Sustained Stability and Activity of Lysozyme in Choline Chloride

Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST

Article

Sustained Stability and Activity of Lysozyme in Choline Chloride against pH Induced Denaturation Indrani Jha, Anjeeta Rani, and Pannuru Venkatesu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02126 • Publication Date (Web): 05 Aug 2017 Downloaded from http://pubs.acs.org on August 7, 2017

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

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

Page 1 of 33

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

ACS Sustainable Chemistry & Engineering

Sustained Stability and Activity of Lysozyme in Choline Chloride against pH Induced Denaturation Indrani Jha, Anjeeta Rani and Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi 110 007, India Mailing address of authors: Indrani Jha: Department of Chemistry, University of Delhi, Delhi 110007, India Anjeeta Rani: Department of Chemistry, University of Delhi, Delhi 110007, India Pannuru Venkatesu: Department of Chemistry, University of Delhi, Delhi 110007, India Corresponding author: e-mail: [email protected]; [email protected]

ABSTRACT The understanding of stability and activity of an enzyme at different experimental conditions is considered as a bottleneck for a successful drug development. Despite of abundant applications of lysozyme (Lyz) in medicine and pharmaceutical industry, fibril formation is major obstacle in development of its biotherapeutic formulations for use in the clinic as Lyz may encounter low pH condition in vitro or in vivo. In this context, the counteraction effects of choline chloride (ChCl) were investigated to offset pH-induced denaturation of Lyz. We employed, UV-vis, fluorescence, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). Furthermore, we showed that the secondary structure, thermal stability and bacteriolytic activity of Lyz at pH 2 are increased in the presence of ChCl to an appreciable extent as compared to that of Lyz in pH 2 buffer only. The highly intensified positive charge on the surface of Lyz under acidic condition is responsible for its denaturation by increasing electrostatic repulsion which is compensated to significant extent on addition of ChCl. Moreover, in ChCl which contains hydroxyl moieties, there may be a high tendency to exhibit the solvophobic effect which helps in retaining Lyz native conformation and activity. This solvophobic effect may offer less competition with water at the specific hydration layer around the Lyz, consequently reducing Lyz–ChCl interactions and maintaining Lyz native structure. It is revealed from the investigation of the results that ChCl is efficiently counteracting pH-undesirable impacts on Lyz. To the best of our knowledge, this study represents the first detailed experimental evidence about the counteraction ability of ChCl. Keywords: Enzyme, Counteraction effect, Protein structure, Transition temperature, Fluorescence spectroscopy, Dynamic light scattering , circular dichroism spectroscopy

---------------------1 ACS Paragon Plus Environment


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

Page 2 of 33

INTRODUCTION Lysozyme (Lyz) is a very well known bacteriolytic enzyme which is synthesized by macrophages throughout the body.1-3 Lyz is present in the liver, articular cartilage, milk and saliva. Lyz is also found in trace amount in body fluids such as serum and cerebrospinal fluid.3 The bacteriostatic and bactericidal properties of Lyz have created abundant applications in medicine and pharmaceutical industry4,5 that necessitate the understanding of its stability and activity at different experimental conditions which is considered as a bottleneck for a successful drug development. Enzymes are generally very sensitive to the changes in pH and temperature of the surrounding environment that extremely influence their activities.4 However, according to Fink et al.,6 Lyz belongs to Type III protein which does not completely unfold even at pH as low as 1. Still, the structure, stability and activity are adversely affected at low pH.1,7 In vivo, Lyz is generally present in the condition with pH ranging from 4.5 to 7.4 in various media. Undoubtedly, pH values can be low (i.e., pH < 3) in intracellular compartments (such as lysosomes),1,8,9 and an acidic environment may be relevant to the situation which augment the proportion of partially folded protein molecules with reduced thermodynamic stability and high propensity to form fibrils.1,10 The formation of fibrils is deemed as one of the most common issues that appreciably affects the quality and effectiveness of therapeutic candidates. Additionally, this aggregation complicates handling and storage of peptides and proteins. In order to squeeze out the benefit from Lyz for its various applications, it becomes mandatory to maintain enzyme stability and function by counteracting the adverse effect of pH. Generally, in biochemical applications, it is preferred to choose the green solvent which is quite effective and biocompatible. In this regards, ionic liquids (ILs) represent very interesting compounds which have been proven as green solvents having a wide variety of applications in various scientific fields, particularly, in biophysical chemistry and bioengineering.11 Ammonium-based ILs are good stabilizing solvents for biomacromolecules due to their water-like properties.11,12 Moreover, according to our very recent reports by Bisht et al.,12,13 ammonium-based ILs maintain the stability and activity of Lyz and are also able to refold urea-induced unfolded Lyz. Strategically, the power of these ILs can be basically amplified by the possibility of the choosing of their ions that exist in nature to design biocompatibility.14,15

2 ACS Paragon Plus Environment

Page 3 of 33

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


Choline, an essential micronutrient, is a prominent example of ammonium-based natural compound.14,15 It is accounted that choline does not permeate cell membranes and serves various physiological roles owing to its assimilation in membrane components, signaling molecules and neurotransmitters.16 Besides, choline has the OH-terminated alkyl chain and this functionalized side chain increases its propensity to form H-bonds.17 Choline ion is the only investigated ion which non specifically binds not only to negatively charged aspartic and glutamic acid residues but also substantially make contacts to arginine (Arg), cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), methionine (Met), proline (Pro), phenylalanine (Phe), serine (Ser) or tyrosine (Tyr) amino acids of peptides and proteins.18 A vast amount of literature is available in past few years which state the importance of cholinium-based ILs in protein stability and function.18-28 Vrbka et al.18 reported that ChCl is highly effective in modifying stability and activity of enzyme. Vijayaraghavan et al.19 and Karimata and Sugimoto20 found choline dihydrogen phosphate (ChDHP) useful as a chemical DNA stabilizer and a nuclease inhibitor. The enhanced solubility and stability of cytochrome c dissolved in ChDHP are also reported by Fujita and his co-workers.21,22 Apparently, according to Rodrigues et al.23 the thermal stability of Lyz is decreased in the presence of cholinium bis(triﬂuoromethylsulfonyl)amide (ChTf2N), whereas it enhanced in the presence of ChDHP. Additionally, the authors reported that the effects of cholinium-based ILs on the stability of the Lyz was also concentration dependent as ChDHP destabilized up to 0.5 M and stabilized above this concentration, whereas ChCl stabilized the protein up to 2 M.23 This reflects anomalous behavior of these IL on the stability of Lyz. In support, Hekmat et al.24 observed that the overall relative enzyme activities of Lyz were increased in the presence of ChDHP over time. It has been also noted that the structural stability of Lyz is found to be decreasing in the order: ChDHP < ChSc < [Bmim][L], where ChSc and [Bmim][L] stand for choline saccharinate and 1-butyl-3-methylimidazolium lactate, respectively. Furthermore, Vrikkis et al.15 observed that the Lyz in ChDHP had a higher activity and stability even after 1 month of storage. Weaver et al.25,26 also investigated ChDHP acting as a biocompatible stabilizer for the Lyz native structure. ChCl, choline acetate (ChCH3COO) and choline methanesulfonate (ChCH3SO3) have been also used to study the crystal growth of the Lyz by Kowacz et al.27 These outcomes recommend that by the use of the cholinium-based ILs, biocompatibility and protein stabilization characteristics can be rationally designed and preserved.



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 4 of 33

Especially, a report by Weaver et al.28 states that ChCl is devoid of cytotoxic effects. Therefore, our choice of system involves ChCl IL. In present study, the potential of cholinium-based IL as a protein stabilizer has been further broadened by its counteraction study against the pH-induced denaturation of Lyz. Despite a noteworthy amount of work done in the field of counteraction by the use of IL in the last few years29-32, there is scarcely any report mentioning counteraction by cholinium-based IL to offset pH-induced denaturation of Lyz. This dearth of knowledge incites us to discover the counteraction ability of cholinium-based IL. In present work, we reveal the efficacy of ChCl for protection of Lyz against the structural and functional damage promoted by pH to maintain the function of the enzyme by employing UV-vis, fluorescence, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). Although there is already huge amount of literature available related to the structure, stability and activity of Lyz using different biophysical methods, no study so far has been able to shed light on the counteraction of the pH-induced denaturation of Lyz which is a major obstacle in pharmaceutical implications. This finding depicts novel properties of ChCl with broad inferences for protein folding/unfolding studies. Any mechanism which offers a generalized protection of proteins against pH-induced denaturation is of crucial significance in biochemistry, biotechnology and evolutionary biology specially bottomed on medicinal grounds. To the best of our knowledge, for the first time, the counteraction against the pHinduced denaturation of Lyz by the use of ChCl has been explored with the help of biophysical techniques. The structures of Lyz and ChCl are represented in Scheme 1.

Scheme 1. (a) The structure of Lyz, which was downloaded from the protein data bank (1DPX) and processed with the PyMOL viewer software and (b) the chemical structure of ChCl.


Page 5 of 33

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


MATERIALS AND METHODS Materials Lysozyme (Lyz) from chicken egg white (≥90% protein lyophilized powder) was purchased from Sigma-Aldrich Company. H3BO3, H3PO4 and CH3COOH used were of high purity and purchased from Sisco Research Lab (SRL), India. Britton–Robinson buffer33 consisting of a mixture of 0.04 M H3BO3, 0.04 M H3PO4 and 0.04 M CH3COOH were prepared for buffer of different pH ranging from 2 to 7. Sample preparation Protein stability was analyzed by incubating protein samples in 2 mL screw-capped vials in different pH buffers as well as in varying concentration of IL at both pH 2 and 7 for 1 h to attain complete equilibrium at 25 oC. The protein concentration was used at 0.5 mg/mL for all measurement except for DLS measurement where 1 mg/mL was used. Different concentration of IL was used such as 0.1, 0.2, 0.3, 0.4 and 0.5 M. All samples were filtered through 0.45 µm pore size disposable syringe filter (Millipore, Millex-GS). All the samples were prepared in distilled deionized water with resistivity of 18.3 Ω cm by using Ultra 370 series water purifier from Rions India Company. For all gravimetric measurements, AND (Japan) balance with a precision of ±0.00001 g and Metler Toledo balance with a precision of ±0.0001 g were used. All samples were also filtered with 0.45 µm disposal filter (Millipore, Millex-GS) through syringe and were incubated for 1 h at 25 oC in order to obtain complete equilibrium before performing experiments. Methods Ultraviolet-visible spectroscopy UV-vis absorption spectra of the protein aqueous solution were recorded between 190 to 600 nm by means of a double beam UV-visible spectrophotometer, UV-1800, from Shimadzu Co., Japan, at room temperature. An aliquot of sample solution (2.5 mL) was transferred uniformly in to the quartz cell of 1.0 cm path length. The spectrophotometer has matched quartz cells, with spectral bandwidth of 1.0 nm with a wavelength accuracy of ± 0.3 nm along with an automatic wavelength correction. Steady-State Tryptophan Fluorescence Experiments The steady state fluorescence measurements were conducted at a constant temperature (25 0

C) using a circulating water bath controlled by a Peltier device attached to the sample holder

of the fluorimeter. The excitation wavelength was set at 295 nm in order to calculate the contribution of the tryptophan to the overall fluorescence emission of proteins throughout the 5 ACS Paragon Plus Environment


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 6 of 33

experiments. The experiments were carried out by using a 1.0 cm sealed cell and both excitation and emission slit width were set at 5 nm, and corrected for background signal. The fluorescence intensity at the emission maximum for the native was recorded (~332 nm for Trp) continuously with increasing temperature starting from 25 to 85 0C at an approximate rate of 2.0 0C/min. The accuracy in the temperature changes were + 0.01 0C. The small increase in the temperature with time allowed complete temperature equilibration for the spectral analysis of the small structural modification in the proteins. Collective data for the changes in the fluorescence intensity and the shift in fluorescence maximum wavelength were recorded and analyzed. Thermodynamic analysis of protein stability using fluorescence spectroscopy The results of the thermal stability of Lyz were analyzed by two-state equilibrium between the folded state (N) and the unfolded state (U): N⇌U

(1)

Sigmoidal fluorescence intensity curves were obtained for Lyz in the presence of ChCl. Details of the experimental procedure has been explained somewhere else.34-36 Circular Dichroism (CD) Spectroscopy All CD spectroscopic studies were performed using a PiStar-180 spectrophotometer (Applied Photophysics, UK) equipped with a Peltier system for temperature control. CD calibration was performed using (1S)-(+)-10-camphorsulphonic acid (Aldrich, Milwaukee, WI), which exhibits a 34.5 M/cm molar extinction coefficient at 285 nm, and 2.36 M/cm molar ellipticity (θ) at 295 nm. The sample was pre-equilibrated at the desired temperature for 15 min and the scan speed was fixed for adaptative sampling (error + 0.01) with a response time of 1 sec and 1 nm bandwidth. Each spectrum was collected by averaging six spectra. Each sample spectrum was obtained by subtracting appropriate blank media containing no protein from the experimental spectrum. Dynamic Light Scattering We have performed the dynamic light scattering (DLS) measurements for the protein samples using MALVERN Zetasizer Nano instrument, U. K. The instrument is equipped with 4 mW He-Ne laser (633 nm); fitted with an automatic laser attenuator with transmission of 100% to 0.0003%. The time-averaged intensities were measured at a scattering angle of 900. Advanced avalanche photodiode, Q.E. > 50% at 633 nm was used as a detector. The temperature accuracy of the instrument was + 0.1 0C. The temperature was set to 25.0 0C and 6 ACS Paragon Plus Environment

Page 7 of 33

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


kept constant until thermal equilibrium was attained. The measurements for each of the temperature was performed and recorded repeatedly for 50 runs to improve the signal-tonoise ratio of the experiment. Enzyme Activity Assay Enzymatic activity of Lyz was determined by using the micrococcus lysodeikticus as the substrate. A stock solution of 0.3 mg/mL micrococcus lysodeikticus cell suspension was prepared in 0.1 M phosphate buffer, pH 7.0. Lyz activity measurements were performed on suspended micrococcus lysodeikticus cells for each of the four ILs at 1% v/v of ILs as reported by Mann et al.37 The decrease in the light scattering intensity of the solution was then measured by following the decrease in apparent absorbance of the solution at 450 nm using a UV-Vis spectrophotometer as stated in above section.

RESULTS AND DISCUSSION We investigate the bio-molecular interactions of cholinium-based IL with Lyz under low pH conditions by applying a series of biophysical techniques. This is resulted by characterizing changes in the structural and thermal stability, conformational size and bacteriolytic activity of the Lyz in the presence of different concentrations of IL. The higher concentrations of any co-solvent may present a different solvent environment in comparison to the dilute solutions, thereby; some kind of the properties must be fairly unlike at diverse concentrations. At this moment, it seems relevant to state the thorough evaluation of all the experimental outcomes at different concentrations of IL by considering the reasonable selection of ChCl in the present work and also rationalizing the existing open literature. UV-Vis Spectral Analysis of Counteracting Effects of ChCl on pH-Induced Conformational Changes in Lyz The aromatic residues absorbance changes can be used as a tool to find out the conformational changes in the protein structure, therefore, we employed UV-vis spectroscopy study. Figure 1 represents the changes in UV absorbance of Lyz at different pH conditions as well as in different concentrations of IL at pH 2 and 7. Pure Lyz in buffer at pH 7 has a characteristic absorbance band with a maximum (Amax) at 280 nm (black spectra in Figure 1). However, as presented in Figure 1 (a), a decrease in the Amax of the Lyz is significantly observed with decrease in pH till 4. The results indicate that the pH changes (≥ 4) lead to the conformational changes which escort to the burial of aromatic residues inside the core of the Lyz, thereby, decreased absorbance of Lyz.



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 8 of 33

Alternatively, both pH 2 and 3 causes increase in the Amax of Lyz in comparison to that for Lyz in pH 7 (Figure 1a). The effect of pH 3 on the Lyz conformation may be more or less similar to that at pH 7 whereas pH 2 resulted in drastic change in Amax of Lyz as can be clearly speculated from red spectra in Figure 1(a). This may be attributed to fairly unstable hydrophobic pocket due to the electrostatic repulsion between increased net positive charge on the Lyz surface under highly acidic conditions (pI of lyz > 10),38 directing the paramount exposure of the aromatic residues to the solvent media, thus, more absorbance. Ultimately, it can be concluded here that Lyz conformation is extremely disturbed at pH 2. Thus, UV-vis study of Lyz conformation at pH 2 in the presence of IL is further considered and compared with that in pH 7 (Figure 1b and c). However, the changes in pH do not allocate the shift to in wavelength maxima (λmax) of UV-absorbance at all pH conditions (Figure 1a). Furthermore, in order to counterbalance this unfavourable effect of pH 2 on Lyz, the changes in UV-absorbance of Lyz at pH 2 in the presence of ChCl are studied. In Figure 1 (b) which is depicting changes in Lyz absorbance at pH 7 in presence of ChCl, the absorption intensities are decreased with increasing concentrations of ChCl till 0.3 M. This may indicate that the aromatic residues are now less exposed to the solvent resulting less absorbance, in turn, Lyz can be presumed in the more compact conformation in the presence of ChCl. At 0.4 and 0.5 M of ChCl, Amax of Lyz at pH 7 is slightly more than that in absence of ChCl. However, it is noteworthy in Figure 1 (b) that values are very near to the control (Lyz at pH 7 only). On the other hand, at pH 2, the presence of ChCl (till 0.3 M) causes decrease in the Amax of Lyz not only in comparison to Amax of Lyz in absence of ChCl at pH 2 (red in Figure 1c), but also that in pH 7 buffer (black in Figure 1c). These observations in Figure 1 (c) symbolize the existence of aromatic residues towards the interior of Lyz. Certainly, from these results, it may be emphasized that ChCl is significantly counteracting the undesirable effects of pH 2 on the Lyz conformation. On contrary, the higher concentrations of ChCl such as 0.4 and 0.5 M, are also resulting in decrease in the absorbance of Lyz at pH 2 with respect to that of Lyz without ChCl in pH 2, however, quite higher than Lyz in buffer at pH 7 (Figure 1c). As a consequence, it may be declared here that ChCl is having efficient counteracting ability against the pH induced denaturation of Lyz at its all concentrations; nevertheless, it is more prominent at intermediate concentration. Fluorescence Spectral Analysis of Counteracting Effects of ChCl on pH-Induced Conformational Changes in Lyz


Page 9 of 33

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


To further confirm the above results, a deep analysis of all samples is executed by using fluorescence spectroscopy method.

The changes in the microenvironment of

tryptophan (Trp) fluorescence are quite important for detection of the conformational changes in the Lyz. Figure 2 portrays fluorescence spectra of Lyz in different pH conditions and also in the presence of ChCl at pH 7 and 2, respectively. The wavelength maximum (λmax) of native Lyz in buffer at pH 7.0 is viewed around 338 nm (black spectra in Figure 2). It is quite clear from the Figure 2(a) that pH 2 is most perturbing the microenvironment of Trp of Lyz where λmax is blue shifted by 3 nm and fluorescence intensity (Imax) is also decreased to an appreciable extent. The large net positive charge on Lyz surface escorts electrostatic repulsions between positive charges resulting destabilization of Lyz conformation, especially, by effecting salt bridges contribution to the stability.38 As a consequence, Lyz molecule is swollen to a large extent. As per the literature survey also,1,10 at very low pH, Lyz exists in a partially folded state which is very prone to the aggregation. Therefore, the observed blue shift and decreased Imax may be attributed to the increased protein-protein interactions leading to decreased exposure of Trp and decrease in the distance between Trp and positively charged quenchers. In order to check the counteraction ability of ChCl against this undesirable effect of low pH, further fluorescence experiment is performed for the Lyz denaturation of at pH 2 which is found to be most effective for the disrupting the Lyz (Figure 2a). Here, Figure 2 (b) indicates the effect of different concentrations of ChCl on conformation of Lyz at pH 7. The varying concentrations of ChCl do not cause any shift in λmax as can be clearly observed in Figure 2 (b). However, Imax for Lyz is found to increase as a function of concentration of ChCl as compared to that in buffer at pH 7 only. It may be stressed here that the burial of Trp in the core of the enzyme describes increased Imax. According to Vrbka et al.,18 chloride is never strongly attracted by the protein surface even though the proteins bear an overall positive charge and exist as Hofmeister neutral anion. Whereas choline cation remains hydrated and have a nonspecific binding to the protein by various types of interactions such as cation-п interaction, hydrophobic interactions, Hbonding and other polar interactions,18 however, not strongly to the positive charged surface of Lyz. This may result in increased Imax of Lyz at pH 7 in the presence of ChCl. However, in Figure 2 (c) which shows fluorescence spectra of Lyz at pH 2 in the presence of various concentrations of ChCl, the significant augment is viewed in Imax of Lyz in ChCl even at pH 2 as compared to that for Lyz in buffer at pH 2. Nevertheless, the Imax of 9 ACS Paragon Plus Environment


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 10 of 33

Lyz at pH 2 approaches to that at pH 7 and also increases than that for pH 7 in some concentrations of ChCl (0.3 M) as shown in Figure 2 (c). This is an indication of the conformational changes which causes internalization of Trp where 0.3 M ChCl is found to be more effective in its counteraction against Lyz denaturation at pH 2. These consequences impressively emphasize on the retaining of Lyz in its native state in the presence of ChCl under acidic conditions also, hence, more stabilized Lyz is formed than Lyz in buffer. This may be attributed to the preferential exclusion of choline ion from highly positive charge surface at low pH and also to the somewhat binding of chloride ion to the surface resulting in compact structure and decrease in electrostatic repulsion within the protein molecule.38 Thus, increase in Imax in Trp fluorescence of Lyz is observed even at pH 2. Hence, all these surveillances depict significant counteraction by ChCl against pHinduced denaturation of Lyz. Still, it is not possible to give a clear unifying statement about counteracting ability of ChCl. Thus, for the more transparency of the obtained results, it is necessary to further evaluate thermal stability of Lyz which is an important thermodynamic parameter. Thermal Analysis of Counteraction of pH-Induced Lyz Denaturation by ChCl Figure 3 reveals the thermal stability parameter for Lyz at various conditions of pH and IL. From transition curves in Figure 3 (a), it is pretty visible that there is drastic shift in the transition curve of Lyz at pH 2 towards the lower temperature in comparison to that in other pH conditions. Therefore, it can be stated here that pH 2 (red colour in Figure 3a) is significantly having adverse effect on the thermal stability of Lyz. Figure 3 (b) depicts more or less no change in the transition curve of the Lyz at pH 7 on addition of varying concentrations of ChCl. However, transition curve of Lyz at pH 2 is largely affected in the presence of choline chloride which now appears very near to that for Lyz in pH 7. This inspection of the Figure 3 (c) depicts that thermal stability of Lyz at pH 2 by the use of ChCl is remarkably increased which explicitly elucidates that ChCl is appreciably attenuating the deleterious effect of pH on Lyz thermal stability. All these results are more apparent from Figure 4 which describes the variation of transition temperature (Tm) for Lyz at different pH and also in presence of different concentrations of ChCl at pH 7 and pH 2. With decrease in pH less than 4, Tm of Lyz is decreasing and it is highly diminished at pH 2 (Tm at pH 2 is ~ 70.0 oC). In Figure 4, it is quite clear that Tm value of Lyz is very low at pH 2 as compared to that in pH 7 whereas Lyz in ChCl at pH 2 is found to have high Tm as compared to that of Lyz in pH 2 as well as in pH 10 ACS Paragon Plus Environment

Page 11 of 33

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


7. Apparently, ChCl is observed to have more or less significant effect on Tm of Lyz at pH 7. On the other hand, ChCl has shown noteworthy potential to counteract the adverse effect of low pH on Tm of Lyz. Also, Table 1 comprises of all the Tm values for Lyz at pH 2 and pH 7 in the presence of ChCl. In Table 1, Tm of Lyz at pH 2 is ~ 60.2 oC which is greatly less than value at pH 7 (Tm ~ 70.0 oC) whereas addition of ChCl leads to increase in Tm of Lyz more or less equal to 72.5 oC which is even more than that of Lyz at physiological pH (Tm ~ 70.0 oC at pH 7). This may again put stress on the counteraction ability of ChCl on the denaturing effects of low pH on the thermal stability of Lyz. The thorough analysis of the apparent pKa values of amino acid residues at lower pH may provide a unique insight into the electrostatic forces stabilizing the folded state. According to Aune and Tanford,39 the stability of Lyz between pH 1 and 4 is primarily due to amino acid groups having abnormally low pKa values in the native state. There is no doubt that the transition of Lyz is two state only even at very low pH also, however, shifts to the lower side of the temperature as can also be visualized from Figure 3 (a).40 At low pH, thermal stability is reduced may be due to the increased positive charge-charge repulsions.38 Moreover, in folded and unfolded states of any protein molecule, amino acids residues are having different pKa depending on the surrounding amino acid environments. Lyz was found to be more thermally stable at pH 4-5 as is obvious from Figure 4 which may be attributed somewhat to salt bridge interactions.39,41 Anderson et al.41 reported that Asp70 is negatively charged and His31 is positively charged in both the folded and unfolded states at pH 4-5 resulting in formation of salt bridge that contributes ~3 kcal/mol in overall thermal stability of Lyz. Apparently, the contribution of His31 and Asp70 as the pH is lowered below pH 4 where the protonation of Asp70 in the unfolded state occurs and continues as pH is lowered until the pH drops below 1.41 Thus, stabilization of the unfolded state occurs as the pH is lowered from 4 to 1. The Asp70 may also contribute to some additional electrostatic interactions. The two Asp or Glu residues may account for an additional 8 kcal/mol of stabilization. Therefore, different types of electrostatic interactions are missing at low pH which could contribute to overall stability of the Lyz.42 However, addition of ChCl may reduce positive charge-charge repulsions by binding of chloride ions to the protein surface contributing more stabilty.38 In addition, there may also be the probability that ChCl changes the pKa value of the amino acid residues present on the surface, thereby, changing their behaviour at pH 2. The poor interaction of choline cation 11 ACS Paragon Plus Environment


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 12 of 33

with Lyz at low pH may also stabilize folded state more. In order to find the exact reason behind the counteraction effect of ChCl for Lyz at very low pH, extensive study of system is required by the use of molecular dynamic simulations. Circular Dichroism Analysis of ChCl Effect on pH-induced Changes in Lyz Secondary Structure Furthermore, the counteracting effect of ChCl is ascertained by performing far-UV CD-spectral analysis. Figure 5 represents secondary structural changes in Lyz at various conditions. Lyz at pH 7 possesses mainly two negative bands, more intense band at 209 nm and shallow band at 222 nm (shown black in Figures 5b and 5c) suggesting that Lyz is belonging to the typical α +β class of enzyme. In Figure 5(a), there is decrease in the secondary structure of Lyz with decrease in pH lower than 5 which are portrayed due to the decreased negative ellipticities at 209 nm. Conversely, pH 2 has shown again most significant impact on secondary structure of Lyz. Haezebrouck et al.40 observed decrease in the helical content of the Lyz at this much low pH. The change in the electrostatic properties with decrease in the pH is the reason behind the diminished secondary structure.40 Intramolecular hydrogen bonding is decreased due to increased charge-charge repulsion at low pH. It is noticeable here in Figure 5 (a) that even this much low pH is not drastically affecting Lyz structure and it is retained in its native structure to a paramount extent. In case of Lyz at pH 7 in the presence of ChCl (Figure 5b), the negative ellipticities of Lyz for all concentration of IL are either more than or almost equal to that in buffer only. This shows that ChCl at all studied concentrations has stabilizing influence on the secondary structure of the Lyz, however, this effect is more pronounced at 0.2 and 0.3 M of ChCl. Now, as is clear from ellipticities for negative bands (Figure 5c), Lyz possesses high amount of secondary structure even at pH 2 if there is presence of ChCl. The negative ellipticities values at this condition are substantially higher than that for Lyz at pH 7. From Figure 5c, it can be undoubtedly accentuated that ChCl is fairly efficient in decreasing the denaturing action at the low pH on secondary structure of Lyz. Again, it may be mentioned here that ChCl diminishes electrostatic repulsion within the Lyz molecule making favourable condition for intramolecular H-bond formation.


Page 13 of 33

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


The close examination of Imax, Tm and ellipticities [Θ] may declare here that there is conclusively change in the conformation of Lyz to the disrupted one under the influence of low pH which is satisfactorily counterbalance by ChCl at its lower concentrations also. DLS Analysis of Counteraction Effect of ChCl on Disruption of the Lyz Structure Figure 6 exemplifies hydrodynamic size (dH) variation of Lyz in different pH and in presence of ChCl at pH 2 and 7. All the peaks of intensity distribution graph are found to be highly monodisperse with PDI < 0.1 for Lyz in buffer at pH 7 and PDI for all other samples also do not exceed 0.5. The dH for Lyz in buffer at pH 7 (shown black in Figures 6b and 6c) is ~3.8 nm at 25 oC and observed to be increased with decrease in the pH < 5 (Figure 6a). The dH of Lyz at pH 2 is monitored ~ 5.2 nm which describe its adverse effect on Lyz that causes swelling of Lyz by disrupting Lyz native state resulting in increased dH. The largely increase in the net positive charge on the Lyz surface leads to the destabilization of Lyz domain by expanding the conformation so that least repulsion is felt by residues of Lyz. Moreover, in the presence of ChCl, it can be clearly examined from Figure 6 (b) as well as Table 2 that dH of Lyz at pH 7 is decreased to a smaller extent at all studied concentration of IL which indicates that conformational size of Lyz is retained. However, at pH 2, addition of ChCl in Lyz leads to a severe decrease in dH and acquires size very similar to that for Lyz at physiological pH (Figure 6 (c) and Table 2). It reveals that ChCl is maintaining Lyz in native conformation even at highly acidic conditions by keeping it compact which point towards the contribution of ChCl to the counteraction of unpleasant effect of pH on Lyz. Further, different concentrations of IL from 0.1 to 0.5 M show more or less similar effects on the size of Lyz. Additionally, the peaks at larger size can be apparently seen in Figures 6 (b) and 6 (c) which may be attributed to the unavoidable aggregates of Lyz formed at experimental concentration (1 mg/ml). At pH 7, the contributions of these large sized particles in total particle size intensity distribution are monitored to be relatively same in absence and presence of different concentrations of ChCl. However, the aggregate size is slightly decreased at pH 7 by ChCl as is clear from Figure 6 (b). On the other hand, solution of Lyz at pH 2 comprises of very elevated amount of large sized aggregates which is somewhat evident from Figure 6 (c). Afterwards, the presence of ChCl in the Lyz at pH 2 results in the appreciable diminishment of percentage contribution of aggregates in the particle size distribution that is quite apparent from Figure 6 (c). Strangely, the size of aggregates of Lyz at pH 2 is increased and amount of aggregates are decreased with increase in the



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 14 of 33

concentration of IL (Figure 6 (c)). The peaks attributed to larger size than 1000 nm are ignored in the present study. These results are found to be in consistent with fluorescence and CD data shown in Figures 1 and 3 that elucidate again that ChCl is efficiently able to counteract the undesirable action of highly acidic conditions on Lyz. Relationship between Stability and Bacteriolytic Activity of Lyz Measuring the activity of Lyz is of overriding importance since amendment in the fluorescence and secondary structure spectra of Lyz do not automatically reflect its enzymatic function. The active site of Lyz is present in a deep crevice dividing the enzyme molecule into two domains i.e., β-sheet domain and α-helix domain linked by α-helix.42,43 The sidechain carboxyl group of Glu35 of α-helical domain projects in the active site and donates proton to the glycosidic oxygen of anomeric carbon in complex sugar of bacterial cell wall.4245

The intermediate formed during hydrolysis is stabilized by the negative charge of Asp52.

According to Kuroki et al.,44 Glu35 plays a crucial role in hydrolytic activity of Lyz. With the intention to verify whether the counteraction of deterioration effects of pH on Lyz structure and stability by ChCl is accompanied with improved activity of Lyz as compared to that in pH 2 only, the initial rate of hydrolysis is examined by Lyz using a micrococcus lysodeikticus cell suspension for all the different solvent conditions is studied. As can be witnessed in Figure 7, Lyz is distinctly more active at physiological pH 7 in comparison to that in pH 2 which is shown by much higher initial rate at pH 7. This may be described to the very loose conformation of Lyz at pH 2 which causes opening of catalytic site resulting unfavourable condition for substrate binding (active site binds hexasaccharides of complex sugar only at one time).42,43,45 In the presence of ChCl, Lyz at pH 7 shows noticeable increase in the activity. There are few reports in the literature which also shows that cholinium-based IL enhances enzyme activity.18,20 Wijaya et al.46 found enhanced Lyz activity arises from electrostatic interactions in protic IL–water systems and salt solutions due to the presence of a net surface charge on Lyz. However, ChCl at 0.2 M is found to be more efficient in increasing the hydrolytic activity of Lyz at pH 7 (blue colour in Figure 7). Afterwards, with further increase in IL concentration, Lyz is comparatively less active), but, still appreciably more than control. Under high concentration of ChCl, reduced flexibility of active site due to the compact structure of Lyz may cause fewer enhancements in activity of Lyz at pH 7. 14 ACS Paragon Plus Environment

Page 15 of 33

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


On the other hand, in the case of pH 2, Lyz possesses more activity on addition of different concentrations of ChCl in comparison to that in pH 2 conditions only as is apparent from initial rate values in Figure 7 (cyan colour). This is an indication of counteracting ability of ChCl against the unpleasant effect of low pH on Lyz activity which may be attributed to conformational change leading slight compacting of Lyz structure which may bring active site residues (Asp52 and Glu35) in a position to work more efficiently for hydrolysis of the glycosidic bond of bacterial cell wall in comparison to loose conformation of Lyz at pH 2. Conversely, higher concentrations of IL are more proficient in counteracting the undesirable impacts of pH 2 on Lyz activity. It is noteworthy here that even though high concentrations of ChCl counteract the unlikeable effects of low pH on Lyz very well, the hydrolytic activity is not fully regained and still considerably less than that in Lyz at physiological pH. This may be attributed to failure of resuming original conformation around the active site, thereby; the little disrupted active site does not lead to perfect binding to the complex sugars of bacterial wall. Ultimately, from the observed decrease in absorbance, increase in intensity of Trp fluorescence spectra, increase in the negative ellipticity in CD spectra, increase in Tm and decrease in the dH of Lyz at pH 2 on addition of ChCl, it can be depicted that there is formation of more compact structure with decreased exposure of non polar groups and increased secondary structures as compared to Lyz in pH 2 buffer only. These inferences escort us to one way thinking i.e. ChCl is having remarkable tendency to offset the denaturing effects of strongly acidic environment on Lyz stability ensuing a more compact Lyz which can be clear depicted from Scheme 2. This finding depicts novel properties of ChCl against the adverse effects of low pH which is a major obstacle in pharmaceutical implications. Moreover, this study may lead to explore the factors responsible for the stabilizing Lyz in adverse condition which is necessity of the time in order to make a successful path from administration site to the target site and also to avoid uninvited consequences in biopharmaceutical formulations as well as in human body. To the best of our knowledge, there is not a single report which tells about this novel property of ChCl to offset the denaturing effects of low pH on Lyz.



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 16 of 33

Scheme 2. Schematic depiction of counteraction effects of ChCl against pH-induced denaturation of Lyz.

CONCLUSION From the assessment of the many researches over the past decades, it is convincingly unequivocal that the counteracting role of the co-solvents is a key for the survival of the organisms under various stresses that might result in severe biological damage. Nowadays, ILs are proven as green solvents in today’s numerous scientific fields. The lack of vast literature regarding the counteracting ability of these green solvents has intrigued our interest in studying the counteracting ability of a well known IL, ChCl, against the adverse effects of highly acidic conditions on the structure, stability and activity of Lyz which may be the barrier for the successful administration of Lyz-based drugs. Our data indicates the existence of crucial contribution of the counteraction proficiency of IL against the critical imbalance in protein stability caused by acidic conditions. By complete analysis of UV-vis, fluorescence, CD spectroscopy and DLS measurements, it can be stated here that ChCl is an efficient IL which not only offset the undesirable effects of highly acidic environment on structure and stability, but also contribute in recovering the lost activity of Lyz to a substantial extent. Eventually, it may be stated after analysis of complete data of stability and activity under all conditions that there is a direct correlation between bacteriolytic activity and conformational stability of Lyz. The electrostatic interactions may be playing an overriding role at low pH conditions. The high positive charge density on the surface of Lyz may be resulting in electrostatic repulsion and thereby, resulting its denatured conformation. Whereas, on 16 ACS Paragon Plus Environment

Page 17 of 33

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


addition of ChCl, preferential exclusion of choline ion as well as decreased electrostatic repulsion due to somewhat binding of chloride ion lead to native like compact Lyz at low pH conditions also. As a concluding remark, our results explicitly elucidates high efficiency of ChCl in counteracting the undesirable impact of acidic conditions on Lyz conformation and activity which may help to resolve most commonly encountered problem in implications of therapeutic application of Lyz. This study should facilitate the development of new strategy in order to avoid the worst fate of Lyz in vivo as a consequence of encountered highly acidic conditions. Moreover, this study has exposed surprising results which, to the best of our knowledge, has never been reported before. It is apparent that this approach can be extended to study other IL and which can be further employed in vivo study in order to regulate protein folding and activity. This kind of research work is speculative at present and, to ascertain the position of these investigations in the clinical laboratories, further research is necessitated. AUTHOR INFORMATION *e-mail: [email protected]; [email protected]; Tel:+91-11-27666646142; Fax: +91-11-2766 6605 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).

REFERENCES (1) Morozova-Roche, L.; Zurdo, A. J.; Spencer, A.; Noppe, W.; Receveur, V.; Archer, D. B.; Joniau M.; Dobson, C. M. Amyloid Fibril Formation and Seeding by Wild-Type Human Lysozyme and its Disease-Related Mutational Variants. J. of Stru. Biol. 2000,

130, 339-351 (DOI: 10.1006/jsbi.2000.4264). 17 ACS Paragon Plus Environment


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 18 of 33

(2) Melrose, J.; Ghosh P.; Taylor, T. K. F. Lysozyme, a Major Low-Molecular-Weight Cationic Protein of the Intervertebral Disc, Which Increases With Ageing and Degeneration. Gerontology 1989, 35, 173–180 (doi.org/10.1159/000213019). (3) Porstmann, B.; Jung, K.; Schmechta, H.; Evers, U.; Pergande, M.; Porstmann, T.; Kramm, H. J.;

Krause, H. Measurement of Lysozyme in Human Body Fluids:

Comparison of Various Enzyme Immunoassay Techniques and Their Diagnostic Application.

Clin.

Biochem.

1989,

22,

349–355

(doi.org/10.1016/S0009-

9120(89)80031-1). (4) Venkataramani, S.; Truntzer J.; Denis, R. Thermal Stability of High Concentration Lysozyme Across Varying pH: A Fourier Transform Infrared Study. J. Pharm. Bioall.

Sci. 2013, 5, 148-153 (DOI: 10.4103/0975-7406.111821). (5) Cegielska-Radziejewska, R.; Lesnierowski G.; Kijowski, J. Properties and Application of Egg White Lysozyme and Its Modified Preparations: A Review. Polish J. Food

Nutr. Sci. 2008, 58, 5-10. (6) Fink, A. L.; Calcino, L. J.; Goto, Y.; Kurotsu T.; Palleros, D. R. Classification of Acid Denaturation of Proteins: Intermediates and Unfolded States. Biochemistry 1994, 33, 12504-12511(DOI: 10.1021/bi00207a018). (7) Aoki, K.; Shiraki K.; Hattori, T. Salt Effects On The Picosecond Dynamics of Lysozyme Hydration Water Investigated by Terahertz Time-Domain Spectroscopy and An Insight Into the Hofmeister Series for Protein Stability and Solubility. Phys. Chem.

Chem. Phys. 2016, 18, 15060-15069 (DOI: 10.1039/c5cp06324h). (8) Ohkuma S.; Poole, B. Fluorescence Probe Measurement of the Intralysosomal pH in Living Cells and the Perturbation of pH by Various Agents. Proc. Natl. Acad. Sci.

U.S.A. 1978, 75, 3327–3331(DOI: 10.1073/pnas.75.7.3327). (9) E. Holtzman, Lysosomes, Plenum Press, New York, 1989. (10) Haezebrouck, P.; Joniau, M.; Van Dael, H.; Hooke, S. D.; Woodruff N. D.; Dobson, C. M. An Equilibrium Partially Folded State of Human Lysozyme at Low pH. J. Mol.

Biol. 1995, 246, 382-387 (DOI: 10.1006/jmbi.1994.0093). (11) Jha I.; Venkatesu, P. Endeavour to Simplify the Frustrated Concept of ProteinAmmonium Family Ionic Liquid Interactions. Phys. Chem. Chem. Phys. 2015, 17, 20466-20484 (DOI: 10.1039/c5cp01735a). (12) Bisht, M.; Kumar A.; Venkatesu, P. Refolding Effects of Partially Immiscible Ammonium-Based Ionic Liquids on the Urea-Induced Unfolded Lysozyme Structure.

Phys. Chem. Chem. Phys. 2016, 18, 12419-12422 (DOI: 10.1039/c6cp01022a).


Page 19 of 33

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


(13) Bisht, M.; Kumar A.; Venkatesu, P. Analysis of the Driving Force That Rule the Stability of Lysozyme in Alkylammonium-Based Ionic Liquids. Int. J. Biol.

Macromol. 2015, 81, 1074-1081(DOI: 10.1016/j.ijbiomac.2015.09.036). (14) Pernak, J.; Syguda, A.; Mirska, I.; Pernak, A.; Nawrot, J.; Pradzynska, A.; Griffin, S. T.; Rogers, R. D. Choline-Derivative-Based Ionic Liquids. Chem. Eur. J. 2007, 13, 6817–6827 (DOI: 10.1002/chem.200700285). (15) Vrikkis, R. M.; Fraser, K. J.; Fujita, K.; MacFarlane, D. R.; Elliott, G. D. Biocompatible Ionic Liquids: A New Approach for Stabilizing Proteins in Liquid Formulation.

J.

Biomech.

Eng.

2009,

131,

0745141-0745144

(DOI:

10.1115/1.3156810). (16) Brock, M.; Nickel, A. C.; Madziar, B.; Blusztajn J. K.; Berse, B. Differential Regulation of The High Affinity Choline Transporter and the Cholinergic Locus by cAMP

Signalling

Pathways.

Brain

Res.

2007,

1145,

1-10

(doi:

10.1016/j.brainres.2007.01.119). (17) Weingärtner, H.; Cabrele, C.; Herrmann, C. How Ionic liquids can Help to Stabilize Native

Proteins.

Phys.

Chem.

Chem.

Phys.

14,

2012,

415–426

(DOI:10.1039/C1CP21947B). (18) Vrbka, L.; Jungwirth, P.; Bauduin, P.; Touraud D.; Kunz, W. Specific Ion Effects at Protein Surfaces: A Molecular Dynamics Study of Bovine Pancreatic Trypsin Inhibitor and Horseradish Peroxidase in Selected Salt Solutions. J. Phys. Chem. B 2006, 110, 7036-7043 (DOI: 10.1021/jp0567624). (19) Vijayaraghavan, R.; Izgorodin, A.; Ganesh, V.; Surianarayanan M.; MacFarlane, D. R. Long-Term Structural and Chemical Stability of DNA in Hydrated Ionic Liquids.

Angew. Chem. Int. Ed. Engl. 2010, 49, 1631-1633 (DOI: 10.1002/anie.200906610). (20) Tateishi-Karimata, H.; Sugimoto, N. Structure, Stability and Behaviour of Nucleic Acids

in

Ionic

Liquids.

Nucl.

Acids

Res.

2014,

42,

8831-8844

(DOI:

10.1093/nar/gku499). (21) Fujita, K.; MacFarlane D. R.; Forsyth, M. Protein Solubilising and Stabilising Ionic Liquids. Chem. Comm. 2005, 4804–4806 (DOI: 10.1039/b508238b). (22) Fujita K.; Ohno, H. Enzymatic Activity and Thermal Stability of Metallo Proteins in Hydrated Ionic Liquids. Biopolymers 2010, 93, 1093-1099 (DOI: 10.1002/bip.21526). (23) Rodrigues, J. V.; Prosinecki, V.; Marrucho, I.; Rebelo, L. P. N.; Gomes, C. M. Protein Stability in an Ionic Liquid Milieu: On the Use of Differential Scanning Fluorimetry.

Phys. Chem. Chem. Phys. 2011, 13, 13614-13616 (DOI: 10.1039/c1cp21187k).



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 20 of 33

(24) Hekmat, D.; Hebel, D.; Joswig, S.; Schmidt M.; Weuster-Botz, D. Advanced Protein Crystallization Using Water-Soluble Ionic Liquids as Crystallization Additives.

Biotechnol. Lett. 2007, 29, 1703−1711 (DOI: 10.1007/s10529-007-9456-9). (25) Weaver, K. D.; Vorst, M. P. V.; Vijayaraghavan, R.; MacFarlane, D. R.; Elliott, G. D. Interaction of Organic Salts with Artificial Biological Membranes: A Model for Elucidating Cellular Interactions. Biochimica et Biophysica Acta 2013, 1828, 18561862 (DOI: 10.1016/j.bbamem.2013.03.017). (26) Weaver, K. D.; Vrikkis, R. M.; Vorst, M. P. V.; Trullinger, J.; Vijayaraghavan, R.; Foureau, D. M.; McKillop, I. H.; MacFarlane, D. R.; Krueger J. K.; Elliott, G. D. Structure And Function Of Proteins in Hydrated Choline Dihydrogen Phosphate Ionic Liquid. Phys. Chem. Chem. Phys. 2012, 14, 790-801(DOI: 10.1039/c1cp22965f). (27) Kowacz, M.; Mukhopadhyay, A.; Carvalho, A. L.; Esperança, J. M. S. S.; Romão M. J.; Rebelo, L. P. N. Hofmeister Effects of Ionic Liquids in Protein Crystallization: Direct and Water-Mediated Interactions.

Cryst. Eng. Comm. 2012, 14, 4912-

4921(DOI: 10.1039/C2CE25129A). (28) Weaver, K. D.; Kim, H. J.; Sun, J.; MacFarlane D. R.; Elliott, G. D. Cyto-toxicity and Biocompatibility of a Family of Choline Phosphate Ionic Liquids Designed for Pharmaceutical

Applications.

Green

Chem.

2010,

507–513

12,

(DOI:10.1039/B918726J). (29) Attri, P.; Venkatesu P.; Kumar, A. Water and a Protic Ionic Liquid Acted as Refolding Additives for Chemically Denatured Enzymes. Org. Biomol. Chem. 2012,

10, 7475-7478 (DOI: 10.1039/c2ob26001h). (30) Attri, P.; Venkatesu P.; Kumar, A.; Byrne, N. A Protic Ionic Liquid Attenuates the Deleterious Actions of urea on α-chymotrypsin. Phys. Chem. Chem. Phys. 2011, 13, 17023-17026 (DOI: 10.1039/c1cp22195g). (31) Kumar, A.; Rani, A.; Venkatesu P.; Kumar, A. Quantitative Evaluation of the Ability of Ionic Liquids to Offset the Cold-Induced Unfolding of Proteins. Phys. Chem. Chem.

Phys. 2014, 16, 15806-15810 (DOI: 10.1039/c4cp01001a). (32) Reddy, P. M.; Umapathi R.; Venkatesu, P.

A Green Approach to Offset the

Perturbation Action of 1-butyl-3-methylimidazolium Iodide on α-Chymotrypsin.

Phys. Chem. Chem. Phys. 2015, 17, 184-190 (DOI: 10.1039/c4cp04180a). (33) Britton, H. T. K.; Robinson, R. A. Universal Buffer Solutions and the Dissociation Constant

of

Veronal.

J.

Chem.

Soc.

1931,

CXCVII,

1456–1462

(DOI:10.1039/JR9310001456). 20 ACS Paragon Plus Environment

Page 21 of 33

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


(34) Jha, I.; Attri, P.; Venkatesu, P. Unexpected Effects of the Alteration of Structure and Stability of Myoglobin and Hemoglobin in Ammonium-Based Ionic Liquids.

Phys. Chem. Chem. Phys. 2014, 16, 5514−5526 (DOI: 10.1039/c3cp54398f). (35) Pace, C. N.; Laurents, D. V. A New Method for Determining the Heat Capacity Change

for

Protein

Folding.

Biochemistry

1989,

28,

2520−2525

(DOI:

10.1021/bi00432a026). (36) Privalov, P. L. Stability of Proteins: Small Globular Proteins. Adv. Protein Chem. 1979, 33, 167−241 (DOI.org/10.1016/S0065-3233(08)60460-X). (37) Mann, J. P.; McCluskey A.; Atkin, R. Activity and Thermal Stability of Lysozyme in Alkylammonium Formate Ionic Liquids-Influence of Cation Modification. Green

Chem. 2009, 11, 785-792 (DOI:10.1039/B900021F). (38) Arakawa, T.; Bhat R.; Timasheff, S. N. Why Preferential Hydration does not Always Stabilize the Native Structure of Globular Proteins. Biochemistry, 1990, 29, 19241931(DOI: 10.1021/bi00459a037). (39) Aune K. C.; Tanford, C. Thermodynamics of the Denaturation of Lysozyme by Guanidine Hydrochloride. II. Dependence on pH at 25 ̊C. Biochemistry, 1969, 8, 45794585. (40) Haezebrouck, P.; Joniau, M.; Van Dael, H.; Hooke, S. D.; Woodruff N. D.; Dobson, C. M. An Equilibrium Partially Folded State of Human Lysozyme at Low pH. J. Mol

Biol. 1995, 246, 382-387 (DOI: 10.1006/jmbi.1994.0093). (41) Anderson, D. E.; Becktel W. J.; Dahlquist, F. W. pH-Induced Denaturation of Proteins: A Single Salt Bridge Contributes 3-5 kcal/mol to the Free Energy of Folding of T4 Lysozyme. Biochemistry 1990, 29, 2403-2408 (DOI: 10.1021/bi00461a025). (42) Held, J.; van Smaalen, S. The Active Site of Hen Egg-white Lysozyme: Flexibility and Chemical Bonding. Acta Cryst. Acta Cryst. 2014, 70, 1136–1146 (DOI: 10.1107/S1399004714001928). (43) Strynadka, N. C. J.; James, M. N. G. Lysozyme Revisited: Crystallographic Evidence for Distortion of an IV-Acetylmuramic Acid Residue Bound in Site D. J. Mol. Biol. 1991, 220, 401-424 (DOI.org/10.1016/0022-2836(91)90021-W). (44) Kuroki, R. Y.; Weaver L. H.; Matthews, B. W. Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site. Proc. Natl. Acad.

Sci. 1999, 96, 8949-8954 (DOI: 10.1073/pnas.96.16.8949). 21 ACS Paragon Plus Environment


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 22 of 33

(45) Kumagail, I.; Sunada, F.; Takeda S.; Miura, K. Redesign of the Substrate-binding Site of Hen Egg White Lysozyme Based on the Molecular Evolution of C-type Lysozymes. J. Biol. chem. 1992, 267, 4608-4612. (46) Wijaya, E. C.; Separovic, F.; Drummond C. J.; Greaves, T. L. Activity and conformation of lysozyme in molecular solvents, protic ionic liquids (PILs) and salt– water systems. J. Biotech. 1999, 75, 1–10 (DOI: 10.1039/c6cp03334b).


Page 23 of 33

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


FIGURE CAPTIONS Figure 1. UV-vis spectral analysis of the Lyz: (a) Lyz at different pH; pH 7 (black), pH 6 (green), pH 5 (blue), pH 4 (cyan), pH 3 (pink) and pH 2 (red), (b) Lyz in buffer at pH 7 (black) and in varying concentration of ChCl at pH 7; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow) and (c) Lyz in buffer at pH 2 (red), at pH 7 (black) and in varying concentration of ChCl at pH 2; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow). Figure 2. Fluorescence spectra analysis of the Lyz conformation at 25 oC: (a) Lyz at different pH, (b) Lyz in buffer at pH 7 (black) and in varying concentration of ChCl at pH 7; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow) and (c) Lyz in buffer at pH 2 (red), at pH 7 (black) and in varying concentration of ChCl at pH 2; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow). Figure 3. Normalized thermal transition curve of the Lyz: (a) Lyz at different pH; pH 7 (black), pH 6 (green), pH 5 (blue), pH 4 (cyan), pH 3 (pink) and pH 2 (red), (b) Lyz in buffer at pH 7 (black) and in varying concentration of ChCl at pH 7; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow) and (c) Lyz in buffer at pH 2 (red), pH 7 (black) and in varying concentration of ChCl at pH 2; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow). Figure 4. The variation of Tm values of Lyz in absence and presence of different concentration of ChCl at pH 7 (blue), pH 2 (cyan) and also in absence of ChCl at different pH (red). Figure 5. Far-UV CD spectra analysis of the Lyz at 25 oC: (a) Lyz at different pH, (b) Lyz in buffer at pH 7 (black) and in varying concentration of ChCl at pH 7; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow) and (c) Lyz in buffer at pH 2 (red) at pH 7 black and in varying concentration of ChCl at pH 2; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow). Figure 6. The hydrodynamic diameter (dH) of Lyz at 25 oC: (a) Lyz at different pH, (b) Lyz in buffer at pH 7 (black) and in varying concentration of ChCl at pH 7; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow) and (c) Lyz in buffer at pH 2 (red) and in varying concentration of ChCl at pH 2; 0.1 M (green), 0.2 M (blue), 0.3 M (cyan), 0.4 M (pink) and 0.5 M (yellow).



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 24 of 33

Figure 7. Rate of hydrolysis from Lyz for micrococcus lysodeikticus cell suspension in absence and presence of varying concentration of ChCl at pH 7 (blue) and pH 2 (cyan), respectively.


Page 25 of 33

(a) Absorbance (a.u.)

2.0 1.6 1.2 0.8 0.4 0.0 250

260

270

280

290

300

Wavelength (nm)

(b)

(c) 2.0

Absorbance (a.u.)

2.0

Absorbance (a.u.)

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


1.6 1.2 0.8 0.4 0.0

1.6 1.2 0.8 0.4 0.0

250

260

270

280

290

300

250

260

Wavelength (nm)

270

280

290

300

Wavelength (nm)

Figure 1.



339 240

338 337

220

336 200

335 334

180

333 160 332 331

2

3

4

5

6

140

7

Fluorescence Intensity (a.u.)

Wavelength Maximum (nm)

(a)

pH

(c)

(b) 300



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 26 of 33

250 200 150 100 50 0

320

340

360

380

400

420

440

300 250 200 150 100 50 0

320

340

Wavelength (nm)

360

380

400

420

440

Wavelength (nm)

Figure 2.


Page 27 of 33

(a)

(b) 1.0

Normalized Fluorescence Intensity (a.u.)

1.0


0.8

0.6

0.4

0.2 20

30

40

50

60

70

80

90

0.8

0.6

0.4

0.2 20

30

o

40

50

60

70

80

90

o

Temperature ( C)

Temperature ( C)

(c) 1.0


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


0.8

0.6

0.4

0.2 20

30

40

50

60

70

80

90

o

Temperature ( C)

Figure 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

Page 28 of 33

74 72 72 70

70

68

68

66

66

64

64

62

62

60

60 0.0

0.1

0.2

0.3

0.4

0.5

Figure 4.


Page 29 of 33

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


-34.0 -34.5 -35.0 -35.5 -36.0 -36.5 -37.0

2

3

4

5

6

0

0

-10

-10

-20

-20

-30

-30

-40

-40 200

7

210

220

230

240

200

210

220

230

240

Figure 5.



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 30 of 33

6.0 5.5 5.0 4.5 4.0 3.5 3.0

2

3

4

5

6

24

24

20

20

16

16

12

12

8

8

4

4

7

0

0 0

2

4

6

8

100

1000

0

2

4

6

8

100

1000

Figure 6.


Page 31 of 33

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


0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

Figure 7.



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 33

Table 1. The Tm values of Lyz in the absence and presence of ChCl at both pH 7 and pH 2. The error in Tm does not exceed 0.2 oC Transition Temperature, Tm (oC) Conc. of IL [M]

Lysozyme

Lysozyme

at pH 7

at pH 2

0.0

70.0

60.2

0.1

69.8

72.5

0.2

69.8

72.4

0.3

70.2

72.5

0.4

70.0

72.5

70.4

72.8

0.5

Table 2. The hydrodynamic diameters (dH) of Lyz in the absence and presence of ChCl at both pH 7 and pH 2. Hydrodynamic Diameter, dH (nm) Conc. of IL [M]

Lysozyme

Lysozyme

at pH 7

at pH 2

0.0

3.8±0.2

5.2±0.3

0.1

3.3±0.2

3.9±0.1

0.2

3.7±0.3

4.0±0.2

0.3

3.5±0.3

3.9±0.2

0.4

3.2±0.1

4.1±0.1

3.4±0.3

4.2±0.3

0.5


Page 33 of 33

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


For Table of Contents Use Only

Synopsis: Choline chloride is having remarkable tendency to offset the the undesirable impact of acidic conditions on Lyz conformation and activity

ACS Paragon Plus Environment

Sustained Stability and Activity of Lysozyme in Choline Chloride

Recommend Documents