Secondary Structure Of Bovine Serum Albumin Research Articles

BSA interaction and DNA cleavage studies of anti-bacterial benzothiazol-2-yl-malonaldehyde

A new benzothiazole derivative, benzothiazol-2-yl-malonaldehyde (BTM) was synthesized and its biological potency has been investigated in terms of its anti-bacterial activity. Its interaction with bovine serum albumin (BSA) and DNA cleavage studies were also carried out. The interaction between BSA and BTM was studied by multi-spectroscopic approach, which included spectrofluorimetry, CD spctrophotometry and FTIR. The experiments were carried out at three different temperatures and the quenching mechanism was observed to be both static as well as dynamic. The binding was found to be moderate as evident by its binding constant values (of the order of 103). A single binding site was observed and site marker experiments showed site III of BSA for BTM. The negative values of ΔG0 suggested the spontaneity of the interaction and positive values for both ΔH0 and ΔS0 indicated that the nature of interaction was hydrophobic. The CD and FTIR studies revealed that the secondary structure of BSA has been altered upon binding of BTM. BTM cleaved the super-coiled DNA in the presence and absence of hydrogen peroxide at micro-molar concentration. It was also found from the antibacterial activity that BTM showed better activity against gram-negative bacteria Pseudomonas aeruginosa than the other bacterial stains used for the study.

Evaluation of Biophysical Interaction between Newly Synthesized Pyrazoline Pyridazine Derivative and Bovine Serum Albumin by Spectroscopic and Molecular Docking Studies

In this research, the pyrazoline pyridazine derivative 7-methyl-2-phenyl-4-(3,4,5-trimethoxyphenyl)-2H-pyrazolo[3,4-d]pyridazine (5d) was studied for its interaction with bovine serum albumin (BSA). Various spectroscopic techniques along with molecular docking analysis were utilized to understand the mechanism of interaction. The quenching of BSA fluorescence by using investigational drug 5d was the basic principle for the methodology. Spectrofluorometric methods and UV-absorption studies were conducted for exploration of the 5d and BSA binding mechanism. The fluorescence quenching mechanism involved in BSA and 5d interaction was static quenching, and a complex formation also occurred between them. Both enthalpy and entropy attained positive values suggesting involvement of hydrophobic forces in BSA and 5d interaction. The Förster distance of 2.23 nm was calculated by fluorescence resonance energy transfer (FRET). An alteration in BSA secondary structure was proven from the conformational studies of BSA-5d interaction. This binding interaction study provided a basis to comprehend the binding interaction between 5d and BSA. These results provided information about sites of BSA involved in its interaction with 5d.

Interaction of repaglinide with bovine serum albumin: Spectroscopic and molecular docking approaches

Repaglinide (RPG) regulates the amount of glucose by stimulating the pancreas to release insulin in the blood. In view of its biological importance, we have examined the interaction between RPG and a model protein, bovine serum albumin (BSA) employing various spectroscopic, electrochemical and molecular docking methods. Fluorescence spectra of BSA were recorded in the presence and absence of RPG in phosphate buffer of pH 7.4. Fluorescence intensity of BSA was decreased upon the addition of increased concentrations of RPG, indicating the interaction between RPG and BSA. Stern-Volmer quenching analysis results revealed that RPG quenched the intensity of BSA through dynamic quenching mechanism. This was further confirmed from the time-resolved fluorescence measurements. The binding constant as calculated from the spectroscopic and voltammetric results was observed to be in the order of 104 M−1 at 298 K, suggesting the moderate binding affinity between RPG and BSA. Competitive experimental results revealed that the primary binding site for RPG on BSA was site II. Absorption and circular dichroism studies indicated the changes in the secondary structure of BSA upon its interaction with RPG. Molecular simulation studies pointed out that RPG was bound to BSA in the hydrophobic pocket of site II.

Biochemical and toxicological investigation of karanjin, a bio-pesticide isolated from Pongamia seed oil

Karanjin, a furanoflavonol from Pongamia pinnata L is used in agricultural practices for its pesticidal, insecticidal and acaricidal activities. It is commercially available as a bio-pesticide targeting a wide variety of pests. The present study was intended to evaluate the biochemical interactions of karanjin with bovine serum albumin (BSA) and study its toxicological effects on mammalian and bacterial cell lines. Karanjin bound to BSA at a single site with a dissociation constant of 19.7 μM. Evaluation of BSA-karanjin interactions at three different temperatures indicated the involvement of static mode of quenching. Binding experiments in the presence of warfarin and computational docking analysis indicated that karanjin bound closer to the warfarin binding site located in the Subdomain IIA of BSA. Using Förster resonance energy transfer analysis the distance between TRP 213 of BSA and karanjin was found to be 20 Å. Collective results from synchronous fluorescence spectra analysis, differential scanning calorimetry, and circular dichroism analysis indicated that binding of karanjin induced conformational changes in the secondary structure of BSA. Karanjin exhibited low toxicity against human cervical cancer cells and normal mouse fibroblast L929 cells and modestly inhibited the growth of B. subtilis and E. coli cells. The data presented in this study provides insights for understanding the binding interactions of karanjin with BSA and its possible toxicological effects on mammalian cell lines and bacteria.

Elucidation of interactions of BSA with [EPMpyr]+[Cl]− using spectroscopic techniques with reference to theoretical thermodynamic and molecular docking studies

On the premise that bovine serum albumin (BSA) is a suitable substitute for Human Serum Albumin (HSA) in experiments, its interactions with ionic liquid (IL), N‑2′,3′‑epoxypropyl‑N‑methyl‑2‑oxopyrrolidinium chloride, ([EPMPyr]{Cl}), was studied. This was done on the basis that the data would give information on the potential role of ionic liquids (ILs) in drug delivery, involved in the treatment of diseases. This was effected by eliciting data from the interactions using UV–visible absorption, emission spectroscopy, circular dichroism (CD), FTIR and molecular docking. The subsequent changes in microenvironment around the tryptophan (Trp) residues in BSA were observed using fluorescence spectroscopy techniques. Furthermore, UV and CD results indicated that the secondary structure of BSA was affected by interaction of IL at high concentrations and temperatures. In addition, the thermophysical and thermodynamic studies were used to evaluate the thermostability of BSA on interaction with IL at various concentrations. These thermophysical and thermodynamic values provided evidence that interactions such as H-bond, dipole, and non-covalent interactions occured in the BSA-IL system. This is attributed, mainly, to hydrophobic and electrostatic interactions resulting in the unfolding of polypeptides within BSA. Therefore, the hydrogen bonding interaction and cation-protein interactions are the driving processes for strong binding between BSA and the IL. Furthermore, molecular docking shows that the entry of IL into subdomains of protein is due to the cationic moiety of oxopyrrolidine interacting with the hydrophobic residues of the domains.

Probing of the binding profile of anti-hypertensive drug, captopril with bovine serum albumin: A detailed calorimetric, spectroscopic and molecular docking studies

Abstract Captopril (CAP), an angiotensin-converting enzyme inhibitor, widely used for the treatment of hypertension. The current study was undertaken to explore the interaction between CAP and the primary plasma protein, bovine serum albumin (BSA) by fluorescence, isothermal titration calorimetry, Forster’s resonance energy transfer, circular dichroism, fourier transform infrared and molecular docking studies. The fluorescence result indicated that CAP quenches the fluorescence intensity of native BSA through a static manner with blue shift in wavelength maxima. Thermodynamic analysis from ITC suggested that hydrogen bonding and van der Waals forces play major role in the association process and it is an enthalpically driven process. The distance between donor (BSA) and acceptor (CAP) has been calculated according to FRET theory. The ITC based displacement experiments concluded that CAP primarily bound to near sub-domain IIA (Sudlow’s site I) of BSA. Alteration in the secondary structure of BSA by CAP is revealed by CD which is further substantiated by synchronous, 3D fluorescence and FTIR spectroscopy. In addition, molecular docking was performed to further confirm the biophysical methods. This study provides an insight into the molecular basis of interaction between CAP and BSA which helps to understand the activity and mechanism of drug binding to protein.

Magnetically triggered release of amoxicillin from xanthan/Fe3O4/albumin patches

This work was motivated by the need of stimuli responsive drug carriers, which can be activated by low cost non-invasive stimuli such as external magnetic field (EMF). Thus, novel antimicrobial materials based on xanthan gum (XG), magnetic nanoparticles (MNP), bovine serum albumin (BSA) and amoxicillin (Amox) were designed in order to promote the release of Amox under magnetic stimuli. Firstly, surfaces with different functionalities were prepared by sequential deposition of thin layers on Si wafers and characterized by means of ellipsometry and atomic force microscopy. Amox adsorbed preferentially onto XG or BSA films. In solution, favorable interactions between Amox and BSA were evidenced by substantial changes in the BSA secondary structure, as revealed by circular dichroism. Patches of XG and XG/MNP/BSA were immersed in 2 g L−1 Amox, yielding 10 ± 3 and 17 ± 4 μg/cm3 Amox loading, respectively. The inclusion of 0.2 wt% Fe3O4 in the patches and their exposure to EMF enabled in vitro release of Amox, at pH 5.5 and 0.02 mol L−1 NaCl, following the quasi-Fickian behavior. Amox diffused from XG/MNP/BSA patches in agar medium containing Staphylococcus aureus and Escherichia coli, inhibiting their growth. The inhibition of E. coli growth was particularly efficient under EMF.

Comparative Binding Analysis of Pyrimidine Derivative to BSA: Equilibrium, FTIR and Acoustical Study

This paper presented the comparative binding interaction of ethyl-4-(4-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4-HP2OTP) and ethyl-4-(2-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2-HP2STP) to bovine serum albumin (BSA) in 1,4-dioxane, DMSO and DMF by equilibrium dialysis, FT-IR and acoustical study at physiological pH. The binding data obtained was interpreted by scatchard plot, which gives the association constants. An increase in association constants is observed with increase in temperature and concentration. FT-IR study explains the binding through shifting in peak positions of amide I and II. It explained the changes in secondary structure of BSA on binding with the drugs. The free energy (∆G), enthalpy (∆H) and entropy (∆S) values were calculated by using van’t Hoff equation. The negative ∆G showed the spontaneous process and positive values of ∆H and ∆S showed endothermic interaction between ligands and BSA. ∆G becomes more negative with increased in temperature, indicated feasibility of binding interaction at high temperature. The positive values of ∆H and ∆S also showed specific electrostatic and hydrophobic interaction between ligand and BSA. DOI: http://dx.doi.org/10.17807/orbital.v10i2.1116

Effect of chain length on the interactions of sodium N-alkyl prolinates with bovine serum albumin: a spectroscopic investigation and molecular docking simulations

The interaction of anionic surfactants with serum albumin has emerged as an important area of research and is considered as a model for gaining fundamental insight into surfactant-protein binding, which is useful in both chemical and biological applications. This study involves the interactions of three synthesized proline-based anionic surfactants of varying chain lengths (C8, C10, and C12) with bovine serum albumin (BSA) using different techniques, including fluorescence, 1H NMR, and FT-IR spectroscopy. The study indicated that the binding of the proline surfactants with BSA followed a static quenching process, with an increase in binding ability upon increasing chain length. FT-IR studies revealed a change in the secondary structure of BSA upon binding with the proline surfactant, while 1H NMR investigations indicated the proximity of the long alkyl chain of the surfactant with tryptophan residues of BSA. Molecular docking studies performed on the three proline surfactants with BSA revealed that the surfactants were able to bind in the vicinity of both tryptophan residues (Trp-213 and Trp-134) with an increase in the free energy of binding while increasing the chain length from C8 to C12. Therefore, this study provided a whole view about the interaction of BSA with anionic proline derived surfactants, which can be used as potential ingredients in pharmaceutical products.

Changes of albumin secondary structure after palmitic acid binding. FT-IR spectroscopic study.

Albumin is an universal transport protein. Plasma pool of free fatty acids arising from triglyceride hydrolysis, critical in energy metabolism and etiology of metabolic disorders is transported by albumin. According to various studies albumin has from seven to nine binding sites with diverse affinity to long chain fatty acids. X-ray diffraction crystallography measurements have provided data only for pure human serum albumin or albumin with fully saturated binding sites. These results have shown that amount of -helices is higher after fatty acids binding. Molecular mechanics simulations suggest that binding of fatty acids in two high-affinity sites leads to major conformational changes in albumin structure. The aim of this research was to investigate albumin secondary structure upon gradually increasing fatty acids to protein mole ratio. Fourier transform infrared spectroscopy was applied to study changes of bovine serum albumin (as an analogue of human serum albumin) -helical structures after binding palmitic acid in a range of 0-20 palmitic acid: albumin molar ratios representing pure protein, partial, full saturation and excess binding sites capacity. Amount of -helices was increasing along with the amount of palmitic acid: bovine serum albumin molar ratio and reached maximum value around 2 mol/mol. Our studies confirmed molecular mechanics simulations and crystallographic studies. Palmitic acid binding in two high-affinity sites leads to major structural changes, filling another sites only slightly influenced bovine serum albumin secondary structure. The systematic study of fatty acids and albumin interactions, using an experimental model mimicking metabolic disorders, may results in new tools for personalized nanopharmacotherapy.

Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy as an Analytical Method to Investigate the Secondary Structure of a Model Protein Embedded in Solid Lipid Matrices.

Protein drugs may encounter conformational perturbations during the formulation processing of lipid-based solid dosage forms. In aqueous protein solutions, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy can investigate these conformational changes following the subtraction of spectral interference of solvent with protein amide I bands. However, in solid dosage forms, the possible spectral contribution of lipid carriers to protein amide I band may be an obstacle to determine conformational alterations. The objective of this study was to develop an ATR FT-IR spectroscopic method for the analysis of protein secondary structure embedded in solid lipid matrices. Bovine serum albumin (BSA) was chosen as a model protein, while Precirol AT05 (glycerol palmitostearate, melting point 58 ℃) was employed as the model lipid matrix. Bovine serum albumin was incorporated into lipid using physical mixing, melting and mixing, or wet granulation mixing methods. Attenuated total reflection FT-IR spectroscopy and size exclusion chromatography (SEC) were performed for the analysis of BSA secondary structure and its dissolution in aqueous media, respectively. The results showed significant interference of Precirol ATO5 with BSA amide I band which was subtracted up to 90% w/w lipid content to analyze BSA secondary structure. In addition, ATR FT-IR spectroscopy also detected thermally denatured BSA solid alone and in the presence of lipid matrix indicating its suitability for the detection of denatured protein solids in lipid matrices. Despite being in the solid state, conformational changes occurred to BSA upon incorporation into solid lipid matrices. However, the extent of these conformational alterations was found to be dependent on the mixing method employed as indicated by area overlap calculations. For instance, the melting and mixing method imparted negligible effect on BSA secondary structure, whereas the wet granulation mixing method promoted more changes. Size exclusion chromatography analysis depicted the complete dissolution of BSA in the aqueous media employed in the wet granulation method. In conclusion, an ATR FT-IR spectroscopic method was successfully developed to investigate BSA secondary structure in solid lipid matrices following the subtraction of lipid spectral interference. The ATR FT-IR spectroscopy could further be applied to investigate the secondary structure perturbations of therapeutic proteins during their formulation development.

Characterization of molecular structures of theaflavins and the interactions with bovine serum albumin.

In this study, theaflavins (TF1, TF2A, TF2B and TF3) were prepared from black tea and their interaction with bovine serum albumin (BSA) was explored by fluorescence and CD spectroscopy. The results showed that the structures of theaflavins exhibited significant effects on the binding/quenching process, and the binding affinity increased with the increase of molecular weight of theaflavins and the presence of galloyl moiety. The quenching effects showed a sequence as TF3>TF2A>TF2B>TF1, demonstrating the important role of the galloyl moiety on the C-3 position of theaflavins. CD spectra indicated that TF3 in high concentration could change the skeleton structure of BSA and induce the unfolding of BSA secondary structure. The present results provide a new perspective for better understanding of the likely physiological fate of theaflavins and help to control the functional characteristics of food.

Exploring the binding of two potent anticancer drugs bosutinib and imatinib mesylate with bovine serum albumin: spectroscopic and molecular dynamic simulation studies.

Bosutinib (BST) and imatinib mesylate (IMT) are tyrosine kinase inhibitors (TKIs). In view of the importance of these inhibitors in cancer treatment, we investigated the mechanism of interaction between BST/IMT and bovine serum albumin (BSA) using various spectroscopic and molecular docking methods. Fluorescence studies indicated that BST/IMT interacted with BSA without affecting the microenvironment around the residue Trp213 of BSA. The quenching mechanism associated with the BST-BSA and IMT-BSA interactions was determined by performing fluorescence measurements at different temperatures. These results suggested that BST and IMT quenched the fluorescence intensity of BSA through static and dynamic processes, respectively, which was confirmed by time-resolved fluorescence measurements. Evaluation of the thermodynamic parameters ∆H°, ∆S°, and ∆G° suggested that hydrophobic and electrostatic interactions played significant roles in the BST-BSA interaction, while IMT-BSA was stabilized by hydrophobic forces. Competitive experimental results revealed that the primary binding sites for BST and IMT on BSA were sites II and I, respectively. This was supported by the results of molecular docking and dynamic simulation studies. The change in the secondary structure of BSA upon binding with BST/IMT was investigated by 3D fluorescence, absorption, and CD spectroscopic studies. In addition, the influences of β-cyclodextrin and metal ions (Cu2+ and Zn2+) on the binding affinities of BST and IMT to BSA were examined. Graphical abstract Binding of BST and IMT in BSA at site II and site I respectively.

Insight into the interaction between chitosan and bovine serum albumin

Exploiting the uses of proteins and polysaccharides is of increasing interest because the combination of the attributes of these two types of biopolymers can produce small emulsion droplets with good physical stability. The objective of this study was to evaluate the effect of adding chitosan to a bovine serum albumin (BSA) solution at different pH values, and the consequent conformational changes were monitored using UV–vis absorption spectroscopy and fluorescence spectroscopy. Spectroscopic observations were further combined with rheological analysis results to illustrate the interaction mechanism. The results showed that the pH and BSA/chitosan ratio significantly affected the interaction between BSA and chitosan. Additionally, the microenvironment and the secondary structure of BSA were changed with the addition of chitosan, and chitosan was found to quench the intrinsic fluorescence of BSA. The experimental results provide theoretical guidance for the design of new ingredients for the food industry.

Secondary structure transitions of Bovine serum albumin induced by temperature variation

Secondary structure transitions of Bovine Serum Albumin (BSA) in H2O and D2O under temperature variation were investigated by in-situ IR spectral analysis and they were found to exhibit the same tendency with the only difference that the significant transition temperature of BSA's secondary structure moved towards a lower temperature in D2O. Before this significant transition temperature, the native secondary structures of BSA were essentially changing in a cooperative fashion with temperature variation, except for the intermolecular β-sheet (high-wavenumber) which increased as a result of thermal denaturation at a later stage. After the transition temperature, the relative contents of α-helix and extended chain continued to drop sharply and they were transformed into random coil, β-turn and β-sheet.

Interaction of Flavonoids from Woodwardia unigemmata with Bovine Serum Albumin (BSA): Application of Spectroscopic Techniques and Molecular Modeling Methods

Phytochemical investigation on the methanol extract of Woodwardia unigemmata resulted in the isolation of seven flavonoids, including one new flavonol acylglycoside (1). The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis and comparison of literature data. The multidrug resistance (MDR) reversing activity was evaluated for the isolated compounds using doxorubicin-resistant K562/A02 cells model. Compound 6 showed comparable MDR reversing effect to verapamil. Furthermore, the interaction between compounds and bovine serum albumin (BSA) was investigated by spectroscopic methods, including steady-state fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular docking approach. The experimental results indicated that the seven flavonoids bind to BSA by static quenching mechanisms. The negative ΔH and ΔS values indicated that van der Waals interactions and hydrogen bonds contributed in the binding of compounds 2–6 to BSA. In the case of compounds 1 and 7 systems, the hydrophobic interactions play a major role. The binding of compounds to BSA causes slight changes in the secondary structure of BSA. There are two binding sites of compound 6 on BSA and site I is the main site according to the molecular docking studies and the site marker competitive binding assay.

Tuning the binding, release and cytotoxicity of hydrophobic drug by Bovine Serum Albumin nanoparticles: Influence of particle size

To elucidate the effect of particle size of albumin nanoparticles on cellular uptake of a hydrophobic drug, herein we report the release kinetics and cytotoxicity of nanoparticle bound dimethylcurcumin (DMC) in A549 tumor cells. The bovine serum albumin (BSA) nanoparticles were prepared by thermal denaturation and characterized by dynamic light scattering (DLS), zeta (ζ) −potential, circular dichroism (CD) and transmission electron microscope (TEM). The preparation conditions were optimized to obtain nanoparticles with mean hydrodynamic diameters 28.0nm (BSAnp1) and 52.0nm (BSAnp2) and corresponding ζ- potential value of∼−7.0 and −6.0mV, respectively. Interaction of DMC with BSA nanoparticles was investigated by UV–vis, fluorescence and CD spectroscopy. CD studies indicated significant changes in the secondary structure of BSA upon particle formation, as revealed by decrease in the helicity. The cellular uptake of DMC increased with increase in particle size and the toxicity of DMC loaded nanoparticles to A549 cells were found to be consistent with their cellular uptake. Between the two formulations studied, BSAnp2 provided enhanced cellular uptake and can be used as an effective delivery system for hydrophobic drugs like DMC.

Exploring thermodynamic parameters and the binding energetic of berberine chloride to bovine serum albumin (BSA): Spectroscopy, isothermal titration calorimetry and molecular docking techniques

The present study reveals a mechanistic approach of binding mode of berberine chloride with bovine serum albumin (BSA) using isothermal titration calorimetry (ITC), fluorescence quenching, circular dichorism (CD), UV–vis absorption spectroscopy and molecular docking techniqes. Thermodynamic profile clearly demonstrated that the binding was endothermic and spontaneous process. The major binding force involved in complexation of berberine chloride to BSA was hydrophobic interactions. Static quenching is the foremost fluorescence quenching mechanism between BSA and berberine chloride and there is one binding site in BSA for berberine chloride. Molecular docking and competitive binding probes denoted that the binding of berberine chloride to BSA primarily took place in site I i.e. sub-domain IIA. Additionally, the alteration of the BSA secondary structure in the presence of berberine chloride were confirmed by UV–vis, circular dichroism spectral analysis. All these results demonstrated that berberine chloride can bind to BSA.

Spectroscopic and molecular docking study on the structure-affinity relationship and mechanism in the interaction of genistein and its derivatives with bovine serum albumin.

In this paper, the interaction of genistein (GEN) and its four derivatives (GEN1-4) with bovine serum albumin (BSA) were investigated by ultraviolet-visible absorption spectra, fluorescence, synchronous fluorescence, three-dimensional fluorescence spectroscopy, circular dichroism and molecular docking techniques. The experimental results showed that the intrinsic fluorescence of BSA was quenched by genisteins and was due to the formation of a genisteins-BSA complex. The quenching constant, binding constants, binding sites, intermolecular distances and thermodynamic properties were calculated at 298K, 306K and 310K. Site marker competitive experiments indicated that the binding site of genisteins to BSA was mainly located in subdomain IIA. The conformational investigation showed that the presence of 0020 genisteins led to changes in the secondary structure of BSA and induced the slight unfolding of protein polypeptides, which confirmed some micro-environmental and conformational changes of BSA molecules. Furthermore, the binding affinity decreased in the order GEN1>GEN>GEN4>GEN3>GEN2, which revealed that different type and position of substituents of genistein significantly influenced the affinity of compounds to BSA. The number of hydroxyl groups on the ring A was the most important factor because increasing the hydroxyl groups on ring A clearly enhanced the binding affinity. However, trifluoromethylation did not much affect the affinity, alkylation, esterification and difluoromethylation slightly enhanced the binding affinity. The results obtained herein will provide valuable information about the pharmacokinetics at a molecular level and be a useful guideline for the further design of much more suitable genistein derivatives.

Good's Buffer Ionic Liquids as Relevant Phase-Forming Components of Self-Buffered Aqueous Biphasic Systems.

A series of new self-buffering ionic liquids (ILs) based on Good's buffers (GBs) anions and the tetrabutylphosphonium cation ([P4444]+) was here synthesized and characterized. The self-buffering behaviour of the GB-ILs was confirmed by measuring their protonation constants by potentiometry. Further, their ability to form aqueous biphasic systems with the biodegradable potassium citrate salt was evaluated, and further investigated for the extraction of proteins, using bovine serum albumin (BSA) as a model protein. If these ionic structures display self-buffering characteristics as well as a low toxicity towards the luminescent bacteria Vibrio fischeri, they were additionally found to be highly effective in the formation of ABS and in the extraction of BSA - extraction efficiencies of 100% to the IL-rich phase obtained in a single-step. The BSA secondary structure in the aqueous IL-rich solutions was evaluated through infrared spectroscopic studies revealing the protein-friendly nature of the synthesized ILs. Dynamic light scattering (DLS), "COnductor-like Screening MOdel for Real Solvents" (COSMO-RS), and molecular docking studies were finally carried out to better understand the main driving forces of the extraction process. The results suggest that van der Waals and hydrogen-bonding interactions are important driving forces of the protein migration towards the GB-IL-rich phase, while the molecular docking investigations demonstrated a stabilizing effect of the studied ILs over the protein.