Unprecedented Improvement in the Stability of Hemoglobin in the

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Unprecedented Improvement in the Stability of Haemoglobin in the Presence of Promising Green Solvent 1-Allyl-3-methylimidazolium Chloride Pannuru Venkatesu, and Indrani Jha ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b00939 • Publication Date (Web): 04 Oct 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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ACS Sustainable Chemistry & Engineering

Unprecedented Improvement in the Stability of Haemoglobin in the Presence of Promising Green Solvent 1-Allyl-3-methylimidazolium Chloride Indrani Jha and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi-110 007, India ABSTRACT In this article, we have explored the influence of peculiar member of imidazolium-based ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([Amim][Cl])

on the stability of

haemoglobin (Hb) by using fluorescence, thermal fluorescence, ANS fluorescence, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) measurements. In the attempt of searching of IL which can provide stability to Hb, for the first time, we have successfully shown the stability of Hb in [Amim][Cl]. Our results show that [Amim][Cl] stabilizes the Hb native structure in concentration dependent manner which indicates to fact that the concentration of IL is very crucial to determine the stabilization/destabilization of the protein in ILs. Here, we have observed that low concentration of IL provides stability to the protein while high concentration destabilizes the protein. The commendatory results obtained from the multi spectroscopic approaches provided some guidance regarding the mechanism of interaction between Hb and [Amim][Cl]. The predicted mechanism may be the accumulation of imidazolium cation on the protein surface and tendency of anion to remain in the bulk phase ultimately resulting in stabilization of the protein since; this can in direct/indirect way affect the hydrogen bonding of protein with the surrounding water. Key words: Ionic liquids, Thermal stability, Fluorescence, Circular dichroism, Dynamic light scattering.

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INTRODUCTION Haemoglobin (Hb) is an important globular protein from physiological point of view.1,2 There are four polypeptides and four haem groups in each porcine Hb (pHb).3 The αsubunit of pHb consists of seven helical (A, B, C, E, F, G and H) and seven non-helical regions however, the β-subunit encloses eight helical segments (A, B, C, D, E, F, G and H) and seven non-helical segments.3 Basically, Hb is a tetramer of two symmetrical αβ dimers. It consists of three tryptophan (Trp) residues in each dimer making it total of 6 Trp residues in the tetramer. Out of these Trp residues, Trp 37 is the main source of fluorescence emission which is situated at the dimer−dimer interface.4 The native conformation of the protein is a result of complex interactions such as Van der Waals interactions, ionic interactions and hydrogen bonds.5 Under physiological conditions, these interactions provide stability to the proteins and prevent ruinous conformational changes which can occur due to perturbations in the environment of the proteins. One of the strategies to stabilize the native conformation of the protein can be to exploit additives which can change the solvent environment. The stability of protein’s structure and its function can be modulated by the balance between rigidity and flexibility of the polypeptide and side chains which can be either improvised or decreased by changing the solvent property or by addition of co-solvents. Persistent studies related to protein-co-solvent interactions yield new insight into protein’s properties and behaviour, particularly, related to protein-solvent interactions, conformational disorder and conformational dynamics. Protein denaturation is a process in which proteins tend to lose their tertiary and secondary structure in the presence of strong stresses such as heat, extreme cold, strong acid or base, high salt level etc. Therefore, there is a need of solvent which could provide


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protection to proteins against these stresses. There are solvents which can play the role of protein stabilizers however, due to environmental related issues; there is a need to search for alternatives of these solvents. Ionic liquids (ILs) have emerged as an important alternative of the usual organic solvents because of certain properties such as non flammability, negligible vapour pressure and high chemical and thermal stability.6-8 Because of their high surface activity, less aqueous toxicity as well as resistance to oxidation and reduction processes, in recent years, the ILs are receiving a very good response from the industrial community.6 These ILs are having wide applications in various fields including electrochemistry7,8, drug delivery9, polymer chemistry10 and many more applications. By taking these properties into consideration, ILs are often being classified as green solvent.11, 12 Nowadays, the awareness regarding the impact of the chemicals on environment is increasing globally. So, the need of the time is to design chemicals consciously, so that safe chemicals with enhanced performance can be delivered. The basic principle of green chemistry is the prevention of hazard generation with encouragement of developments which could improve novel processes.13 ILs have the potential to fulfil the requirement of environment sustainability, however, the concept of environment sustainability have to be conceptually considered for the majority of ILs. Particularly taking protein stability in ILs into consideration, we aim to achieve overall a high degree of protein stabilization in ILs. The behavioural changes occurring in proteins in the presence of ILs have been frequently reported.14-18 The investigation related to studies of interaction between proteins and different ILs in aqueous solution are comparatively more important when compared to those in neat ILs. These investigations suggest that there is no fixed rule which can predict the behaviour of protein in ILs. There are enough evidences in literature which conclude that



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the interaction between protein and IL depends on the protein as well as on the cations and anions present in the ILs.19-23 Studies have shown ammonium-based ILs to act as better stabilizing agent for proteins as compared to imidazolium-based ILs.24 Our research group has out carried protein stability studies in both ammonium and imidazolium-based ILs.16, 25-29 Recently, Jha et al.27 showed the destabilization of Mb and Hb in ammonium-based ILs which was much unexpected result because of the fact that ammonium-based ILs are known as good protein stabilizers. Again, Attri et al.28 showed ammonium-based ILs to act as both stabilizer as well as destabilizer for Mb depending on the viscosity of the IL. Recently, imidazolium-based ILs were found to be acting as destabilizer for the stability of Mb16 and Hb29 where destabilization tendency of the ILs increased with increasing chain length of the cation of ILs. From our studies of heme protein (Hb and Mb), it can be seen that only very few of the ammonium-based ILs stabilized heme proteins while imidazolium-based ILs proved to be destabilizer. Hence, the search for the IL which could provide stability to Hb was prime aim of our study. The literature survey about the interaction of imidazolium-based ILs with different proteins suggests the frequent use of ILs based on 1-alkyl-3-methylimidazolium chloride ILs where the alkyl group is saturated group.30-37 The studies carried out by Zhang and coworkers38,

39

proved 1-allyl-3-methylimidazolium chloride ([Amim][Cl])

to be non-

derivatizing solvent for cellulose. Thus, this IL serves as novel solvent for cellulose. In [Amim][Cl], one of the substituent on the nitrogen is not saturated alkyl rather it is an alkenyl group. Since this substitution on one nitrogen atom makes this IL having relatively low melting point with high thermal stability compared to IL containing alkyl substituent having the same number of carbon atoms.39 All these unique characteristics of this particular IL intrigued our interest to investigate its impact on the stability of proteins. Also, studies related 4 ACS Paragon Plus Environment

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to structure and function of Hb is well studied, however, research related to its interaction with co-solvent is limited. Therefore, both of these factors induced us to dig deep into our understanding of the impact of alkenyl substituted IL on protein structure and stability. Here, we have studied the stability of Hb protein in the presence of [Amim][Cl] which is peculiar member of imidazolium family IL by using different spectroscopic techniques and dynamic light scattering (DLS). We are able to describe the conformations as well as size distribution for the Hb molecule in buffer as well as in the presence of different concentration of [Amim][Cl]. Depending on IL concentration, Hb can show multi-facetted behaviour. In this work, we try to focus concentration effect on structural and stability changes in Hb brought about by [Amim][Cl]. MATERILAS AND METHODS Hb porcine, lyophilized powder (molecular weight: 66.7 kDa), [Amim][Cl] (≥97.0%) and Tris-base, Tris(hydroxymethyl)aminomethane (99.9 %) were obtained from Sigma-Aldrich chemical company USA. Tris-HCl buffer solution (10 mM) at pH 7.2 was prepared using distilled deionized water with a resistivity of 18.3 Ωcm. All the samples were prepared gravimetrically using a Mettler Toledo balance with a precision of ±0.0001 g. Protein stability was analyzed by incubating 2 mL screw-capped vials in 10 mM Tris-HCl buffer at pH 7.2 in presence and absence of ILs at 25 °C for 4 h to attain complete equilibrium. For all fluorescence experiments the protein concentration was 0.5 mg/mL. For far UV-CD experiments 0.15 mg/mL protein concentration was used since at higher protein concentration we were unable to obtain the secondary spectra due to high voltage however, for near UV-CD experiments 1 mg/mL concentration of Hb was used. After completely dissolving the protein in the solution, the mixture was filtered with a 0.45 µm disposal filter (Millipore, Millex-GS) through a syringe prior to measurements.



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

(1)

A sigmoidal fluorescence intensity curves were obtained for Hb in the presence of [Amim][Cl]. Further details of the thermal analysis have been described elsewhere.27, 40, 41 8-Anilino-1-Naphthalene–Sulphonic Acid (ANS) Fluorescence Measurements We performed the ANS binding measurements using fluorescence emission spectra with excitation at 380 nm and emission was recorded from 400 to 600 nm using same fluorescence spectroscopy. Circular Dichroism (CD) Spectroscopy Circular dichroism (CD) spectroscopic studies were performed on a PiStar-180 spectrophotometer (Applied Photophysics, U.K.) equipped with a Peltier system for temperature control. CD calibration was performed using (1S)-(+) - 10-camphorsulfonic acid 6 ACS Paragon Plus Environment

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(Aldrich, Milwaukee, WI), which exhibits a 34.5 M cm-1 molar extinction coefficient at 285 nm and 2.36 M cm-1 molar ellipticity (Θ) at 295 nm. All the samples were pre equilibrated at the desired temperature for 15 min, and the scan speed was fixed for adaptative sampling (error F 0.01) with a response time of 1 s and 1 nm bandwidth. The secondary and tertiary structures of Hb were investigated by using far-UV (190-240 nm) (0.1 cm path length cuvette) and near-UV (250-300 nm) (1.0 cm path length cuvette) spectra, respectively. Each of the sample spectra was subtracted from the blank media. Dynamic Light Scattering (DLS) Measurements The hydrodynamic diameter (dH) has been determined by means of a Zetasizer Nano ZS90 dynamic light scattering (DLS) (Malvern Instruments Ltd, UK). The instrument measures the time-dependent fluctuation in the intensity of light scattered from particles in the solution at a fixed scattering angle of 90°. The instrument was equipped with 4 mW He–Ne laser with a fixed wavelength, λ = 633 nm as a light source and utilized to characterize the obtained aggregate sizes. All of the DLS experiments were carried out at constant temperature of 25 °

C. A filtered bubble free sample of around 1.5 mL was transferred into a quartz sample cell,

which was sealed with a Teflon-coated screw cap to protect from dust. Then the air tight sample was introduced into the sample holder of the sample chamber of the DLS instrument. The Brownian motion of particles was detected by the DLS and it was correlated to the particle size. The relationship between the size of a particle and its speed due to Brownian motion is defined by the Stokes-Einstein equation. All data obtained were analyzed by Malvern Zetasizer Software version 7.01. RESULTS AND DISCUSSION Different spectroscopic results show that IL-induced transitions in Hb are dependent on the concentration of IL and suggest that this IL has profound influence on the structure and stability of Hb. To ascertain the impact of [Amim][Cl] as a function of concentration on the 7 ACS Paragon Plus Environment


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behaviour of Hb, we have studied fluorescence, thermal fluorescence, ANS fluorescence, near-UV CD, far-UV CD spectroscopy and DLS measurements of Hb in eight different concentrations such as 0.01, 0.03, 0.05, 0.10, 0.15, 0.25, 0.50 and 1.0 M of [Amim][Cl]. Fluorescence Analysis of the Effect of [Amim][Cl] on the Structure and Stability of Hb Intrinsic fluorescence is a sensitive index for studying the alteration in the conformation of proteins. Generally, the intrinsic fluorescence of proteins containing Trp and tryrosine (Tyr) can be used as an important probe for estimation of some of the factors such as protein conformational transition, biomolecular binding, denaturation and so on.42 The fluorescence properties such as maximum intensity (Imax) of fluorescence and maximum emission wavelength (λmax) are highly sensitive to the polarity of microenvironment around the fluorophore. These basically indicate the tryptophanyl environment. The fluorescence studies show that this IL has profound influence on Hb. Generally, the Hb fluorescence spectra are highly quenched because of efficient energy transfer from the β-37 Trp (the main source of emission) to the porphyrin system43 however; we observed a substantial increase of the emission intensity of fluorescence on addition of IL at all concentrations. The reason behind this increase in intensity at various concentrations of IL could be different based on the diverse structural alterations brought about by this IL at different concentration range. The first aspect to be considered is, prevention of quenching effects produced due to non-radiative energy transfer from the β-37 Trp to its nearest heme group. Another major reason accounting for the increase in intensity can be increased hydrophobicity around the main fluorophore. Hb possess emission maximum at 327 nm in native state.

From Figure 1, Hb

fluorescence spectra showed blue shift of the λmax up to 0.10 M of [Amim][Cl], suggesting stabilization of the native conformation of the Hb in [Amim][Cl] up to this concentration


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which is evidence for the presence of Trp in a hydrophobic environment. Therefore, the reason for the increase in intensity in the presence of 0.01 to 0.10 M [Amim] [Cl]

was

obviously the increased hydrophobicity around the main fluorophore. In the concentrations of 0.15 and 0.25 M of [Amim][Cl], there was diminutive shift in the λmax with slight broadening of band. On further increasing the concentration of IL to 0.50 and 1.0 M, the fluorescence spectra showed tremendous increase in intensity. There was red shift in spectra at 0.50 M. At 1.0 M [Amim][Cl], the red shift of λmax was to 333 nm. Here, the reason for the increase in intensity was that on partial unfolding of the protein, the distance between β-37 Trp and porphyrin may be increased which blocked the efficient energy transfer from Trp to porphyrin hence resulting in increased intensity. This clearly indicated that very high concentration of [Amim][Cl] has negative impact on the stability of Hb. However, the significant feature of the spectra was that even at concentration of 1.0 M, the λmax shifted to only 333 nm, which is indicative of the presence of Trp in limited contact with water. Detailed Inspection of the Thermal Stability of Hb in [Amim][Cl] The fluorescence analysis results compelled us to observe the thermal stability of Hb in [Amim][Cl]. The Tm values of Hb in buffer and in [Amim][Cl] are collected in Table 1. The Tm values presented in Figure 2 have been obtained from the fluorescence intensity curves (Figures S1-S7) of thermal analysis of Hb in [Amim][Cl]. The addition of [Amim][Cl] indeed shifted Tm of Hb from 67.5 °C (in buffer) to 72.6, 71.5, 71.1, 68.3 0C in 0.01, 0.03, 0.05 and 0.10 M of [Amim][Cl], respectively. Our results clearly indicate that the thermal stability of Hb was enhanced in the presence of [Amim][Cl] at 0.01, 0.03, 0.05, 0.10 M of IL. Later, with increasing concentration of IL the Tm values were 68.1 and 65.9 °C for 0.15 and 0.25 M, respectively. This shows that the protein destabilized at high concentration of 0.25 M. The notable feature was that even at 0.25 M IL, the decrease in Tm was not that much effective. The Tm values are in accordance with fluorescence results which predict that in low 9 ACS Paragon Plus Environment


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concentration of [Amim][Cl] the Hb is stabilized, however, the capacity of IL to stabilize the Hb kept on decreasing with the increasing concentration of the IL as evident from thermal data analysis. The thermal stability of the Hb in low concentration of IL (72.6 °C at 0.01 M) was much higher with respect to the Tm of Hb (65.9 °C at 0.25 M) in high concentration of IL. We were unable to obtain Tm values of Hb in 0.50 and 1.0 M [Amim][Cl] since we did not observe any fluorescence transition as a function of temperature at these concentrations of IL. ANS Fluorescence Studies of Hb in Presence of [Amim][Cl] Further, to bestow a much deeper understanding to these conformational changes brought by [Amim][Cl], we performed ANS fluorescence studies. Since, fluorescent probing can prove to be extremely informative tool in the studies related to the conformational states of different proteins.44 1, 8-Anilinonaphthalene sulfonate (ANS) is dye molecule which gives high fluorescence intensity when it binds to hydrophobic region of a protein.45 The ANS fluorescence intensity gradually decreased as the concentration of IL increased from 0.01 to 0.10 M IL which is evident from Figure 3. This decreased ANS fluorescence intensity indicated the burial of hydrophobic patches. Therefore, the exhibition of relatively less ANS fluorescence intensity in the presence of 0.01 to 0.10 M IL as compared to intensity of ANS fluorescence without IL indicates the absence of hydrophobic environment around the ANS molecule which gave further confirmation of Hb stability in 0.01 to 0.10 M [Amim][Cl]. With further increase in the concentration of [Amim][Cl] to 0.15 and 0.25 M, there was blue shift in the λmax with increase in intensity. This increased intensity suggested that the dye is binding slowly to hydrophobic patches of Hb on slow destabilization of Hb which is again consistent with our fluorescence and thermal analysis. However, a marked increase in intensity was observed upon addition of 0.50 and 1.0 M IL. This increase in ANS fluorescence intensity at 0.50 and 1.0 M of [Amim][Cl]


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suggested the unfolding of Hb at such a high concentration. Furthermore, ANS binding showed increased ANS fluorescence intensity demonstrating the binding of ANS to the hydrophobic patches of the Hb on partial unfolding. Note that here partial unfolding has taken place which implies that protein is not completely unfolded. In view of the fact that ANS is having fewer tendencies to bind to native protein and completely unfolded protein. Since, in both the cases the hydrophobic patches are unavailable for dye binding. The large increase in intensity suggested that hydrophobic patches of the protein is now available for ANS binding giving rise to high intensity. Changes in the Secondary Structure of Hb in the Presence of [Amim][Cl] We applied far UV-CD spectroscopy to follow the changes in the secondary structure of Hb in [Amim][Cl]. It is well known that different types of secondary structures exhibited by proteins give rise to characteristic CD spectra in the far-UV region. The band at 208 nm corresponds to Π to Π* transitions of the α-helix, while the band at 222 nm corresponds to Π to Π* transition for both α-helix and random coil.46,47 As can be seen from Figure 4 that native Hb shows characteristic bands at 222 and 208 nm. Only at 0.01 M of IL CD spectra showed characteristic bands as in case of native Hb with increase in the negative ellipticity values which indicate stabilization of Hb. However, as the concentration of the IL is increased, due to the interfering effects of IL on the α-helical region of the far-UV CD spectra, the CD spectra did not provide any accurate information. Changes in the Tertiary Structure of Hb in the Presence of [Amim][Cl] In CD spectroscopy apart from secondary structure prediction, there is tertiary structure finger print region 260-320 nm. Here, in this region each amino acid tends to have a characteristic wavelength profile. This is known as the near-UV CD region which gives valuable information about the tertiary structure of the protein. Trp shows peak close to 290



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nm, Tyr shows a peak between 275 and 282 nm while phenylalanine (Phe) shows weak bands. To probe the asymmetry of the aromatic amino acids environments in Hb, near-UV spectra were used to evaluate the extent of the conformational changes in Hb in the ILs in comparison to the native protein.48, 49 As can be seen from Figure 5 that spectrum is complex and therefore it is difficult to divide the spectra into particular regions. For the sake of simplicity, here we will discuss only the overall tertiary structure instead of emphasizing particularly Trp, Tyr and Phe residues. From Figure 5, the [Amim][Cl] has led to enhancement of the tertiary structures of Hb as compared to Hb in buffer which is evident from the increased positive ellipticity values of band in the presence of different concentration of IL. However, the amount of tertiary structure induced by the IL decreases with the increasing concentration. Influence of [Amim][Cl] on the dH of Hb by DLS Measurements To further ascertain our results, we performed DLS measurements for computing the dH of Hb in the presence of different concentrations of [Amim][Cl]. DLS is one of the most important as well as popular method to determine the size of the particles.50 The dH is highly influenced by the shape of the protein molecule.51 We have used DLS for the characterization of size distribution of Hb in the presence of different concentrations of [Amim][Cl] at a constant temperature of 25 °C. The dH of Hb in the presence of buffer and different concentrations of [Amim][Cl] along with polydispersity of the solution are shown in Table 2. The lower values of polydispersities are indication of presence of homogenous species in solution. Figure 6 depicts the intensity distribution graph which illustrates the relative intensity of scattered light from the DLS measurement of Hb in buffer as well as in different concentrations of ILs. In buffer, the dH of Hb was centred around 6.404 nm (Figure 6). These results are in good agreement with the results of previous measurement.52 Our results elucidate that the dH values of Hb in buffer and in 0.01 to 0.25 M [Amim][Cl] are obtained 12 ACS Paragon Plus Environment

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from the major population (native state). As can be seen from the intensity distribution graph that Hb in buffer and in 0.10, 0.15 0.25 M [Amim][Cl] give rise to two peaks where the second peaks are in the range of 100 nm and above . For the second peaks (minor peaks) we observed lower percentage of intensity when compared to first peak (major peaks). This implies that the population of the second peaks are negligibly small. Consequently, the second peak could be due to aggregated Hb molecules. Figure 7 depicts the dH obtained from the intensity distribution graph as a function of IL concentration. For the sake of clarity we have presented the trend in dH values of Hb in 0.01 to 0.25 M [Amim][Cl] in the inset graph of Figure 7. It is observed that relatively constant shape persists in the concentration range of 0.01 to 0.15 M IL. Therefore, Hb maintains its compact structure up to 0.15 M in IL. The observed sizes in the concentration range of 0.01 to 0.15 M of IL are nearly same as those in buffer, which indicates that the addition of IL at these concentrations does not cause a measurable alteration in the size of Hb which is again confirmation of spectroscopic results. The dH of Hb in 0.25 M IL was found to be 7.835 nm. This increase in size of Hb was only nominal. However, on further increasing the concentration to 0.50 and 1.0 M of IL a typical protein peak couldn’t be detected. This can be due to significant amount of aggregation in Hb in the presence of these high concentrations of IL which has masked the signal from protein. Therefore, DLS gave an affirmation of our conclusion of Hb stability in the 0.01 to 0.15 M IL. Apparently, the structure of Hb stabilized at the lower concentration of [Amim][Cl] however, this stabilization behaviour decreased with increasing concentration of this IL such as 0.25, 0.50 and 1.0 M. Even at 1.0 M, the [Amim][Cl] only feebly destabilized the Hb as evident from λmax value at 1.0 M IL which is 333 nm. It was found that the presence of [Amim][Cl] provided stability to Hb up to 0.15 M, however at higher concentrations this IL decreased

its

stability.

All

these

observations

give

clear

picture

that

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stabilization/destabilization behaviour of Hb in IL depends on IL concentration (Scheme 1). An IL which is stabilizing a protein at lower concentrations can show opposite effect at high concentrations. As a result, it is quite essential to use the appropriate amount of IL as far as protein stability studies are concerned. From the above results, it can be stated that interaction of IL with protein is dependent on the interaction of IL with the amino acids present in the protein. In Hb, the majority of the residues which are polar are on exposed surface and the non-polar ones being internally present. The anion present in the imidazolium-based ILs plays a crucial role in the determining the type of interaction between IL and protein. In Hofmeister series of ILs, the position of Cl- is on the border line of series of anions which predicts the protein stability. The increased stability of Hb in [Amim][Cl] may be due to exclusion of anion from the surface of the protein. This may result in tight binding of cation with protein. Some of the MD simulation studies

53, 54

predicted that the imidazolium cation have great tendency to

accumulate on protein’s surface which results in exclusion of water molecules from the surface of protein. While, the anions have a tendency to stay in the bulk solution and are expelled from the protein surface. This ultimately results in protection of carbonyl group of backbone of the protein from the hydrogen bonding with water which is in an indirect manner.53, 54 Many studies related to other solvents and water solutions have come up with important role of hydrogen bonding between water and protein in destabilization of protein structure stability.55,

56

Also possibility of direct electrostatic or hydrophobic interaction is

there. Both of the direct/indirect interaction between Amim+ and protein leads to protein structure stabilization. Since the tendency of water molecules due to their small size can insert between the carbonyl and amide groups of the backbone. This insertion of water molecules can result in disruption of intra-molecular hydrogen bonding.55,

57

However, the

accumulation of cation around the protein surface could be helpful in removing the 14 ACS Paragon Plus Environment

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surrounding water molecules which decreases the probability of hydrogen bonding from water to protein. This accumulation of cation on the surface of the protein is in consistence with the study by Haberler et al.53,

58

where they predicted that the concentration of low

charge density ions i.e. cations prefers protein surface whereas the high charge density ions i.e. anions favours the hydration with water molecules which are in bulk phase. Also, proteins carry a net positive charge at pH below their pI, and above their pI they carry a net negative charge. pI of porcine Hb is ~ 6.7, therefore, at neutral pH which is our experimental condition the surface of Hb carries overall net negative charge. Therefore, there is greater tendency of cation to bind to the surface of Hb. All these, result in decrease in the quantity of anion and water at the protein surface. In the bulk solution, the anions contribute in the hydrogen bonding with the water molecules. Small water clusters to big water networks also exist in addition to anion-water hydrogen bonding. The imidazolium cation does not seem to play any role in water-anion network. Therefore, from the above discussion, it can be stated that the role of both cation and anion is important in determining the stability of the protein. Our results show increased thermal stability of Hb in the presence of [Amim][Cl] are consistent with the studies carried out by Tamura et al.59 which showed the dissolution of cytochrome c (cyt c), in [Amim][Cl] with the keeping of redox activity of dissolved cyt c up to 140 °C. Homogeneous acetylation of cellulose was carried out in [Amim][Cl] which showcased the potential of this IL as promising green solvents. Also, Zhang et al.39 showed the dissolution and regeneration of cellulose in [Amim][Cl] that proved it to be a novel and non-polluting process for the manufacture of regenerated cellulose materials which also consistent with our studies. Our results are in accordance with these studies which give further encouragement for the use of [Amim][Cl] as promising green solvent. The interaction of ILs with the proteins is a complex matter since this interaction plays important role in the water structure around the protein and interaction with the residues of the proteins. Therefore, 15 ACS Paragon Plus Environment


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on the basis of our results [Amim][Cl] seems to be novel solvent for the stability of Hb, however, further studies with other proteins need to be carried out for further information. CONCLUSION Our results illustrate that interactions of ions of ILs with proteins are important for understanding the effects shown by them on proteins whether stabilization/destabilization. These interactions are governed by the ions present as well as the concentrations used. In this work [Amim][Cl] has been identified as a compatible solvent for Hb structure, nevertheless, the stabilization tendency being concentration dependent. Low concentration of [Amim][Cl] stabilized Hb while higher concentration destabilized Hb. Therefore, the results obtained here facilitate a much better understanding of protein folding/unfolding in IL. Hence, this IL was found to be stabilizing agent for Hb. Since, [Amim][Cl] is thermally stable as well as non-volatile, consequently it can be supposed to be promising green/biocompatible solvent for the proteins. However, further research with other proteins in the presence of this IL is required. Also, from our research this becomes completely clear that the role of alkyl group of imidazolium ILs is vital in determining the protein stability. Therefore, this work can encourage designing of novel ILs which can provide stability to many other proteins. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website Thermal fluorescence spectra analysis of the Hb in buffer and in different concentrations of [Amim][Cl] (Figures S1-S7).


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AUTHOR INFORMATION *e-mail: [email protected]; [email protected]; Tel:+91-11-27666646142; Fax: +91-11-2766 6605 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We are grateful for the support from the Department of Biotechnology (DBT), New Delhi, through the Grant Ref. /File No. BT/PR5287/BRB/10/1068/2012 for financial support and I J is grateful to CSIR, New Delhi for awarding Senior Research Fellowship (SRF).



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59. Tamura,

K.;

Nakamura,

N.;

Ohno,

H.

Cytochrome

Page 24 of 35

c

dissolved

in

1-allyl-3-

methylimidazolium chloride type ionic liquid undergoes a quasi-reversible redox reaction up to 140 0C. Biotechnol Bioeng. 2012, 109 (3), 729–735.

FIGURE CAPTIONS


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Figure 1. Fluorescence spectra analysis of Hb in buffer (black) and in [Amim][Cl] with red (0.01 M), green (0.03 M), blue (0.05 M), cyan (0.10 M), magenta (0.15 M), yellow (0.25 M), dark yellow (0.50 M) and navy (1.0 M) at 25 0C. Figure 2. The variation in Tm values of Hb in buffer (black) and in [Amim][Cl] which is obtained from fluorescence analysis with red (0.01 M), green (0.03 M), blue (0.05 M), cyan (0.10 M), magenta (0.15 M) and yellow (0.25 M). (We did not obtain fluorescence transitions for 0.50 and 1.00 M of [Amim][Cl]). Figure 3. ANS Fluorescence spectra analysis of Hb in buffer (black) and in [Amim][Cl] with red (0.01 M), green (0.03 M) blue (0.05M), cyan (0.10 M), magenta (0.15 M), yellow (0.25 M), dark yellow (0.50 M) and navy (1.0 M). Figure 4. Influence of [Amim][Cl] on the structure of Hb in buffer (black), from far-UV CD analysis with red (0.01 M), green (0.03 M), blue (0.05 M), cyan (0.10 M), magenta (0.15 M), yellow (0.25 M), dark yellow (0.50 M) and navy (1.0 M). Figure 5. Influence of [Amim][Cl] on the structure of Hb in buffer (black), from near-UV CD analysis with red (0.01 M), green (0.03 M), blue (0.05 M), cyan (0.10 M), magenta (0.15 M), yellow (0.25 M), dark yellow (0.50 M) and navy (1.0 M). Figure 6. The distribution of the intensity of light scattered by Hb in buffer and in [Amim][Cl] with red (0.01 M), green (0.03 M), blue (0.05 M), cyan (0.10 M), magenta (0.15 M), yellow (0.25 M), dark yellow (0.50 M) and navy (1.0 M). Figure 7. Hydrodynamic diameter (dH) obtained from the intensity distribution graph for Hb in buffer and different concentrations of [Amim][Cl]. Scheme1. Mechanism depicting effect of different concentrations of [Amim][Cl] on Hb native structure.



150

Fluorescence Intensity

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

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120 90 60 30 0 300

350

400

450

500

Figure 1.


70 60 50 40 30 20 10 0

M 0.25

M 0.15

M 0.10

M 0.05

M 0.03

r

M 0.01

Buffe

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


Transition Temperature (Tm) oC

Page 27 of 35

Figure 2.



100

Fluorescence Intensity

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 28 of 35

80 60 40 20 0 400

450

500

550

600

Wavelength (nm) Figure 3.


Page 29 of 35

80

[ө]( deg cm2dmol-1) 10-3

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


60

40

20

0

-20

200

210

220

230

240

250

260

Figure 4.


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

[ө]( deg cm2dmol-1) 10-3


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3

2

1

0 260

280

300

320

340

Figure 5.


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20 18 16 14 12 10 8 6 4 2 0 -2 6

8

10

200

400

600

800 1000

Figure 6.



250

8.0 7.8 7.6

200

7.4 7.2 7.0

150

6.8 6.6 6.4

100

6.2 6.0

0

0.01 0.03 0.05 0.10 0.15 0.25 [Amim][Cl]/ M

50 0 Hb in 1.0 M

M 0.50 Hb in ][Cl]

][Cl]

[Amim

][Cl]

l]

][Cl]

im][C

[Amim

[Am

[Amim

][Cl]

l]

][Cl]

im][C

[Amim

[A m

[Amim

[Amim

M 0.25 Hb in

M 0.15 Hb in

M 0.10 Hb in

M 0.03

M 0.01

M 0.05 Hb in

Hb in

Hb in

r Buffe Hb in

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 32 of 35

Figure 7.


Page 33 of 35

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Table 1. Transition temperature (Tm) of the Hb in different concentrations of [Amim][Cl].

Hb in Buffer

Transition Temperature (Tm)/ (°C) 67.5

0.01 M [Amim][Cl]

72.6

0.03 M [Amim][Cl]

71.5

0.05 M [Amim][Cl]

71.1

0.10 M [Amim][Cl]

68.3

0.15 M [Amim][Cl]

68.1

0.25 M [Amim][Cl]

65.9

Table 2. Hydrodynamic diameter (dH) and polydispersity (Pd) of the Hb in different concentrations of [Amim][Cl]. Hb in Buffer 0.01 M [Amim][Cl] 0.03 M [Amim][Cl] 0.05 M [Amim][Cl] 0.10 M [Amim][Cl] 0.15 M [Amim][Cl] 0.25 M [Amim][Cl] 0.50 M [Amim][Cl] 1.0 M [Amim][Cl]

Hydrodynamic diameter (dH )/(nm) 6.404 6.300 6.309 6.428 6.440 6.950 7.835 241.1 251.5

% Polydispersity (Pd) 5.6 6.6 8.9 5.4 8.8 8.5 8.6 13.2 27.2



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Scheme 1.

j


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Graphical Abstract

Unprecedented Improvement in the Stability of Haemoglobin in the Presence of Promising Green Solvent 1-Allyl-3-methylimidazolium methylimidazolium Chloride

Indrani Jha and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi Delhi, Delhi-110 007

Synopsis: The native structure of Hb stabilized at low concentration while destabilized at high concentration of [Amim][Cl]

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Unprecedented Improvement in the Stability of Hemoglobin in the

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