Sustained Stability and Activity of Lysozyme in Choline Chloride

Research Article pubs.acs.org/journal/ascecg

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 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 abundant applications of lysozyme (Lyz) in medicine and the pharmaceutical industry, fibril formation is a major obstacle in the development of its biotherapeutic formulations for use in the clinic, as Lyz may encounter low pH conditions 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 conditions is responsible for its denaturation by increasing electrostatic repulsion, which is compensated to a significant extent on the 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’s 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

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high propensity to form fibrils.1,10 The formation of fibrils is deemed 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 regard, 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

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 amounts in body fluids such as serum and cerebrospinal fluid.3 The bacteriostatic and bactericidal properties of Lyz have created abundant applications in medicine and the pharmaceutical industry4,5 that necessitate the understanding of its stability and activity under different experimental conditions, which is considered 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 under conditions 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 augments the proportion of partially folded protein molecules with reduced thermodynamic stability and © 2017 American Chemical Society

Received: June 28, 2017 Revised: July 31, 2017 Published: August 5, 2017 8344

DOI: 10.1021/acssuschemeng.7b02126 ACS Sustainable Chem. Eng. 2017, 5, 8344−8355

Research Article

ACS Sustainable Chemistry & Engineering

In the present work, we reveal the efficacy of ChCl for the 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 a 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 pH-induced 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.

unfolded Lyz. Strategically, the power of these ILs can be basically amplified by the possibility of choosing ions that exist in nature to design biocompatibility.14,15 Choline, an essential micronutrient, is a prominent example of an 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 In addition, choline has the OH-terminated alkyl chain, and this functionalized side chain increases its propensity to form H bonds.17 The choline ion is the only investigated ion which not only nonspecifically binds to negatively charged aspartic and glutamic acid residues but also substantially makes 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 has become available in the past few years that states 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 the stability and activity of enzymes. Vijayaraghavan et al.19 and Tateishi-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 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 is enhanced in the presence of ChDHP. Additionally, the authors reported that the effects of cholinium-based ILs on the stability of the Lyz were 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 the anomalous behavior of these ILs 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 also been 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. 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 the 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 past few years,29−32 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.

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

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MATERIALS AND METHODS

Materials. Lysozyme (Lyz) from chicken egg white (≥90% protein lyophilized powder) was purchased from Sigma-Aldrich Company. The H3BO3, H3PO4, and CH3COOH used were of high purity and purchased from Sisco Research Lab (SRL), India. A Britton−Robinson buffer33 consisting of a mixture of 0.04 M H3BO3, 0.04 M H3PO4, and 0.04 M CH3COOH was prepared for buffers of different pH’s 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 concentrations of IL at both pH 2 and 7 for 1 h to attain complete equilibrium at 25 °C. The protein concentration was used at 0.5 mg/mL for all measurement except for DLS measurement where 1 mg/mL was used. Different concentrations of IL were used such as 0.1, 0.2, 0.3, 0.4, and 0.5 M. All samples were filtered through a 0.45 μm pore size disposable syringe filter (Millipore, Millex-GS). All the samples were prepared in distilled deionized water with a resistivity of 18.3 Ω cm by using an Ultra 370 series water purifier from Rions India Company. For all gravimetric measurements, an AND (Japan) balance with a precision of ±0.00001 g and a Metler Toledo balance with a precision of ±0.0001 g were used. Methods. Ultraviolet−Visible Spectroscopy. UV−vis absorption spectra of the protein aqueous solution were recorded between 190 and 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 into a quartz cell of 1.0 cm path length. The spectrophotometer has matched quartz cells, with a spectral bandwidth of 1.0 nm with a wavelength accuracy of ±0.3 nm along with an automatic wavelength correction. 8345


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Figure 1. UV−vis spectral analysis of the Lyz. (a) Lyz at different pH’s: 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 concentrations 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). (c) Lyz in buffer at pH 2 (red), at pH 7 (black), and in varying concentrations 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). Steady-State Tryptophan Fluorescence Experiments. The steady state fluorescence measurements were conducted at a constant temperature (25 °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 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 °C at an approximate rate of 2.0 °C/min. The accuracy in the temperature changes was ±0.01 °C. 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

temperature control. CD calibration was performed using (1S)-(+)-10camphorsulfonic 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 s 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 a MALVERN Zetasizer Nano instrument, U. K. The instrument is equipped with a 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 90°. An advanced avalanche photodiode, Q.E. > 50% at 633 nm, was used as a detector. The temperature accuracy of the instrument was ±0.1 °C. The temperature was set to 25.0 °C and kept constant until thermal equilibrium was attained. The measurements for each of the temperatures were performed and recorded repeatedly for 50 runs to improve the signal-to-noise ratio of the experiment. Enzyme Activity Assay. Enzymatic activity of Lyz was determined by using 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, at pH 7.0. Lyz activity measurements were performed on suspended Micrococcus lysodeikticus cells for varying concentration of IL.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 the above section.

(1)

Sigmoidal fluorescence intensity curves were obtained for Lyz in the presence of ChCl. Details of the experimental procedure have 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 8346


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Figure 2. Fluorescence spectra analysis of the Lyz conformation at 25 °C. (a) Lyz at different pH’s. (b) Lyz in buffer at pH 7 (black) and in varying concentrations 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). (c) Lyz in buffer at pH 2 (red), at pH 7 (black), and in varying concentrations 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).

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RESULTS AND DISCUSSION We investigate the biomolecular interactions of choliniumbased 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 cosolvent may present a different solvent environment in comparison to the dilute solutions, thereby some kinds 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 a UV−vis spectroscopy study. Figure 1 represents the changes in UV absorbance of Lyz under 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 1a, a decrease in the Amax of the Lyz is significantly observed with a decrease in pH until 4. The results indicate that the pH changes (≥4) lead to the conformational changes which escort the burial of aromatic residues inside the core of the Lyz, thereby, decreased absorbance of Lyz.

Alternatively, both pH 2 and 3 cause an 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 a drastic change in Amax of Lyz, as can be clearly speculated from the red spectra in Figure 1a. This may be attributed to a fairly unstable hydrophobic pocket due to the electrostatic repulsion between the 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, a 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 in wavelength maxima (λmax) of UV absorbance under all pH conditions (Figure 1a). Furthermore, in order to counterbalance this unfavorable 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 the presence of ChCl, the absorption intensities are decreased with increasing concentrations of ChCl until 0.3 M. This may indicate that the aromatic residues are now less exposed to the solvent, resulting in 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, the Amax of Lyz at pH 7 is slightly more than that in the absence of ChCl. However, it is noteworthy in Figure 1b that values are very near to the control (Lyz at pH 7 only). 8347


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Figure 3. Normalized thermal transition curve of Lyz. (a) Lyz at different pH’s: 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 concentrations 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). (c) Lyz in buffer at pH 2 (red), pH 7 (black), and in varying concentrations 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).

On the other hand, at pH 2, the presence of ChCl (until 0.3 M) causes a decrease in the Amax of Lyz not only in comparison to the Amax of Lyz in the 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 1c symbolize the existence of aromatic residues toward 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 the contrary, the higher concentrations of ChCl such as 0.4 and 0.5 M also result in a decrease in the absorbance of Lyz at pH 2 with respect to that of Lyz without ChCl in pH 2; however, it is quite higher than Lyz in buffer at pH 7 (Figure 1c). As a consequence, it may be declared here that ChCl has an 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. To further confirm the above results, a deep analysis of all samples is executed by using a 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 under different pH conditions and also in the presence of ChCl at pH 7 and 2. 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 Figure 2a that pH 2 is 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 the Lyz surface escorts electrostatic repulsions between positive charges, resulting in destabilization of the Lyz conformation, especially by effecting salt bridge contribution to the stability.38 As a consequence, the 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 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 a 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, a further fluorescence experiment is performed for the Lyz denaturation at pH 2, which is found to be most effective for disrupting the Lyz (Figure 2a). Here, Figure 2b indicates the effect of different concentrations of ChCl on the 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 2b. However, Imax for Lyz is found to increase as a function of concentration of ChCl as 8348


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ACS Sustainable Chemistry & Engineering 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 a Hofmeister neutral anion. Whereas the choline cation remains hydrated and has a nonspecific binding to the protein by various types of interactions such as the cation−Π interaction, hydrophobic interactions, H-bonding, and other polar interactions,18 however, it does not do so strongly to the positive charged surface of Lyz. This may result in an increased Imax of Lyz at pH 7 in the presence of ChCl. However, in Figure 2c, which shows fluorescence spectra of Lyz at pH 2 in the presence of various concentrations of ChCl, a significant augmentation is viewed in the Imax of Lyz in ChCl, even at pH 2, as compared to that for Lyz in buffer at pH 2. Nevertheless, the Imax of Lyz at pH 2 approaches that at pH 7 and also increases compared to that for pH 7 in some concentrations of ChCl (0.3 M) as shown in Figure 2c. This is an indication of the conformational changes which cause 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 the retaining of Lyz in its native state in the presence of ChCl under acidic conditions also; hence, a more stabilized Lyz is formed than Lyz in buffer. This may be attributed to the preferential exclusion of choline ions from highly positive charged surfaces at low pH and also to the somewhat binding of chloride ions to the surface, resulting in a compact structure and decrease in electrostatic repulsion within the protein molecule.38 Thus, an increase in Imax in Trp fluorescence of Lyz is observed even at pH 2. Hence, all of this surveillance depicts significant counteraction by ChCl against pH-induced denaturation of Lyz. Still, it is not possible to give a clear unifying statement about the counteracting ability of ChCl. Thus, for greater transparency of the obtained results, it is necessary to further evaluate the 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 under various conditions of pH and IL. From transition curves in Figure 3a, it is pretty visible that there is a drastic shift in the transition curve of Lyz at pH 2 toward the lower temperature in comparison to that under other pH conditions. Therefore, it can be stated here that pH 2 (red color in Figure 3a) is significantly having an adverse effect on the thermal stability of Lyz. Figure 3b depicts more or less no change in the transition curve of the Lyz at pH 7 on the addition of varying concentrations of ChCl. However, the transition curve of Lyz at pH 2 is largely affected in the presence of choline chloride, which now appears very near that for Lyz at pH 7. This inspection of Figure 3c depicts that the 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’s and also in the presence of different concentrations of ChCl at pH 7 and pH 2. With a decrease in pH less than 4, Tm of Lyz is decreasing, and it is highly diminished at pH 2 (Tm at pH 2 is ∼60.0 °C). In Figure 4, it is

Figure 4. Variation of Tm values of Lyz in the absence and presence of different concentrations of ChCl at pH 7 (blue), pH 2 (cyan), and also in absence of ChCl at different pH’s (red).

quite clear that the 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 a high Tm as compared to that of Lyz in pH 2 as well as in pH 7. Apparently, ChCl is observed to have a more or less significant effect on the Tm of Lyz at pH 7. On the other hand, ChCl has shown noteworthy potential to counteract the adverse effect of low pH on the Tm of Lyz. Also, Table 1 Table 1. Tm Values of Lyz in the Absence and Presence of ChCl at Both pH 7 and pH 2a transition temperature, Tm (°C)

a

conc. of IL [M]

lysozyme at pH 7

lysozyme at pH 2

0.0 0.1 0.2 0.3 0.4 0.5

70.0 69.8 69.8 70.2 70.0 70.4

60.2 72.5 72.4 72.5 72.5 72.8

The error in Tm does not exceed 0.2 °C.

comprises all of the Tm values for Lyz at pH 2 and pH 7 in the presence of ChCl. In Table 1, the Tm of Lyz at pH 2 is ∼60.2 °C, which is much less than the value at pH 7 (Tm ∼ 70.0 °C), whereas the addition of ChCl leads to an increase in Tm of Lyz more or less equal to 72.5 °C, which is even more than that of Lyz at physiological pH (Tm ∼ 70.0 °C 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, it shifts to the lower side of the temperature as can also be visualized from Figure 3a.40 At low pH, thermal stability is reduced and may be due to the increased positive charge− charge repulsions.38 Moreover, in folded and unfolded states of any protein molecule, amino acid residues have different pKa’s depending on the surrounding amino acid environments. Lyz was found to be more thermally stable at pH 4−5 as is obvious 8349


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Figure 5. Far-UV CD spectra analysis of the Lyz at 25 °C. (a) Lyz at different pH’s. (b) Lyz in buffer at pH 7 (black) and in varying concentrations 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). (c) Lyz in buffer at pH 2 (red), at pH 7 (black) and in varying concentrations 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).

more, the counteracting effect of ChCl is ascertained by performing far-UV CD-spectral analysis. Figure 5 represents secondary structural changes in Lyz under various conditions. Lyz at pH 7 possesses mainly two negative bands, a more intense band at 209 nm and a shallow band at 222 nm (shown black in Figure 5b and c), suggesting that Lyz belongis to the typical α + β class of enzymes. In Figure 5a, there is a decrease in the secondary structure of Lyz with a decrease in pH lower than 5, which is portrayed due to the decreased negative ellipticities at 209 nm. Conversely, pH 2 has shown again the most significant impact on secondary structure of Lyz. Haezebrouck et al.40 observed a decrease in the helical content of the Lyz at this low pH. The change in the electrostatic properties with a 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 5a that even this low of a pH is not drastically affecting the Lyz structure, and it is retained in its native structure to a paramount extent. In the case of Lyz at pH 7 in the presence of ChCl (Figure 5b), the negative ellipticities of Lyz for all concentrations of IL are either more than or almost equal to that in buffer only. This shows that ChCl at all studied concentrations has a stabilizing influence on the secondary structure of the Lyz; however, this effect is more pronounced at 0.2 and 0.3 M ChCl. Now, as is clear from ellipticities for negative bands (Figure 5c), Lyz possesses a high amount of secondary structure even at pH 2 if there is a presence of ChCl. The negative ellipticities values under this condition are substantially higher than that for Lyz at

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 the formation of a 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 the overall stability of the Lyz.42 However, the addition of ChCl may reduce positive charge− charge repulsions by binding 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 behavior at pH 2. The poor interaction of the choline cation with Lyz at low pH may also stabilize the folded state more. In order to find the exact reason behind the counteraction effect of ChCl for Lyz at very low pH, an extensive study of the system is required by the use of molecular dynamic simulations. Circular Dichroism Analysis of ChCl Effect on pHInduced Changes in Lyz Secondary Structure. Further8350


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Figure 6. Hydrodynamic diameter (dH) of Lyz at 25 °C. (a) Lyz at different pH’s. (b) Lyz in buffer at pH 7 (black) and in varying concentrations 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). (c) Lyz in buffer at pH 2 (red) and in varying concentrations 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).

pH 7. From Figure 5c, it can be undoubtedly accentuated that ChCl is fairly efficient in decreasing the denaturing action at a low pH on the secondary structure of Lyz. Again, it may be mentioned here that ChCl diminishes electrostatic repulsion within the Lyz molecule, making conditions favorable for intramolecular H-bond formation. A close examination of Imax, Tm, and ellipticities [Θ] may declare here that there is conclusive change in the conformation of Lyz compared to the disrupted one under the influence of low pH, which is satisfactorily counterbalanced 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 at different pH’s and in the presence of ChCl at pH 2 and 7. All the peaks of the intensity distribution graph are found to be highly monodisperse with PDI < 0.1 for Lyz in buffer at pH 7, and PDIs for all other samples also do not exceed 0.5. The dH for Lyz in buffer at pH 7 (shown black in Figure 6b and c) is ∼3.8 nm at 25 °C and observed to increase with a decrease in the pH < 5 (Figure 6a). The dH of Lyz at pH 2 is monitored at ∼5.2 nm, which describes its adverse effect on Lyz that causes swelling of Lyz by disrupting the Lyz native state, resulting in increased dH. The large increase in the net positive charge on the Lyz surface leads to the destabilization of the Lyz domain by expanding the conformation so that the least repulsion is felt by residues of Lyz. Moreover, in the presence of ChCl, it can be clearly examined from Figure 6b as well as Table 2 that the dH of Lyz at pH 7 is decreased to a smaller extent at all studied

Table 2. 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] 0.0 0.1 0.2 0.3 0.4 0.5

lysozyme at pH 7 3.8 3.3 3.7 3.5 3.2 3.4

± ± ± ± ± ±

0.2 0.2 0.3 0.3 0.1 0.3

lysozyme at pH 2 5.2 3.9 4.0 3.9 4.1 4.2

± ± ± ± ± ±

0.3 0.1 0.2 0.2 0.1 0.3

concentrations of IL, which indicates that the conformational size of Lyz is retained. However, at pH 2, the addition of ChCl in Lyz leads to a severe decrease in dH and acquires a size very similar to that for Lyz at physiological pH (Figure 6c and Table 2). It reveals that ChCl is maintaining Lyz in a native conformation even under highly acidic conditions by keeping it compact, which points toward the contribution of ChCl to the counteraction of unpleasant effects 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 Figure 6b and 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 the same in the absence and presence of different concentrations of ChCl. However, the aggregate size is slightly 8351


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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 a noticeable increase in the activity. There are few reports in the literature which also show that cholinium-based IL enhances enzyme activity.18,20 Wijaya et al.46 found that 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 color in Figure 7). Afterward, with a further increase in IL concentration, Lyz is comparatively less active, but still appreciably more than the control. Under a high concentration of ChCl, reduced flexibility of the active site due to the compact structure of Lyz may cause fewer enhancements in activity of Lyz at pH 7. On the other hand, in the case of pH 2, Lyz possesses more activity on the addition of different concentrations of ChCl in comparison to that under pH 2 conditions only, as is apparent from initial rate values in Figure 7 (cyan color). 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 to a slight compacting of the 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 the 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 the failure of resuming the original conformation around the active site; thereby the little disrupted active site does not lead to perfect binding to the complex sugars of the 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 the addition of ChCl, it can be depicted that there is formation of a more compact structure with decreased exposure of nonpolar groups and increased secondary structures as compared to Lyz in pH 2 buffer only. These inferences escort us to one way of thinking, i.e., ChCl has a remarkable tendency to offset the denaturing effects of a strongly acidic environment on Lyz stability, ensuring a more compact Lyz which is clearly depicted in 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 exploring the factors responsible for stabilizing Lyz under adverse conditions, which is a necessity in order to make a successful path from the administration site to the target site and also to avoid uninvited consequences in biopharmaceutical formulations as well as in the 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.

decreased at pH 7 by ChCl, as is clear from Figure 6b. On the other hand, a solution of Lyz at pH 2 is comprised of very elevated amounts of large sized aggregates, which is somewhat evident from Figure 6c. Afterward, the presence of ChCl in the Lyz at pH 2 results in the appreciable diminishment of the percentage contribution of aggregates in the particle size distribution that is quite apparent from Figure 6c. Strangely, the size of aggregates of Lyz at pH 2 is increased, and the amount of aggregates is decreased with an increase in the concentration of IL (Figure 6c). The peaks attributed to a larger size than 1000 nm are ignored in the present study. These results are found to be inconsistent with fluorescence and CD data shown in Figures 2 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 does 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., the β-sheet domain and α-helix domain linked by an α-helix.42,43 The side-chain carboxyl group of Glu35 of the α-helical domain projects into the active site and donates a proton to the glycosidic oxygen of anomeric carbon in the complex sugar of the bacterial cell wall.42−45 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 by 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 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 a much higher initial rate at pH 7. This may be described by the very loose conformation of Lyz at pH 2 which causes opening of the catalytic site, resulting in unfavorable conditions for substrate

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CONCLUSION From an assessment of the research over the past decades, it is convincingly unequivocal that the counteracting role of the

Figure 7. Rate of hydrolysis from Lyz for Micrococcus lysodeikticus cell suspension in the absence and presence of varying concentrations of ChCl at pH 7 (blue) and pH 2 (cyan), respectively. 8352


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ACS Sustainable Chemistry & Engineering Scheme 2. Schematic Depiction of Counteraction Effects of ChCl against pH-Induced Denaturation of Lyz

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.

cosolvents 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 wellknown 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 Lyzbased drugs. Our data indicate the existence of a 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 a 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 the complete data of stability and activity under all conditions that there is a direct correlation between the bacteriolytic activity and conformational stability of Lyz. The electrostatic interactions may be playing an overriding role under low pH conditions. The high positive charge density on the surface of Lyz may be resulting in electrostatic repulsion and, thereby, resulting in its denatured conformation. Whereas, on the addition of ChCl, preferential exclusion of the choline ion as well as decreased electrostatic repulsion due to somewhat binding of chloride ions leads to native-like compact Lyz at low pH conditions also. As a concluding remark, our results explicitly elucidate 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 a 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 ILs, which can be further employed in an in vivo study in order to regulate

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +91-11-27666646-142. Fax: +91-11-2766 6605. E-mail: [email protected], [email protected]. ORCID

Pannuru Venkatesu: 0000-0002-8926-2861 Present Addresses †

Department of Chemistry, University of Delhi, Delhi 110007, India ‡ Department of Chemistry, University of Delhi, Delhi 110007, India § Department of Chemistry, University of Delhi, Delhi 110007, India Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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 a Senior Research Fellowship (SRF).

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