Implications of Imidazolium-Based Ionic Liquids as Refolding Additives for Urea-Induced Denatured Serum Albumins

et al. 2020

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Human serum albumin (HSA) attracts a great deal of scientific interest for drug discovery and development of novel biologically active molecules. Over the past few years, there has been an increased demand to consider ionic liquids (ILs) as novel biocompatible cosolvents for proteins. While there exist multiple studies on HSA with ILs, HSA remains greatly unexplored with respect to the variation of chain length and functional group of a variety of cholinium-based ILs and their ability for refolding and counteraction of crowding-induced unfolding of HSA. Furthermore, most of the studies are performed in dilute buffers, which is highly inconsistent with in vivo conditions. The intracellular environment is extremely crowded owing to the existence of a variety of macromolecules such as proteins, nucleic acids, ribosomes, carbohydrates, etc. Thus, this article explores the biocompatibility of a series of cholinium-based ILs, namely, choline malonate (Mal), choline propionate (Prop), choline bitartrate (Bit), choline citrate (Cit), and choline dihydrogen phosphate (Dhp) on HSA using various spectroscopic techniques in combination with dynamic light scattering studies. On the basis of the outcome of the current study, most of the biocompatible ILs, i.e., Bit and Dhp, are selected to revoke the effect of a crowding agent, namely, dextran 6 kDa (dex6) on HSA at a fixed concentration. Both the ILs Bit as well as Dhp successfully counteract the crowding effects on the HSA structure. Furthermore, Bit and Dhp showed positive results for refolding of the dex6perturbed HSA structure. Intriguingly, it was also observed that the stabilizing effect of ILs was more profound under crowded conditions compared to an IL alone.

Section: ■ Results and Discussionmentioning

confidence: 99%

Exploring the Counteracting and Refolding Ability of Choline-Based Ionic Liquids toward Crowding Environment-Induced Changes in HSA Structure

Bhakuni

Sindhu

et al. 2020

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“…1−5 Recently, many researchers have demonstrated that the addition of ionic liquids (ILs) to enzymes can improve the enzymatic activity and stability. 6,7 In 2019, Paẗzold et al 9 explained about deep eutectic solvents (DESs) as alternatives to ILs due to the lack of biodegradability, toxicity, lower sustainability, complexity in preparation, and high cost of ILs. Zhang et al 10 introduced DESs as emerging cosolvents to replace the anciently used ILs because of recyclability and availability of DESs compared to ILs.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Enzymes are the biocatalysts that perform various biochemical transformations and industrial processes. , However, extreme processing conditions and the inherent instability of enzymes confines their potential applications. Addition of cosolvent is a simple and efficient method to improve the stability and reusability of enzymes against multiple stresses like high temperatures, pH, and nonaqueous solvents. − Recently, many researchers have demonstrated that the addition of ionic liquids (ILs) to enzymes can improve the enzymatic activity and stability. , In 2019, Pätzold et al explained about deep eutectic solvents (DESs) as alternatives to ILs due to the lack of biodegradability, toxicity, lower sustainability, complexity in preparation, and high cost of ILs. Zhang et al introduced DESs as emerging cosolvents to replace the anciently used ILs because of recyclability and availability of DESs compared to ILs.…”

Section: Introductionmentioning

confidence: 99%

Expanding the Potential Role of Deep Eutectic Solvents toward Facilitating the Structural and Thermal Stability of α-Chymotrypsin

Yadav

Bhakuni

et al. 2020

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Since the introduction of deep eutectic solvents (DESs), numerous reports have surfaced describing its tunable properties and environmentally benign nature. Due to such favorable properties of DESs, they have found a wide variety of applications. Moreover, in order to harness the potential of proteins in numerous industries, there is an emergent need to find a suitable cosolvent that is biocompatible with protein and is also environmentally safe. In this context, this work presents a systematic evaluation of effect of two deep eutectic solvents (DESs), namely, choline chloride-urea (ChCl-urea) and choline chloride-glycerol (ChCl-gly) on the structural and thermal stability along with activity of enzyme α-chymotrypsin (CT) using circular dichroism (CD), UV–visible, steady state, and thermal fluorescence spectroscopy. It was observed that the presence of DESs does lead to enhancement in the thermal stability of CT along with the preservation of activity. The enzymatic activity was well maintained in both the DESs, and the deleterious effect of urea was overcome by ChCl-urea on the enzyme. Also, desirable results were observed for ChCl-urea, despite having urea as one of its major components. Thus, the negative outcome of urea was overpowered by the combination of ChCl and urea. Furthermore, all the biomolecular studies were also performed with the individual constituents of DESs. It was found that the effect imparted by both the ChCl-based DESs on CT is by the virtue of DES itself rather than its individual constituent. Overall, both the DESs can be described as potential biocompatible, sustainable, and promising cosolvents for CT with enhanced structural and thermal stability along with preservation of its activity.

“…Figures and show the specific binding of cholinium, acetate, dihydrogen phosphate, and dihydrogen citrate ions with F ab and F c fragments of IgG through molecular docking. However, the docking results for chloride could not be displayed as the Cl anion does not form a H-bond with the protein molecules Figures c,d and c,d show that hydrophobic interactions in the case of dihydrogen citrate and dihydrogen phosphate are higher with three amino acid residues of F ab , whereas three amino acid residues of F c fragments show hydrophobic interactions with dihydrogen phosphate, which has also been seen in the FT-IR studies.…”

Section: Resultsmentioning

confidence: 86%

Cholinium-Based Ionic Liquids as Efficient Media for Improving the Structural and Thermal Stability of Immunoglobulin G Antibodies

Dhiman

Tavares

et al. 2022

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Among antibody-based biopharmaceuticals, immunoglobulin G (IgG) plays a central role. However, IgG is a protein and as such easily loses stability, thus compromising its use as an effective therapeutic. To overcome this drawback, in this work, we evaluated cholinium-based ionic liquids (ILs) as effective stabilizers of IgG. We have investigated four ILs in aqueous solution, namely, cholinium acetate ([Ch][Ac]), cholinium chloride ([Ch]Cl), cholinium dihydrogen citrate ([Ch][Dhc]), and cholinium dihydrogen phosphate ([Ch][Dhp]). We used a quantitative approach using different spectroscopic and chromatographic techniques to evaluate the thermal and structural stability of IgG. Thermal fluorescence spectroscopy studies were performed to obtain the transition temperature (T m ) and the change in Gibbs free energy (ΔG) of IgG in all cholinium-based IL aqueous solutions. The results indicate an appreciable increase in T m in the presence of [Ch][Ac] and [Ch]Cl. The thermodynamic parameters obtained from thermal fluorescence are compared with the structural stability of IgG by UV, fluorescence, CD, and FT-IR spectroscopies, as well as with size exclusion high-performance liquid chromatography and sodium dodecyl-sulfate polyacrylamide gel electrophoresis, showing that all results are in good agreement. Furthermore, the hydrodynamic diameter (d H ) of the IgG as a function of the concentration of IL was analyzed using dynamic light scattering, showing favorable interactions between protein residues. Finally, molecular docking studies for IgG in ILs using Molegro virtual docker have been performed, reinforcing the main interactions ruling the IgG stability. Time dependent studies were also performed for one month to study the long-term stability of IgG at room temperature in presence of ILs. This work highlights the potential validity of using cholinium-based ILs in IgG formulations for enhancing its thermal and structural stability and thus the preservation of IgG antibodies.