Studies on the interaction between benzidine and bovine serum albumin by spectroscopic methods

The interaction between bovine serum albumin (BSA) and benzidine (BD) in aqueous solution was investigated by fluorescence spectroscopy, circular dichroism (CD) spectra and UV–Vis spectroscopy, as well as resonance light scattering spectroscopy (RLS). It was proved from fluorescence spectra that the fluorescence quenching of BSA by BD was a result of the formation of BD–BSA complex, and the binding constants (K _a) were determined according to the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be ?34.11 kJ mol^?1 and ?25.89 J mol^?1 K^?1, respectively, which implied that van der Waals force and hydrogen bond played predominant roles in the binding process. The addition of increasing BD to BSA solution caused the gradual enhancement in RLS intensity, exhibiting the forming of the aggregate. Moreover, the competitive experiments of site markers suggested that the binding site of BD to BSA was located in the region of subdomain IIA (sudlow site I). The distance (r) between the donor (BSA) and the acceptor (BD) was 4.44 nm based on the Förster theory of non–radioactive energy transfer. The results of synchronous fluorescence and CD spectra demonstrated the microenvironment and the secondary conformation of BSA were changed.     

Spectroscopic Investigation of the Interaction Between Copper (II) 2-oxo-propionic Acid Salicyloyl Hydrazone Complex and Bovine Serum Albumin

The interaction between copper (II) 2-oxo-propionic acid salicyloyl hydrazone (Cu^IIL) and bovine serum albumin (BSA) under physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-Vis absorption, and circular dichroism spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by Cu^IIL was the result of the formation of the BSA–Cu^IIL complex. The apparent binding constants (K _a) between Cu^IIL and BSA at four different temperatures were obtained according to the modified Stern–Volmer equation. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), for the reaction were calculated to be ?80.79 kJ mol^?1 and ?175.48 J mol^?1 K^?1 according to van’t Hoff equation. The results indicated that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. The binding distance (r) between Cu^IIL and the tryptophan residue of BSA was obtained to be 4.1 nm according to Förster’s nonradioactive energy transfer theory. The conformational investigation showed that the application of Cu^IIL increased the hydrophobicity of amino acid residues and decreased the α-helical content of BSA (from 62.71% to 37.31%), which confirmed some microenvironmental and conformational changes of BSA molecules.     

Combined spectroscopies and molecular docking approach to characterizing the binding interaction of enalapril with bovine serum albumin

The binding interaction between bovine serum albumin (BSA) and enalapril (ENPL) at the imitated physiological conditions (pH = 7.4) was investigated using UV–vis absorption spectroscopy (UV–vis), fluorescence emission spectroscopy (FES), synchronous fluorescence spectroscopy (SFS), Fourier transform infrared spectroscopy (FT‐IR), circular dichroism (CD) and molecular docking methods. It can be deduced from the experimental results from the steady‐state fluorescence spectroscopic titration that the intrinsic BSA fluorescence quenching mechanism induced by ENPL is static quenching, based on the decrease in the BSA quenching constants in the presence of ENPL with increase in temperature and BSA quenching rates >10¹⁰ L mol^?1 sec^?1. This result indicates that the ENPL–BSA complex is formed through an intermolecular interaction of ENPL with BSA. The main bonding forces for interaction of BSA and ENPL are van der Waal's forces and hydrogen bonding interaction based on negative values of Gibbs free energy change (ΔG ⁰), enthalpic change (ΔH ⁰) and entropic change (ΔS ⁰). The binding of ENPL with BSA is an enthalpy‐driven process due to |ΔH °| > |T ΔS °| in the binding process. The results of competitive binding experiments and molecular docking confirm that ENPL binds in BSA sub‐domain IIA (site I) and results in a slight change in BSA conformation, but BSA still retains its α‐helical secondary structure.     

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi‐spectroscopic and computational investigation

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH 7.4) by multi‐spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, K_b, value was found to lie between 2.69 × 10³ and 9.55 × 10³ M^?1 at experimental temperatures (293, 298, 303, and 308 K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub‐domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH⁰) and entropy change (ΔS⁰) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril‐BSA interaction, and 8‐anilino‐1‐naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3‐dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction.     

Photochemical studies on the binding of an organic fluoride to bovine serum albumin

The interaction of fipronil (FPN), a pesticide containing fluorine, to bovine serum albumin (BSA) was studied by spectroscopy including fluorescence spectra, UV–Visible absorption, scattering spectra, circular dichroism (CD) spectra, synchronous and three-dimensional fluorescence spectra. The number of binding sites n and observed binding constant Kb was measured by fluorescence quenching method. The thermodynamic parameters ΔH, ΔG, ΔS at different temperatures were calculated and the results indicate that hydrophobic forces played major role in the reaction. The distance r between donor (BSA) and acceptor (FPN) was obtained according to the Förster theory of non-radiation energy transfer. The structural change of BSA molecules with addition of FPN was analyzed and the results may be helpful to biologists, chemists and therapeutists.     

Probing the behavior of bovine serum albumin upon binding to atenolol: insights from spectroscopic and molecular docking approaches

Molecular interaction of atenolol, a selective β₁ receptor antagonist with the major carrier protein, bovine serum albumin (BSA), was investigated under imitated physiological conditions (pH 7.4) by means of fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular modeling studies. The steady-state fluorescence spectra manifested that static type, due to formation of the atenolol-BSA complex, was the dominant mechanism for fluorescence quenching. The characteristic information about the binding interaction of atenolol with BSA in terms of binding constant (K_b) were determined by the UV–vis absorption titration, and were found to be in the order of 10³ M^?1 at different temperatures, indicating the existence of a weak binding in this system. Thermodynamic analysis revealed that the binding process was primarily mediated by van der Waals force and hydrogen bonds due to the negative sign for enthalpy change (ΔH⁰), entropy change (ΔS⁰). The molecular docking results elucidated that atenolol preferred binding on the site II of BSA according to the findings observed in competitive binding experiments. Moreover, via alterations in synchronous fluorescence, three-dimensional fluorescence and FT-IR spectral properties, it was concluded that atenolol could arouse slight configurational and micro-environmental changes of BSA.     

Chemicobiological effects of herbicide MCPA-Na on plasma proteins

Under physiological conditions, the potential hematological toxicity of herbicide 2-methyl-4-chlorophenoxyacetic acid sodium (MCPA-Na) was discussed by fluorescence probe technology and spectroscopy methods including three-dimensional (3D) fluorescence, UV absorption and circular dichroism (CD) spectra. In vitro, MCPA-Na bound with bovine serum albumin (BSA) and formed new complex at ground state by electrostatic force and hydrogen bond. During the process, non-radiation energy transfer from BSA to MCPA-Na occurred and the distance r between donor and acceptor was obtained based on Förster theory. The binding site was investigated by fluorescence probe method and the results implied MCPA-Na was absorbed on domain II of BSA molecule. The enthalpy change (ΔH ^θ), Gibbs free energy change (ΔG ^θ) and entropy change (ΔS ^θ) were calculated at four different temperatures according to Van’t Hoff isobar equation and Gibbs–Helmholtz equation. Negative value of ΔG ^θ indicated the process of binding was a spontaneous and irreversible process, which gave a broad hint that MCPA-Na was likely to be poisonous. CD spectra exhibited significant changes of secondary structures in BSA molecule and three-dimensional fluorescence spectra indicated the tryptophan residue in BSA was placed in a less hydrophobic environment, which presented additional evidence to caution the danger of MCPA-Na residue in food. Meanwhile, the mechanism and geometry of the binding was analyzed at molecular level.     

The investigation of the interaction between ribavirin and bovine serum albumin by spectroscopic methods

The interaction between ribavirin (RIB) with bovine serum albumin (BSA) has been investigated by fluorescence quenching technique in combination with UV–vis absorption and circular dichroism (CD) spectroscopies under the simulative physiological conditions. The quenching of BSA fluorescence by RIB was found to be a result of the formation of RIB–BSA complex. The binding constants and the number of binding sites were calculated at three different temperatures. The values of thermodynamic parameters ?H, ?S, ?G at different temperatures indicate that hydrophobic and hydrogen bonds played important roles for RIB–BSA association. The binding distance r was obtained according to the theory of FÖrster’s non–radiation energy transfer. The displacement experiments was performed for identifying the location of the binding site of RIB on BSA. The effects of common ions on the binding constant of RIB and BSA were also examined. Finally, the conformational changes of BSA in the presence of RIB were also analyzed by CD spectra and Synchronous fluorescence spectra.     

Fluorescence spectroscopic studies on the interaction of Gemini surfactant 14‐6‐14 with bovine serum albumin

The interaction of the cationic Gemini surfactant hexamethylene‐1,3‐bis (tetradecyldimethylammonium bromide) (14‐6‐14) with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectra and three‐dimensional (3D) fluorescence spectra. The Stern–Volmer quenching constants K_SV and the corresponding thermodynamic parameters ΔH, ΔG and ΔS have been estimated by the fluorescence quenching method. The results indicated that hydrophobic forces were the predominant intermolecular forces between BSA and the surfactant. Competitive experiments and the number of binding sites calculation show that 14‐6‐14 can be inserted in site‐II (in subdomain IIIA) of BSA. The effect of 14‐6‐14 on the conformation of BSA was evaluated by synchronous fluorescence spectroscopy and 3D fluorescence spectral methods. The results show that the conformation of BSA was changed dramatically in the presence of 14‐6‐14, by binding to the Trp and Try residues of BSA. The investigation provides interaction between BSA and 14‐6‐14 as a model for molecular design and industrial research. Copyright © 2011 John Wiley & Sons, Ltd.     

Underlying molecular interaction of bovine serum albumin and linezolid: a biophysical outlook

Linezolid, one of the reserve antibiotic of oxazolidinone class has wide range of antimicrobial activity. Here we have conducted a fundamental study concerning the dynamics of its interaction with bovine serum albumin (BSA), and the post binding modification of the later by employing different spectroscopic (absorption, fluorescence and circular dichroism (CD) spectroscopy) and molecular docking tools. Gradual quenching of the tryptophan (Trp) fluorescence upon addition of linezolid to BSA confirms their interaction. Analysis of fluorescence quenching at different temperature indicates that the interaction is made by static complex formation and the BSA has one binding site for the drug. The negative Gibbs energy change (ΔG⁰), and positive values of enthalpy change (ΔH⁰) and entropy change (ΔS⁰) strongly suggest that it is an entropy driven spontaneous and endothermic reaction. The reaction involves hydrophobic pocket of the protein, which is further stabilized by hydrogen bonding and electrostatic interactions as evidenced from 8-anilino-1-napthalene sulfonic acid, sucrose and NaCl binding studies. These findings also support the molecular docking study using AutoDock 4.2. The influence of this interaction on the secondary structure of the protein is negligible as evidenced by CD spectroscopy. So, from these findings, we conclude that linezolid interacts with BSA in 1:1 ratio through hydrophobic, hydrogen bonding and ionic interactions, and this may not affect the secondary structure of the protein.     

Fluorometric probing on the binding of hematoxylin to serum albumin

The binding of a cell nucleus stain, hematoxylin (HTL), to bovine serum albumin (BSA) was studied by spectroscopy including fluorescence spectra, UV–Visible absorption, circular dichroism (CD) spectra, synchronous and three-dimensional fluorescence spectra. The results indicated that the binding had led to static fluorescence quenching, with non-radiation energy transfer happening within single molecule. The observed binding constant was calculated to be 10^5.588 l mol^?1 at 311 K and one binding site had formed. The thermodynamic parameters of the interaction complied with ΔG ^θ < 0, ΔH ^θ < 0, ΔS ^θ < 0 and the results indicate that hydrogen bonds played major role in the reaction. The distance r between donor (BSA) and acceptor (HTL) was obtained according to the Förster theory of non-radiation energy transfer. The structural change of BSA molecules with addition of HTL was analyzed and the optimized geometry of HTL–BSA was investigated by fluorescence probe method.     

Synthesis of Morin–Zinc(II) Complex and its Interaction with Serum Albumin

A morin–zinc(II) complex (MZ) was synthesized and its interaction with bovine serum albumin (BSA) were studied by molecular spectroscopy including fluorescence emission spectra, UV-visible spectra, circular dichroism (CD) spectra, three-dimensional fluorescence spectra, and synchronous fluorescence spectra. The interaction mechanism of BSA and MZ was discussed by fluorescence quenching method and Förster non-radiation energy transfer theory. The thermodynamic parameters ΔH ^θ, ΔG ^θ, ΔS ^θ at different temperatures were calculated and the results indicate the interaction is an exothermic as well as entropy-driven process. Hydrogen bond forces played the most important role in the reaction. The fluorescence probe experiment showed that the binding site of MZ is in subdomain IIA of BSA and the distance between BSA and MZ is 3.17 nm at normal body temperature. The conformation changes of BSA in presence of MZ were investigated by CD spectra and three-dimensional fluorescence spectra.     

Synthesis of an octupolar compound and its biological effects on serum albumin

A special rigid planar structural octupolar molecule titled 2,4,6-tris(p-methylstyryl)-s-triazine (TMT) was synthesized and its interaction with bovine serum albumin (BSA) were studied by molecular spectroscopy. The quenching mechanism of fluorescence of BSA by TMT was discussed. The thermodynamic parameters ΔH ^θ, ΔG ^θ, ΔS ^θ at different temperatures were calculated and the results indicate hydrogen bond forces played major role in the reaction and the reaction was mainly enthalpy-driven. The distance r between donor (BSA) and acceptor (TMT) was obtained according to Förster theory of non-radiation energy transfer. Circular dichroism (CD) spectra, synchronous and three-dimensional fluorescence spectra were used to investigate the structural change of BSA molecules with addition of TMT, the result indicates that the α-helical structures of BSA molecules reduced in the presence of TMT. Sketch map of the interaction process was analyzed at molecular level.     

Spectroscopic and molecular docking studies on the interaction between N‐acetyl cysteine and bovine serum albumin

The interaction between N‐acetyl cysteine (NAC) and bovine serum albumin (BSA) was investigated by UV–vis, fluorescence spectroscopy, and molecular docking methods. Fluorescence study at three different temperatures indicated that the fluorescence intensity of BSA was reduced upon the addition of NAC by the static quenching mechanism. Binding constant (K_b) and the number of binding sites (n) were determined. The binding constant for the interaction of NAC and BSA was in the order of 10³ M^?1, and the number of binding sites was obtained to be equal to 1. Enthalpy (ΔH), entropy (ΔS), and Gibb's free energy (ΔG) as thermodynamic values were also achieved by van't Hoff equation. Hydrogen bonding and van der Waals force were the major intermolecular forces in the interaction process and it was spontaneous. Finally, the binding mode and the binding sites were clarified using molecular docking which were in good agreement with the results of spectroscopy experiments. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 638–645, 2015.     

Fluorescent bovine serum albumin interacting with the antitussive quencher dextromethorphan: a spectroscopic insight

The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV–vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, K_A, are 7.159 × 10³, 9.398 × 10³ and 16.101 × 10³ L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV–vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM. Copyright © 2015 John Wiley & Sons, Ltd.     

Insights into the binding interaction between copper ferrite nanoparticles and bovine serum albumin: An effect on protein conformation and activity

The binding affinity between bovine serum albumin (BSA) and copper ferrite (CuFe₂O₄) nanoparticles in terms of conformation, stability and activity of protein was studied using various spectroscopic methods. The quenching involved in BSA–CuFe₂O₄ NP interaction was static quenching as analysed by different techniques (steady‐state and time‐resolved fluorescence along with temperature‐dependent fluorescence measurements). Among all types of possible interactions, it was revealed that the major binding forces were van der Waals interaction and hydrogen bonding, which were explored from negative values of enthalpy change (?H = ?193.85 kJ mol^?1) and entropy change (?S = ?588.88 J mol^?1 K^?1). Additionally, synchronous, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy measurements confirmed the conformational changes in BSA upon the addition of CuFe₂O₄ NP. Furthermore, thermal denaturation observations were consistent with the circular dichroism results. The interaction of CuFe₂O₄ NP with BSA decreased the esterase activity in the BSA assay, revealing the affinity of copper ferrite towards the active site of BSA.     

Exploration of interaction of canthaxanthin with human serum albumin by spectroscopic and molecular simulation methods

The interaction between the food colorant canthaxanthin (CA) and human serum albumin (HSA) in aqueous solution was explored by using fluorescence spectroscopy, three‐dimensional fluorescence spectra, synchronous fluorescence spectra, UV–vis absorbance spectroscopy, circular dichroism (CD) spectra and molecular docking methods. The thermodynamic parameters calculated from fluorescence spectra data showed that CA could result in the HSA fluorescence quenching. From the K_SV change with the temperature dependence, it was concluded that HSA fluorescence quenching triggered by CA is the static quenching and the number of binding sites is one. Furthermore, the secondary structure of HSA was changed with the addition of CA based on the results of synchronous fluorescence, three‐dimensional fluorescence and CD spectra. Hydrogen bonds and van der Waals forces played key roles in the binding process of CA with HSA, which can be obtained from negative standard enthalpy (ΔH) and negative standard entropy (ΔS). Furthermore, the conclusions were certified by molecular docking studies and the binding mode was further analyzed with Discovery Studio. These conclusions can highlight the potential of the interaction mechanism of food additives and HSA.     

Interaction between felodipine and bovine serum albumin: fluorescence quenching study

The fluorescence quenching spectrum of bovine serum albumin (BSA) was investigated in the presence of felodipine (FLD) by spectroscopic methods including fluorescence spectroscopy and UV–Vis absorption spectroscopy. Stern–Volmer quenching was successfully applied and the corresponding thermodynamic parameters, namely enthalpy change (ΔH), free energy change (ΔG) and entropy change (ΔS) at different temperatures (304, 314 and 324 K) were calculated according to the Van't Hoff relation. This revealed that the hydrophobic interaction plays a major role in stabilizing the complex. The fluorescence spectrum of BSA was studied in presence of various concentrations of SDS surfactant. The distance (r) between donor (BSA) and acceptor (FLD) was obtained according to fluorescence resonance energy transfer (FRET). The synchronous fluorescence spectroscopy was used to investigate the effect of FLD on BSA molecule. The result shows that the conformation of BSA was changed in the presence of felodipine. Copyright © 2009 John Wiley & Sons, Ltd.     

Study on the interaction between anti‐tuberculosis drug ethambutol and bovine serum albumin: multispectroscopic and cyclic voltammetric approaches

The binding of bovine serum albumin (BSA) to ethambutol (EMB) was investigated using spectroscopic methods, viz., fluorescence, Fourier transform infrared (FTIR), ultraviolet (UV)/vis absorption and cyclic voltammetry techniques. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of serum albumin by EMB is static, which was also confirmed by lifetime measurements. The number of binding sites, n, and binding constant, K, were obtained at various temperatures. The distance, r, between EMB and the protein was evaluated according to the Förster energy transfer theory. Based on displacement experiments using site probes, viz., warfarin, ibuprofen and digitoxin, the site of binding of EMB in BSA was proposed to be Sudlow's site I. The effect of EMB on the conformation of BSA was analyzed by using synchronous fluorescence spectra (SFS) and 3D fluorescence spectra. The results of fluorescence, UV/vis absorption and FTIR spectra showed that the conformation of BSA was changed in the presence of EMB. The thermodynamic parameters including enthalpy change (ΔH⁰), entropy change (ΔS⁰) and free energy change (ΔG⁰) for BSA–EMB were calculated according to the van't Hoff equation and are discussed.     

Using resonance light scattering and UV/vis absorption spectroscopy to study the interaction between gliclazide and bovine serum albumin

At different temperatures (298, 310 and 318 K), the interaction between gliclazide and bovine serum albumin (BSA) was investigated using fluorescence quenching spectroscopy, resonance light scattering spectroscopy and UV/vis absorption spectroscopy. The first method studied changes in the fluorescence of BSA on addition of gliclazide, and the latter two methods studied the spectral change in gliclazide while BSA was being added. The results indicated that the quenching mechanism between BSA and gliclazide was static. The binding constant (K_a), number of binding sites (n), thermodynamic parameters, binding forces and Hill's coefficient were calculated at three temperatures. Values for the binding constant obtained using resonance light scattering and UV/vis absorption spectroscopy were much greater than those obtained from fluorescence quenching spectroscopy, indicating that methods monitoring gliclazide were more accurate and reasonable. In addition, the results suggest that other residues are involved in the reaction and the mode ‘point to surface’ existed in the interaction between BSA and gliclazide. Copyright © 2015 John Wiley & Sons, Ltd.