. All chemicals and reagents used were of analytical grade. The structure of h-cyt c, [C8mim][Cl] and [C10mim][Cl] is depicted in Scheme 1 (a, b & c). Millipore water was used throughout the experiments. Preparation of Stock Solution. The h-cyt c was oxidized completely to ferric form by adding 0.1% potassium ferriccyanide to the solution of h-cyt c and was dialysed against several changes of 0.1 M NaCl, at pH 7.0

25

. The oxidized h-cyt c solutions were filtered by using a 0.22 µm

Millipore filter. Concentration of the dialyzed solution of h-cyt c were determined experimentally by using molar absorbance coefficient (ε) values of 1.06 x 105 M-1cm-1 at 410 nm 26

. All the measurements were carried in the degassed sodium phosphate buffer at pH 7.0. The

samples were incubated in the dark for a period of four hours before each experiment. The stock solutions of [C8mim][Cl] and [C10mim][Cl] ILs were made in analytical grade millipore water and stored in glass vials. The critical micelle concentration (cmc) of [C8mim][Cl] and [C10mim][Cl] ILs were 220 mM and 55 mM, respectively 27. Circular Dichroism Measurements. Far-UV CD spectra of h-cyt c were carried out through Jasco spectropolarimeter (J-1500), equipped with peltier type temperature controller (PTC-100). CD spectra of native h-cyt c were taken in the absence and presence of [C8mim][Cl] and [C10mim][Cl] at 25.0 ± 0.1 °C and pH 7.0. For near and Soret spectral measurement cuvette having 1 cm path length were utilized, while 0.1 cm pathlength was used for far-UV


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measurements. The raw CD data were converted into [θ]λ, the mean residue ellipticity (MRE) (deg cm2 dmol-1) at a given wavelength λ using the relation28: [θ ]λ = θλ Mo /10lc

(1)

where θλ is the observed ellipticity (millidegrees) at wavelength λ, Mo is the mean residue weight of the protein, c is the protein concentration (mg/cm3), and l is the path length (centimetres). The α-helical content of h-cyt c was calculated from the [θ]λ value at 222 nm using following equation 29.

%α =

[ θ ] 222 − 2340

× 100

30300

(2)

FT-IR Spectroscopy Measurements. FT-IR spectra were recorded with Specac Golden Gate diamond ATR sampler fitted to a Bruker Tensor 27 with an MCT detector and MIRacle, a single reflection horizontal ATR diamond crystal plate at 25 °C. A total of 128 background-subtracted scans at 2 cm-1 resolution were averaged of pure h-cyt c and with [C8mim][Cl] and [C10mim][Cl]. The concentration of h-cyt c used was 5 mg/ml and the ILs concentration utilized for the experiments were 10 х 10-4 M. In addition, the FT-IR spectra were also utilized to estimate the secondary structure compositions of pure h-cyt c and with [C8mim][Cl] and [C10mim][Cl] by curve fitting results of the amide I band41. The subtraction of the reference spectrum was carried out in accord with the criteria described by Dong et al30.

Absorbance Measurements. Soret absorption spectra of h-cyt c in the absence and presence of [C8mim][Cl] and [C10mim][Cl] were obtained with the help of Specord 210 plus spectrophotometer (Analytik Jena, Jena, Germany) equipped with a peltier controlled temperature controller JUMO dTRON 308 to maintain the constant temperature at 25.0 ± 0.05 °C throughout experiments.



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Dynamic Surface Tension Measurements. Dynamic surface tension measurements were performed using Du Nouy-Padday method on DeltaPi-4 (Kibron, Helsinki, Finland), equipped with four parallel microbalances and having a small diameter (0.51 mm) special alloy wire (Dyne Probes, cleaned by Blazer piezo micro torch). The dynamic surface tension of h-cyt c were monitored in buffer when spread at air/water interface31. The temperature control plate was attached with an external circulating water bath (Grant GD120 water thermostat) to control the subphase temperature at 25.0 ± 0.1 °C. The Delta Graph software was utilized to record the data on the computer.

Time Resolved Fluorescence Measurements. Fluorescence lifetimes were calculated from time resolved fluorescence intensity decays by the single-photon counting spectrometer equipped with pulsed nanosecond LED excitation heads at 280 nm (Horiba, Jobin Yvon, IBH Ltd, Glasgow, UK) at temperature 25 °C. Lamp profiles were measured at the excitation wavelength using Ludox (colloidal silica) as a scatterer. The fluorescence lifetime data were measured to 10,000 counts in the peak, otherwise indicated. The instrumental response function was recorded sequentially using a scattering solution and a time calibration of 114 ps/channel. All experiments were performed using excitation and emission slits with a band pass of 8 nm. The emission wavelength was set to 340 nm and the goodness of fit was judged in terms of both a chi-squared (χ2) value and weighted residuals. Data was analyzed using a sum of exponentials, employing a nonlinear least square reconvolution analysis from Horiba (Jobin Yvon, IBH Ltd). The impulse response functions (IBH DAS6 software) were used to analyze decay curves 32. The mean fluorescence lifetimes for tri-exponential iterative fittings and pre-exponential factors were calculated using the following relation 33:


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τ

∑a τ = ∑a τ

2

i i

(3)

i i

where ai and τi are the relative contribution and life time of different components to the total decay. A fit was considered acceptable when plots of the weighted residuals, and the autocorrelation function showed random deviation about zero with a minimum χ2 values.

Steady-State Fluorescence Measurements. Steady-state fluorescence measurements were performed on a Cary Eclipse spectrofluorimeter (Varian, USA) equipped with a 150W xenon lamp using 1 cm path length quartz cuvette at 25 °C. Temperature was controlled during experiments using constant-temperature cell holder connected to constant-temperature water circulator (Varian,USA). The excitation and emission slits with a band pass of 5 nm for all experiments. Fluorescence spectra of the h-cyt c was measured at an excitation wavelength of 280 nm and the emission spectra were recorded in the wavelength range of 300-400 nm. The 8Anilino-1-napthalenesulfonic acid (ANS) is a extrinsic fluorescent dye and was performed to detect the presence of partially folded intermediates. The excitation wavelength was set at 360 nm for ANS fluorescence measurements and the protein to ANS molar ratio of 1:20 was used for fluorescence measurements. The concentration for fluorescence measurements of h-cyt c was kept between 5-7 µM.

Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) measurements of pure h-cyt c and in presence of 1mM [C8mim][Cl] and [C10mim][Cl] were performed in MicroCal (USA) VP differential scanning calorimeter having a cell volume of 0.5 mL under a constant pressure of 34 psi, in order to avoid the formation of gas bubbles during the experiment. The scanning rates of 1°C per minute in the temperature range of 20–110°C for all experiments were used. The protein and buffer solutions were degassed for 20 min prior to loading into the



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DSC cells. The protein concentration used was 1mg/ml. The data was analysed using the Origin computer program supplied by MicroCal, Inc. The instrumental baseline was subtracted from the sample data, and the data were normalized from calories per degree Celsius to calories per mole per degree Celsius. The base-lines were created with the software progress baseline function.

Peroxidase Activity of h-cyt c. The peroxidase activity of the h-cyt c in absence and presence of [C8mim][Cl] and [C10mim][Cl] at 1 mM were investigated. The samples of h-cyt c (2 µM) were preincubated for 5 minutes having guaiacol in 50 mM potassium phosphate buffer, pH 7.0 and use the catalytic activity of guaiacol as substrate and H2O2 as an oxygen donor at 25 °C. The hcyt c catalyzes the oxidation of guaiacol to tetraguaiacol and produces orange colour that shows absorption band at 470 nm

34

. The rate of formation of tetraguaiacol was observed from the

appearance of orange colour and qualitatively measured using molar absorption coefficients of 26,678 mM

-1

cm-1

35-36

. H2O2 solution was prepared from its 30% stock solution (9.79M), and

working solution was prepared form it of 100 mM. The concentration of guaiacol and H2O2 used for the activity studies were 2 mM and 10 mM, respectively.

Molecular Docking. The docking of ILs with h-cyt c was performed by using molecular docking software AutoDock4.237. The 3-D structure of h-cyt c (PDB ID: 1hrc) was downloaded from the Protein Data Bank. Binding structure of [C8mim][Cl] and [C10mim][Cl] was generated on Chem3D Ultra 8.0. Blind docking was performed for docking of both [C8mim][Cl] and [C10mim][Cl] with h-cyt c. The possible binding conformation of the ILs with h-cyt c was computed by using Lamarckian Genetic Algorithm (LGA) implemented in AutoDock4.2. For blind docking, the grid volume of the protein was set in such a way that it covers whole surface of the h-cyt c. Grid volume was set as 86 × 82 × 98 points with a grid spacing of 0.375 Å and grid center of 46.775, 22.633, 4.362. In the genetic algorithm parameters file, 10 numbers of GA


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runs, 2.5 × 106 maximum number of energy evaluations, 27000 maximum numbers of generations and 150 population size were set for docking of both ILs. After completion of the docking experiment, 10 different conformers were obtained for binding of ILs with h-cyt c. Out of 10 conformers; one with the lowest energy (selected on the basis of moldock rank scores based on the lowest binding energy and highest H-bonding score) was used for analysis of the results.

RESULTS The following study of h-cyt c interaction and stability with [C8mim][Cl] & [C10mim][Cl] ILs were divided and presented in two separate sections (1 & 2). First one dictates the interaction of residues of h-cyt c with the alkyl chain of ILs and stability induced as a result of the interaction, in respect to the techniques performed. The effect of interaction on structure, surface activity and thermal behaviour of protein with ILs ([C8mim][Cl] and [C10mim][Cl]) was probed through techniques mentioned. Later one presents the time dependent stability of protein at the structural level of protein (secondary & tertiary) and activity of h-cyt c, when stored for six months at RT and it also discuss its aspects. This section is followed by a brief discussion of comparative long term stability due to the difference in alkyl chain of ILs and probable mechanism involved. 1) Interaction of Long Chain ILs with h-cyt c shows Stabilizing Nature.

Circular Dichroism Spectroscopy. Far-UV spectrum investigates the alterations in the secondary structure of proteins in the range of 200–250 nm. Any change in the backbone orientation will affect the optical transition and hence indicate the changes in the secondary structure of the protein. Helical proteins give characteristic negative peaks at 208 nm (π–π* transition) and 222 nm (n–π* transition). The secondary structure of h-cyt c was first estimated by the far-UV CD upon interaction with [C8mim][Cl] and [C10mim][Cl] shown in Figure 1 (a &



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b), respectively. In native conformation h-cyt c possess dominant α-helical structure with minimal sheet content. The secondary structural content of h-cyt c at different concentrations of [C8mim][Cl] and [C10mim][Cl] were calculated by the equation 2 listed in Table 138. The variation of helical content as a function of [C8mim][Cl] and [C10mim][Cl] was complimentary to the results observed in UV for 409 nm peak, time resolved studies as well as near and Soret UV CD (discussed later). In the far-UV range at high concentrations (10 & 50 х 10-3M) spectra were not recorded owing to high tension voltage. The observed increase in helical content of the hydrophobic ILs alkyl chain interact with the side chains of the hydrophobic amino acid around the heme pocket of h-cyt c and play important role in its stabilization. The interaction of ILs were located through the help of docking at respective sites of h-cyt c. Figure S1 a & b, shows the image of the docked sites of the h-cyt c at respective domains bound to long chain ILs ([C8mim][Cl] and [C10mim][Cl], respectively) as a result of interaction. The residues involved are the mostly placed around the loops of h-cyt c lying adjacent to the heme crevice. Results obtained after docking were analyzed and interpreted, in order to understand the effect of interaction with different chain length ILs. The near-UV spectrum has been used for studying the change in tertiary structure of h-cyt c in the (270-300 nm). The structure is maintained by non-specific interactions of side chains of amino acid in peptide backbone by hydrophobic forces. The near-UV spectrum of the native hcyt c gives two sharp minima near 282 and 289 nm 39 which resembles to tight tertiary structural packing of tryptophan residue at position 59 (Trp59) and tyrosine residues (Tyr) 39-40. The Figure 1 (c & d) shows the stability of tertiary structure upon addition of ILs, respectively. The bands at 282 and 289 nm in the presence of ILs show increase and the tertiary structure of h-cyt c is preserved by tight packing of sidechains39. The tertiary structure is preserved even at high


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concentration of ILs due to the conserved interaction of Trp59 with the heme propoinate39 that suggest the increased stability in presence of alkyl chain of ILs to h-cyt c which maintains its tertiary structure. The ILs having long alkyl chain are hydrophobic and contain positively charged imidazolium ring that interacts with the hydrophobic side chains of various amino acids in protein backbone, residues like lysine having hydrophobic side chain and isoleucine that is itself hydrophobic41-42. Thus, the interaction increases the stability of the tertiary structure and also maintains the heme crevice, hence preserves the structure of h-cyt c. The Soret region of CD spectrum provides insight into the integrity of heme crevice i.e. heme polypeptide interactions. The Soret spectrum gives information regarding the immediate conformational environment of the heme group, and any change in the spectrum reflects change in heme pocket with distinct heme environment. The Soret region is mainly observed due to the coupling of heme π-π* electric dipole transition moments with those of nearby aromatic residues in the protein. The h-cyt c spectrum shows a bisignate pattern i.e. one negative at 416 and the other one positive at 405 nm. The positive peak at 405 nm quantifies the heme-globin interaction and the spin state of heme-Fe, so under progressive disruption increase and shifts towards lower wavelength occur. The 416 nm negative band correspond to Met80-Fe and Phe82-heme interaction43-44. The band also corresponds to the distance and orientation of the Met80 side of heme plane. The Soret CD spectra of both ILs show no change in spin state in heme Fe and Met80-Fe as shown in Figure 1 (e & f), respectively. This shows the ILs maintains heme-globin interaction and the spin state of heme-Fe, both and stabilizes it.

FT-IR Spectroscopy.

FT-IR spectroscopy serves well for investigating the structural

characteristic of proteins that range from gross aspects to subtle rearrangement upon ligand binding. The amide band I peak is located in the 1600–1700 cm-1region is associated with the



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mainly C=O stretching and quite sensitive to study the secondary structure changes of protein. The amide peaks have been shown in the Figure 2 & S2 (a) of h-cyt c with ILs, show the effect of ILs on interaction with h-cyt c on the secondary structure of h cyt c. The strong band appears at the 1652 cm-1, the dominant peak show the presence of major α-helix with the presence of some different structures represented by small peak45. The major peak at 1652 cm-1 shifted slightly towards 1654 cm-1with increased intensity shows increase in content of helix with the addition of ILs to it. The [C10mim][Cl]

showed greater increase in the intensity than

[C8mim][Cl]. Thus, upon ILs interaction with h-cyt c the increase in structure occurs (helical content). The curve fitted spectra shown in the Figure S2 (b, c & d) depicts the changes in peak position of bands with the respective ILs ([C8mim][Cl] & [C10mim][Cl]). The peak in spectral region 1700 to 1600 cm-1 were resolved and the range in which the peak lies and corresponds to the their respective secondary structural content reported elsewhere46, the free h-cyt c spectra obtained were consistent with the far-UV CD data and earlier reported data

6, 47

. The squares of

the correlation coefficient (R2) shows good fitting and the data is mentioned in the Table S1. These results conclude that the [C10mim][Cl] shows greater interaction and serves better additive to maintain the enhance structural content of the h-cyt c than the [C8mim][Cl].

UV-vis Spectroscopy. The h-cyt c in its native conformation shows absorption bands at 280 nm (n–π* transition of aromatic amino acids), Soret band at 409 nm (π–π* transition a position reflecting low spin state iron, a form imposed by the heme’s axial ligands, His18 and Met80 of the heme group). Figure 3 (a & b) gives the absorbance spectra for the pure h-cyt c and h-cyt c in the presence of increasing concentration of both ILs. The absorbance of the peak at 409 nm shows an increase with no peak shift that suggests no changes in the ground state properties of the protein in the presence of ILs and the heme group in the protein remains intact. The heme of


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h-cyt c is buried inside the packed hydrophobic core of hydrophobic amino acids, with some lysine residues at the adjacent position. The increase in the absorbance of the Soret peak indicates that the transition towards a non-polar environment around the heme cleft as the hypochromic shifts in UV-vis spectra are observed due to the lowering in the energy of the π* orbital. The slight unfolding of h-cyt c have reported with decreasing absorbance around 409 nm having peak at the same position48. The inset in the Figure 3 (a & b) shows the 695 nm (charge transfer band). This peak corresponds to the Met80-Fe interactions and measures local stability of protein (appears due to axial ligation of heme with Met80 that yields a six-coordinated low spin form), it is enhanced in presence of ILs. This depicts the stability conferred by the ILs to the protein structure in solution. The UV-vis spectra at relatively high concentration of ILs (2 х 101

M) were also performed in Figure S3, shows no significant change in spectral characteristics

i.e. the heme crevice remained intact and spin state of metal ion in heme group remains same with no perturbation of existing bonds (also the charge transfer band at 695 nm). The hemeglobin interactions are not perturbed i.e the 409 nm peak is maintained even at such higher ILs concentration, this shows maintained structure of protein. The DSC thermograms for the h-cyt c and with both ILs at 10 х 10-4 M concentration are shown in the Figure S4. The graph shows the midpoint of denaturation temperature (Tm) of 82.6 °C for h-cyt c in agreement to reported literature 11. The h-cyt c with [C8mim][Cl] and [C10mim][Cl] at 10 х 10-4 M concentration do not perturb the Tm value of the h-cyt c and is 82.2 °C and 82.6 °C, respectively. This suggests that both ILs maintains stability of h-cyt c at 10 х 10-4 M. The respective Tm values are listed in the Table S2.

Time Resolved Spectroscopy. Fluorescence lifetime is a very sensitive technique utilized to monitor molecular interactions and motions useful in analyzing the structure and dynamics of



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biological macromolecules. Here we have measured the decay of the discrete lifetimes of h-cyt c upon interaction with ILs. The h-cyt c is a single Trp containing protein, and it shows a multimodal form of distribution due to the existence of different rotameric conformations of tryptophan side chain that interacts differently with the environment12. The lifetime h-cyt c is believed to be dependent on the relative orientation and distance between tryptophan and the heme

33

. The lifetime experiments of h-cyt c with [C8mim][Cl] and [C10mim][Cl] fitted triple

exponential decay at pH 7.0 shown in Table 2. The average lifetime (τav) shows decrease accompanied with decrease in longer lifetime of the h-cyt c with addition of increasing concentration of ILs shown in decay curve spectra in Figure 4 (a & b), respectively. Earlier studies of protein with ILs

11, 49

have also shown similar trend in which decrease in longer

lifetime (τ3) relates to change in polarity. The results corroborate with those of CD & FTIR in which increase in the helical content of the protein in its secondary structure is observed, and protein becomes more compact. Precisely, the fast and medium components are from the vicinity of heme region so change in polarity around heme and the conformation can easily be monitored50. The results obtained at 50 х 10-3 M of both ILs showed greater increase of fast and medium components with their pre-exponential factors. The decrease in longer lifetime correlates to the increase in hydrophobicity around the fluorophore. The overall contribution of the individual components show greater pre-exponential factor (αi) for shorter lifetimes (τ1). The αi relates to the individual rotamer population and also corresponds to the secondary structure of the tryptophan residue by main chain on rotamer populations. A very interesting observation was noted at relatively high concentration (10 & 50 х 10-3 M) of [C10mim][Cl], where drastic increase in longer lifetime (τ3) with an exponential decrease in its contribution occurs (α3). It was assumed due to the increase in hydrophobicity around Trp59, it


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becomes part of regular secondary structure

51

. As the Trp59 resides around the hydrophobic

core placed in the loop region of the h-cyt c and at this concentration it becomes part of the secondary structure due to greater increase in helical content with IL concentration. Here, the longer lifetime probably correlates to the increase of hydrophobicity around Trp59 that is placed in the hydrophobic core around the heme region, the decrease in longer lifetime occurs mainly attributed to the increase in hydrophobicity around the fluorophore corresponds to the secondary structural increase that makes protein become more compact (decrease relates to the optimum packing and efficient electron transfer)11, 50. This confirms greater interaction of [C10mim][Cl] to nearby residues of Trp59 that might help for long term stability and decrease the mobility of the loops. Moreover, dynamic surface tension (DST) of proteins shows different adsorption behaviour on air/water interface dependent on important factors as conformational stability and hydrophobicity 52

. Results shown in Figure S5 suggests the pure h-cyt c have equilibration time much higher

compared to h-cyt c with [C8mim][Cl] and [C10mim][Cl] which clearly suggests the interaction of ILs with h-cyt c as a result of faster adsorption occurs with conformational changes. The greater surface activity shown by the complex (more by [C10mim][Cl]) confirms the interaction of h-cyt c with the ILs.

2) Influence of [C8mim][Cl] and [C10mim][Cl] on Time Dependent Stability of h-cyt c Secondary Structural Stability. The secondary structural features of protein were checked through far-UV CD to analyze the structural stability maintained by the ILs when the h-cyt c was stored at RT for 6 months. The loss of helical content for fresh, 3 month and 6 month stored sample at RT for both ILs are shown in Figure 5, respectively. Most of the aliquots of different concentrations of [C8mim][Cl] and [C10mim][Cl] stored for 3 months preserved helical content



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when measured at [θ]222. The long chain ILs preserves the secondary structure of h-cyt c from unfolding also presented in shown Figure S6 (a & b) Table S3. Our observation for 6 months stored samples reveal unprecedented results. Although the secondary structural content is not preserved at lower concentrations of both ILs ( up to 50 х 106

M) , but little high concentration of the [C8mim][Cl] (100 х 10-6M) preserved helical content

when compared to h-cyt c without IL. Most amino acid residues involved in the interaction with [C8mim][Cl] are located in the helix. Striking consequences were obtained from h-cyt c with [C10mim][Cl] that depicts the preserved secondary structure even when stored for long term period (helical content was maintained corresponding to increase) at RT shown in Figure S6 (c & d), Table S3. This was probably due to the interaction pattern of both hydrophobic ILs having long chain with the side chains of the hydrophobic amino acids around the heme pocket53 (docking results Figure S1 a & b ).

Tertiary Structure Stability. The h-cyt c stored for 3 months showed loss of structure by absence of charge transfer band at 695 nm in UV-vis results with decreased intensity around 409 nm due to disrupted heme-globin interaction (reduction in efficiency of electronic transition and decreased stability of heme) but consist of the Q band (530 nm) showed in Figure S7 (a & b). The optical transitions are quite sensitive regarding ligand exchange reactions

6, 44

. Samples

stored with both ILs for 3 months show almost no change in spectral characteristics. The Soret CD spectra of samples showed loss of negative peak at 416 nm of h-cyt c (data not shown), while stored samples having 1mM [C8mim][Cl] and [C10mim][Cl] showed bisignate pattern with both peaks conserved. Lifetime results in Figure S7 (c & d) & Table S4 (a) shows stable protein samples with minimal change in lifetimes were seen at several concentrations of both ILs. Little perturbation in fast and medium lifetimes were observed.


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However, extending our observations to 6 months in Figure 6 (a & b), UV-vis result shows the hcyt c without any IL showed broad Soret band, this is due to exposure of heme to aqueous solution after dissociation (disrupted heme-globin interaction moves heme out of its pocket). The loss in the structure of the h-cyt c is due to the loss of axial heme ligand after peptide chain shows extended configuration due to denaturation 54. Sample having lower concentrations ( up to 50 х 10-6M) of both ILs, showed similar trends (Table S4 a & b). To our surprise, concentrations beyond as shown Figure 6 (a & b) the structural integrity of h-cyt c with both ILs was maintained with noted 409 nm, 530 nm & 695 nm distinctly visible bands in the spectra shown. The stabilization effect of the h-cyt c by ILs has been strongly evident. The maintenance of tertiary structure and heme packing (heme stability) of h-cyt c for long time at RTs was perhaps due to small water content in direct interaction with the protein. Concomitant results have been shown by Fujita et al.

6

by storing protein in short chain ILs but comparatively at very high

concentrations (80 wt% Dhp ILs). The near-UV CD spectra showed distinct bands of 282 and 289 nm only samples having relatively higher concentration of ILs (10 х 10-4M & 10 х 10-5M for [C8mim][Cl] and [C10mim][Cl], respectively) (same as UV-vis spectra) in Figure 6 (c & d) showing stable heme-trp59 interactions (compact structure) due to maintained packing of residues around heme. Again, the Soret CD spectra showed in Figure 6 (e), depicts conserved bisignate pattern of peaks at respective positions of both the ILs. This showed conserved structure of h-cyt c at 1mM concentration by both imidazolium ILs to h-cyt c even after 6 months being stored at RT. The heme-globin interaction at 405 nm shows no change in the spin state of heme Fe and Met80-Fe (also shown by 695nm in UV-vis) and Phe82-heme interaction at negative peak at 416 nm were preserved which signifies stability of h-cyt c.



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Fluorescence emission was utilized to probe the structural changes in aqueous environment of hcyt c strored with long chain imidazolium ILs. The emission spectra is a characteristic of aromatic Trp59, which is quenched by heme group through foster energy transfer, so native h-cyt c produces no fluorescence. While, upon protein unfolding the distance between Trp59 and the heme moiety increases, that further reduces the extent of quenching and increase in tryptophan fluorescence is observed, this goes in hand with loss in protein structure. To further warrant the conformational changes we recorded the fluorescence spectra of stored samples. The fluorescence spectra of h-cyt c in Figure 6 (f) shows distinguished emission maximum at 340 nm having higher intensity. This hints towards the loss of tertiary structure of the protein during storage at RT with exposure of Trp59. The samples with 1mM concentration of ILs showed preserved structure and produced almost no fluorescence. 8-Anilino-1-naphthalene sulfonic acid (ANS) is a hydrophobic fluorescent molecule that is commonly used for the characterization of partially folded intermediates

55-56

. They are minimally fluorescent in polar environments but

exposure to nonpolar environment their fluorescence emission increases dramatically57.The ANS fluorescence were also utilized as probe to inspect the presence of exposed hydrophobic patches on the protein58. The presence of these exposed hydrophobic patches are confirmed by the binding of ANS to it producing a blue shift in its emission maximum, different from when ANS is present in aqueous solution55. The spectra of h-cyt c stored for 6 months (denatured h-cyt c) showed in Figure 6 (g) have almost same emission maximum as the native h-cyt c i.e. no hydrophobic patch of proteins are exposed for binding to ANS. The results obtained confirms that no shift in stored sample denotes preserved structure of h-cyt c by ILs that restrains unfolding and provide stabilization at RT for long storage periods. The lifetime results also


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augmented above results as the average lifetimes listed in Table 3, also differs from the stored hcyt c without IL due to the different conformational fluctuations, Figure 7 (a & b).

Influence of [C8mim][Cl] and [C10mim][Cl] on Peroxidase Activity of h-cyt c The activity of h-cyt c is useful to study its unfolding and it was also utilized as an indicator to monitor the conformational changes. The spectra at 470 nm by UV-vis spectroscopy were recorded to monitor formation of tetraguaiacol from guaiacol in presence of H2O2 with increase in absorbance 35-36. The activity of fresh samples of h-cyt c as well as the one with [C8mim][Cl] and [C10mim][Cl] were recorded. The h-cyt c showed peroxidase activity slight higher in presence of both ILs as in Figure 8. The samples subjected to storage for 6 months were also checked for their activity. The stored h-cyt c peroxidase activity was enhanced due to altered conformation. The h-cyt c preserved with ILs for over 6 months showed slightly reduced activity than fresh samples. The formation of tetraguaiacol has been shown for formation of product during reaction shown in Figure S8. The results depict preserved structure of h-cyt c by ILs for 6 months at RT. The ILs have hydrophobic chain through which they tend to interact with amino acid residue which increases the hydrophobicity around heme. The ILs stabilizes the tertiary structure of h-cyt c by interacting around the flexible structures at protein surface that restrains the unfolding of polypeptide chain and disallows the disruption of hydrogen bonds and thus hold back the loops to cooperatively unfold by excluding from excessive water content and maintain the structural integrity53. The primary unfolding hotspot shown by groups which triggers sequential unfolding of h-cyt c which are concentrated in the loop region

59-60

, thus interaction of ILs confers greater

stability to the native oxidized form of h-cyt c.



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DISCUSSION Now we wondered why ILs stabilize the protein for a longer period and does not perturb the heme crevice even at higher concentrations? We analyzed the results in accordance to the specificity of IL ions as the protein stability by ILs is result of a subtle balance between ion– protein and ion-solvent interaction

53

. The anionic moiety of ILs play important role, as in

Hofmeister series Cl- is placed in midst of range for stabilizing/destabilizing effect on proteins, so they have smaller tendency towards protein denaturation. The Cl- anion is excluded from the protein surface that equips cation to interact and promote stability. The Cl- anion is strongly hydrated thus it have limited interaction with the cationic moiety in aqueous solution and with the charged or polar residues of the protein backbone which restrains dehydration of the protein surface. The internal mobility of h-cyt c (mostly in loop region) in presence of IL is also much restricted than in aqueous solutions that rigidifies the segments. Although, Cl- ion can show weak interactions in area with positively charged patches at the protein surface61. So, Clenhances the stability by preserving structure of h-cyt c and indirectly the heme packing (heme stability) for longer period of time. Thus availing the advantage by Cl- the imidazolium IL cation ([C10mim]+ and [C8mim]+) interacts at the protein surface (SI Figure S1 a & b). The cationic part having imidazolium head group is amphiphillic in nature as it consist of a high charge density part (polar head group) and low charge density (non polar tail). The accumulation of imidazolium cation on surface is useful in removing the surrounding water molecules that reduces the probable hydrogen bonding of water from the protein surface9, 53, 62. Owing to different chain length of both ILs, [C10mim]+ and [C8mim]+, different specificity to protein residues is shown. The [C10mim]+ interacts with accessible residues of 60's α-helix and with unstructured loop regions, infrared loop and red loop


Page 23 of 37

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later (having Met-80 and considered "hotspot" for unfolding59-60) at the protein surface and thus the affinity of imidazolium IL cation for protein surface depends upon hydrophobicity, and accessibility (size of cation). In results, the presence of charge transfer band at 695 nm (Figure 6 a & b) of h-cyt c with both ILs shows preserved structural integrity of h-cyt c and restrains denaturation. The charged residues at pH 7.0 show cation binding thus the more electrostatic contribution after interaction but limited with the case of longer alkyl chain. The [C10mim][Cl] interacts at structures located on the protein surface and showed prolonged effect for maintaining structural integrity (tertiary and secondary) of h-cyt c. This shows significant role of both anion and cation in determining stability of protein. Recent literature suggests important function of lys73 along with other lysine residues (lys72 and lys79) required for stabilizing axial coordination of Met80-heme as well as the tertiary structure41. It maintains two salt bridges with Glu66 and Glu69 and in absence of lys73 both are lost41. It is also involved in molecular recognition when binding with cardiolipin41. Another study provides hydrophobic binding pocket for long chain ligands that binds creating small shifts in position of lys55, val57 and tyr74 (least stable infrared loop)

63

. The accommodation of

hydrophobic chain slightly affects the tertiary structure of h-cyt c, and tyr67 occurs at nearest to it lying adjacent to the heme

63

. These findings corroborate docking results obtained for

[C10mim][Cl] after interaction with h-cyt c (SI Figure S1 b) and comprehends its long term stability in aqueous solution of IL.

CONCLUSION This work reveals unusual potential of the long chain imidazolium ILs to maintain the stability of h-cyt c for longer period at RT at low concentrations (1 mM) never been reported before. The long chain IL [C10mim][Cl] interacts around 60's helix and unstructured loops present at protein



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Page 24 of 37

surface having Met-80 that reduces mobility of loop region and excludes excessive water content. This enhances stability to local protein regions which are first prone to unfolding during denaturation. So, the study provides promising and compatible solvent for maintaining protein structural stability and also prompts us further for evaluation of parameters controlling stability. We propose development of long chain imidazolium ILs can cause major advances in maintenance of the cyt c stability for long term period.

Supporting Information Docking figures, FT-IR, UV-vis, DSC, Far-UV CD, Time-Resolved Fluorescence spectra, Surface Tension, Peroxidase activity (long term stability) results. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS Dr. Rajan Patel greatly acknowledges the financial supports from Science and Engineering Research Board (EEQ/2016/000339 and SR/S1/PC-19/2011) New Delhi, India. Authors also thank DST for providing the FIST grant with Sanction Order No. (SR/FIST/LS-541/2012). Upendra Kumar Singh is thankful to UGC, New Delhi for SRF fellowship.

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Figures & Tables Scheme 1 (a) Structure of h-cyt c Showing Helices and Loops (PDB ID: 1HRC). The Figure Depicts the Two Axial Ligands His18 and Met80 Covalently Linked to Heme, its and the Single Trp Residue at Position.

Scheme 1 (b) Structure of 1-Methyl-3-Octylimidazolium Chloride [C8mim][Cl], having Positively Charged Imidazolium and Negatively Charged Chloride (Green).



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Scheme 1 (c) Structure of 1-Decyl-3-Methylimidazolium Chloride [C10mim][Cl], having Positively Charged Imidazolium and Negatively Charged Chloride (Green).

Figure 1 (a & b) Far-UV CD spectra of h-cyt c (18 х 10-6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples. (c & d) Near-UV CD spectra of h-cyt c (18 х 10-6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples. (e & f) Soret UV-CD spectra of h-cyt c (18 х 10-6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples.

Figure 2 FT-IR spectra of h-cyt c in absence and presence of 10 х 10-4 M [C8mim][Cl] & [C10mim][Cl].


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Figure 3 (a & b) UV-Vis Spectra of h-cyt c (18 х 10-6 M) with [C8mim][Cl] and [C10mim][Cl] for Fresh Prepared Samples. Inset in (a) & (b) shows plots of the presence of charge transfer band (Met80–Fe axial bond) at 695 nm.

Figure 4 (a & b) Time-resolved fluorescence decay spectra of the h-cyt c (18 х 10-6 M) with [C8mim][Cl] and [C10mim][Cl] of freshly prepared samples, respectively.



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Figure 5 Far-UV CD spectra of h-cyt c (18 х 10-6 M) with [C8mim][Cl] and [C10mim][Cl] of fresh, 3 month and 6 month stored sample at room temperature.

Figure 6 (a & b) UV-vis spectra of h-cyt c (18 х 10-6 M) with [C8mim][Cl] and [C10mim][Cl] of samples stored for 6 months at RT. Inset in (a) & (b) shows plots of the presence of charge transfer band (Met80–Fe axial bond) at 695 nm. (c & d) Near UV CD spectra of h-cyt c (18 х 106 M) with 10 х 10-4 M [C8mim][Cl] and [C10mim][Cl] of samples stored for 6 months at RT. (e) Soret CD spectra of h-cyt c (18 х 10-6 M) in 10 х 10-4 M [C8mim][Cl] and [C10mim][Cl] stored samples for 6 months at RT. (f & g) Fluorescence and ANS Fluorescence spectra of h-cyt c in 10 х 10-4 M [C8mim][Cl] and [C10mim][Cl] stored samples for 6 months at RT.


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Figure 7 (a & b) Time-resolved fluorescence decay spectra of h-cyt c (18 х 10-6 M) with 10 х 10-4 M [C8mim][Cl] and [C10mim][Cl of samples stored for 6 months at RT, respectively.

Figure 8 Peroxidase activity of h-cyt c in presence of 10 х 10-4 M [C8mim][Cl] and [C10mim][Cl]. The graph shows samples freshly prepared samples and stored h-cyt c for 6 months at RT.


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Table 1: Far UV CD data of h-cyt c with ILs Shows the Mean Residue Ellipticity at θ222 and αhelical Content (%) at Respective Concentration.

Concentration [C10mim][Cl] ( х 10-6M ) θ222 (deg cm2 dmol-1) 0 -11444 ± 343

[C8mim][Cl] θ222 (deg cm2 dmol-1) -11444 ± 343

[C10mim][Cl] [C8mim][Cl] α-helical α-helical content (%) content (%) 30 30

5

-11507 ± 345

-12635 ± 379

30

34

10

-12083 ± 423

-13032 ± 456

32

35

50

-12622 ± 379

-12939 ± 388

34

35

100

-13395 ± 536

-13682 ± 547

37

37

1000

-14123 ± 381

-13859 ± 374

39

38

Table 2. Fluorescence Lifetime Measurements and the Population Distribution of h-cyt c with [C8mim][Cl] and [C10mim][Cl] of Freshly Prepared Samples at Room Temperature. Samples h-cyt c

τ1 (ns)* 0.39

τ2 (ns) * 1.98

τ3 (ns) * 6.87

α1 (%) α2 (%) α3 (%) τ av (ns) * χ2 29 46 25 4.99 1.12

[C8mim][Cl] 5 х 10-6M 10 х 10-6M 5 х 10-5M 10 х 10-5M 10 х 10-4M 10 х 10-3M

0.39 0.35 0.37 0.32 0.36 0.37

2.02 2.01 2.10 1.94 1.98 1.93

6.19 6.17 6.70 6.12 5.93 5.64

29 29 30 28 30 29

46 46 45 45 43 48

25 26 25 27 27 24

4.43 4.49 4.84 4.55 4.37 3.96

1.12 1.04 1.20 1.16 1.15 1.08

50 х 10-3M

1.63

2.76

5.19

53

29

18

3.26

1.06

[C10mim][Cl] 5 х 10-6M -6

10 х 10 M

0.39

2.31

6.86

26

50

24

4.83

1.14

0.31

1.90

6.09

27

45

28

4.57

1.19

-5

5 х 10 M 0.41 2.22 5.89 25 45 30 4.41 0.90 -5 10 х 10 M 0.35 1.91 5.66 29 43 28 4.22 1.07 -4 10 х 10 M 0.37 1.94 5.94 33 44 23 4.19 1.13 -3 10 х 10 M 0.34 1.19 14.44 37 58 5 7.19 1.47 -3 50 х 10 M 0.43 2.02 12.39 39 50 11 7.44 1.21 * The mean error estimated for the lifetime parameters: τ1 is ± 0.02 ns, τ2 is ± 0.07ns, τ3 is ± 0.21 ns and τav is ± 0.15. 35 ACS Paragon Plus Environment


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Table 3: Fluorescence Lifetime Measurements and the Population Distribution of h-cyt c with [C8mim][Cl] and [C10mim][Cl] of 3 and 6 Months Stored Samples at Room Temperature. Samples

τ1 (ns) * τ2 (ns) *

τ3 (ns) *

α1 (%)

α2 (%)

α3 (%)

τ av (ns) *

χ2

3 months h-cyt c

0.36

1.82

6.39

24

47

29

4.81

1.26

10 х 10-4M [C8mim][Cl]

0.36

2.05

5.84

19

44

37

4.62

1.30

10 х 10-4M [C10mim][Cl]

0.42

2.22

6.09

19

48

33

4.64

1.27

6 months h-cyt c

0.18

1.00

4.44

60

33

7

2.43

0.99

10 х 10-4M [C8mim][Cl]

0.47

2.46

7.18

25

49

26

5.12

1.10

10 х 10-4M [C10mim][Cl] 0.48 2.29 7.12 24 52 24 4.98 1.05 * The mean error estimated for the lifetime parameters: τ1 is ± 0.02 ns, τ2 is ± 0.07ns, τ3 is ± 0.1 ns and τav is ± 0.15.


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Graphical Abstract Synopsis Long-term stability of h-cyt c sustained by long chain imidazolium ionic liquids at low concentration.

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Mechanism and Dynamics of Long-Term Stability of Cytochrome c

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