Studies on the interaction between scopoletin and two serum albumins by spectroscopic methods

The interactions of scopoletin to bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated by spectroscopic methods. The fluorescence tests indicated that the formation mechanism of scopoletin–BSA/HSA complexes belonged to the static quenching. The displacement experiments suggested that scopoletin primarily bound to tryptophan residues of BSA/HSA within site I (subdomain IIA). The binding distance of scopoletin to BSA/HSA was 2.38/2.34 nm. The thermodynamic parameters (ΔG, ΔH and ΔS) calculated on the basis of different temperatures revealed that the binding of BSA–scopoletin was mainly depended on van der Waals interaction and hydrogen bond, and yet the binding of HSA–scopoletin was strongly relied on the hydrophobic interaction and electrostatic interaction. The results of synchronous fluorescence, 3D fluorescence, UV–vis absorption, and FT-IR spectra showed that the conformations of BSA and HSA altered with the addition of scopoletin. In addition, the effects of some common ions on the binding constants of scopoletin to proteins were also investigated.     

Study of interaction of butyl p-hydroxybenzoate with human serum albumin by molecular modeling and multi-spectroscopic method

Study of the interaction between butyl p-hydroxybenzoate (butoben) and human serum albumin (HSA) has been performed by molecular modeling and multi-spectroscopic method. The interaction mechanism was predicted through molecular modeling first, then the binding parameters were confirmed using a series of spectroscopic methods, including fluorescence spectroscopy, UV-visible absorbance spectroscopy, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. The thermodynamic parameters of the reaction, standard enthalpy ΔH⁰ and entropy ΔS⁰, have been calculated to be −29.52 kJ mol⁻¹ and −24.23 J mol⁻¹ K⁻¹, respectively, according to the Van’t Hoff equation, which suggests the van der Waals force and hydrogen bonds are the predominant intermolecular forces in stabilizing the butoben-HSA complex. Results obtained by spectroscopic methods are consistent with that of the molecular modeling study. In addition, alteration of secondary structure of HSA in the presence of butoben was evaluated using the data obtained from UV-visible absorbance, CD and FT-IR spectroscopies.     

The binding of 3-(p-bromophenyl)-5-methyl-thiohydantoin with human serum albumin: Investigation by fluorescence spectroscopy and molecular model

The binding of 3-(p-bromophenyl)-5-methyl-thiohydantoin (BPMT) with human serum albumin (HSA) was investigated by fluorescence spectroscopy in combination with UV absorption spectrum under physiological conditions. The intrinsic fluorescence of HSA was quenched by BPMT through static quenching mechanism and the fluorescence emission spectrum of HSA exhibited appreciable hypsochromic shift with increasing concentration of BPMT. The binding constants (K) of HSA with BPMT and the number binding sites (n) at different temperatures, thermodynamic parameter enthalpy changes (ΔH) and entropy changes (ΔS) of HSA-BPMT have been calculated according to the relevant fluorescence data, indicating that the hydrophobic interaction played a major role, which was consistent with the result of molecular modeling study.     

Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding.     

Interaction of human serum albumin with N-(4-ethoxyphenyl)-N′-(4-antipyrinyl) thiourea using spectroscopies and molecular modeling method

The interaction between N-(4-ethoxyphenyl)-N′-(4-antipyrinyl) thiourea (EPAT) and human serum albumin (HSA) was studied by fluorescence spectroscopy in combination with UV absorption spectroscopy. The intrinsic fluorescence of human serum albumin was quenched by EPAT through a static quenching procedure. The binding constants of EPAT with HSA were estimated according to the fluorescence quenching results at different temperatures. The binding distance was obtained and the binding force was suggested to be mainly hydrophobic force, which was in accordance with the study of molecular model. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented, and results were very satisfactory.     

Spectroscopic study on interaction between bisphenol A or its degraded solution under microwave irradiation in the presence of activated carbon and human serum albumin

In this study, the interaction between bisphenol A (BPA) or its degraded solution under microwave irradiation after their adsorption on activated carbon (AC/MW) and human serum albumin (HSA) was investigated by UV-vis and fluorescence spectroscopy techniques. The results showed that BPA could bind to HSA molecule, which could cause the stretch of peptide chains. Also, the degraded BPA solution with a few residues could still interact with HSA. Otherwise, the influences of pH and ionic strength on the interaction were estimated. The fluorescence quenching modes of HSA initiated by BPA at three temperatures (298, 310 and 315 K) were all obtained using Stern-Volmer and Lineweaver-Burk equations. The number of binding sites (n), binding constants (K_D) and energy transfer efficiency (E) were all calculated. The thermodynamic parameters (ΔH, ΔG and ΔS) and binding distances (r) were all measured at the three temperatures, respectively. Synchronous fluorescence spectroscopy was also carried out.     

Transport of a Cancer Chemopreventive Polyphenol,Resveratrol: Interaction with Serum Albumin and Hemoglobin

Resveratrol is a natural phytoalexin with pharmacologic effects on several human diseases: carcinogenesis, coronary heart disease and neurodegenerative disease. Due to its poor water solubility, resveratrol must be bound to proteins to keep it at a high concentration in serum. In our work, the bindings of resveratrol to plasma proteins, human serum albumin (HSA) and hemoglobin (Hb), have been investigated systematically by fluorescence quenching technique, synchronous fluorescence, UV–vis absorption spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling method. The fluorescence data show that the binding of resveratrol to HSA or Hb is a static quenching procedure and each protein has only one binding site for the drug. The binding constant of resveratrol to HSA is larger than that of resveratrol to Hb at corresponding temperature, which indicates that the affinity of HSA toward the drug is higher than that of Hb. The CD spectroscopy indicates that the secondary structures of the proteins are changed in the presence of resveratrol with the reduction of α-helices, which decreased about 18.75% for HSA and 9.43% for Hb at the drug to proteins molar ratio of 2. Thermodynamic analysis and molecular modeling suggest that hydrophobic interaction plays a major role in the binding of resveratrol to HSA, and hydrogen bonding is the mainly binding force in the binding of resveratrol to Hb. The study of molecular modeling shows that resveratrol is located in the hydrophobic cavity between subdomain IB and IIA of HSA (the entrance of site I), or located in the central cavity of Hb (partial to the subunit A).     

An alternate mode of binding of the polyphenol quercetin with serum albumins when complexed with Cu(II)

Polyphenols find wide use as antioxidants, cancer chemopreventive agents and metal chelators. The latter activity has proved interesting in many aspects. We have probed the binding characteristics of the polyphenol quercetin–Cu(II) complex with human serum albumin (HSA) and bovine serum albumin (BSA). Fluorescence studies reveal that the quercetin–Cu(II) complex can quench the fluorescence of the serum albumins. The binding constant (K_b) values are of the order of 10⁵ M^?1 which increased with rise in temperature in case of HSA and BSA interacting with the quercetin–Cu(II) complex. Displacement studies reveal that both the ligands bind to site 1 (subdomain IIA) of the serum albumins. However, thermodynamic parameters calculated from temperature dependent studies indicated that the mode of interaction of the complexes with the proteins differs. Both ΔH° and ΔS° were positive for the interaction of the quercetin–Cu(II) complex with both proteins but the value of ΔH° was negative in case of the interaction of quercetin with the proteins. This implies that after chelation with metal ions, the polyphenol alters its mode of interaction which could have varying implications on its other physicochemical activities.     

Complexes between C.I. Acid Orange 6 and human serum albumin, a multi-spectroscopic approach to investigate the binding behavior

The interaction mechanism of Acid Orange 6 (AO6) with human serum albumin (HSA) was investigated firstly by using fluorescence quenching technique, UV absorbance, circular dichroism (CD), Fourier transform infrared (FT-IR), three-dimensional fluorescence spectroscopy in combination with molecular modeling method under simulative physiological conditions. Fluorescence data indicated that there is a single class of binding sites between AO6 and HSA, and the alterations of HSA secondary structure in the presence of AO6 was confirmed by synchronous fluorescence, UV, CD, FT-IR and three-dimensional fluorescence spectra. The efficiency of fluorescence resonance energy transfer provided the binding distance (r) of 2.83 nm for AO6-HSA system. Furthermore, the thermodynamic parameters enthalpy change (ΔH⁰) and entropy change (ΔS⁰) for the reaction were calculated to be −5.77 kJ mol⁻¹ and 109.42 J mol⁻¹ K⁻¹, respectively, according to Van't Hoff equation, these data suggested that both hydrophobic forces and hydrogen bonding play a major role in the binding of AO6 to HSA, which agrees well with the results of molecular modeling study. Experimental results showed that the interaction between AO6 and HSA induced a conformational change of HSA, which was proved by the qualitative and quantitative analysis data of different spectroscopic techniques under simulative physiological conditions.     

光谱法和分子对接法研究盐酸氨溴索与人血清白蛋白的相互作用

在模拟生理条件下,用光谱法和分子对接技术研究了盐酸氨溴索与人血清白蛋白的相互作用.不同温度下的Steen-Volmer曲线和紫外-可见吸收光谱表明:盐酸氨溴索主要以动态猝灭方式使人血清白蛋白的荧光猝灭.由热力学数据确定了两者的主要作用力类型为疏水作用力.根据F(o)rster非辐射能量转移理论,得到给体-受体间的作用距...     

Spectrophotometric studies on the interaction between pazufloxacin mesilate and human serum albumin or lysozyme

The binding of pazufloxacin mesilate (PZFX) to human serum albumin (HSA) or lysozyme (Lys) was investigated using spectrophotometric techniques. The intrinsic fluorescence of both HSA and Lys was strongly quenched by PZFX. This effect was rationalized in terms of a static quenching procedure. Negative values of ΔH⁰ and ΔS⁰ for the formation of PZFX-HSA or PZFX-Lys complex implied that both hydrogen bonds and hydrophobic interactions might play a significant role in PZFX binding to HSA or Lys. The binding distances deduced from the efficiency of energy transfer were 4.04 and 3.21 nm for PZFX-HSA and PZFX-Lys systems, respectively. Furthermore, association constants and binding mechanism were successfully derived from the synchronous fluorescence spectra. Circular dichroism (CD) spectra and UV/vis detections supported a change in the secondary structure of proteins caused by the interaction of PZFX with HSA or Lys.     

Effect of temperature and pH on interaction between bovine serum albumin and cetylpyridinium bromide: Fluorescence spectroscopic approach

The fluorescence spectroscopic technique has been efficiently employed to investigate the interaction between bovine serum albumin (BSA) and cetylpyridinium bromide (CPB) under different pH and temperature conditions. The binding constant, number of binding sites, thermodynamic parameters such as ΔG, ΔH, ΔS, and nature of binding forces between BSA and CPB were obtained by measuring the steady state fluorescence quenching of BSA by CPB. The experimental results showed that the fluorescence quenching of BSA by CPB was a result of the formation of CPB-BSA complex. The static quenching was confirmed from the Stern-Volmer quenching constant at different temperatures. The effect of CPB on the conformation of BSA was analyzed using synchronous and three-dimensional fluorescence spectroscopy. pH dependence complex formation between BSA-CPB is due to the interaction between cationic side chain of CPB and the net charge developed on BSA. The distance ‘r’ between BSA and CPB was obtained according to the fluorescence resonance energy transfer.     

多西环素与人血清白蛋白相互作用的研究

将经典光谱法与内滤光校正、取代实验和分子对接等技术相结合,较全面地研究了多西环素(DC)与人血清白蛋白(HSA)之间的相互作用。通过荧光猝灭实验测得在298和310 K时,DC与HSA的结合常数分别为2.73×105和0.74×105 L·mol-1,二者有一个结合位点,表明DC与HSA间具有较强的结合作用,属于静态猝灭。根据Vant’Hoff公式计算的热力学参数(ΔH=-83.55 kJ·mol-1,ΔS=-176.31 J·mol-1·K-1)表明,两者间主要作用力为氢键和范德华力。根据Fster能量转移定律求得DC与HSA的Trp-214之间的结合距离为4.98 nm。取代反应结果表明,DC键合在HSA的亚域IIA内。三维荧光光谱结果显示,DC使HSA疏水性增加,改变了HSA的构象。DC与HSA作用前后红外光谱二级结构的定量分析结果表明,DC能使HSA结构松散。分子对接技术进一步表明DC通过氢键和范德华力等键合在HSA的亚域IIA疏水腔中,结合距离与光谱法计算结果相近。实验结果为研究药物小分子与人血清白蛋白的相互作用提供了理论依据和可靠数据。     

Interaction of the flavonoid hesperidin with bovine serum albumin: A fluorescence quenching study

The interaction between the flavonoid hesperidin and bovine serum albumin (BSA) was investigated by fluorescence and UV/Vis absorption spectroscopy. The results revealed that hesperidin caused the fluorescence quenching of BSA through a static quenching procedure. The hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The binding site number n, and apparent binding constant K_A, corresponding thermodynamic parameters ΔG^o, ΔH^o, ΔS^o at different temperatures were calculated. The distance r between donor (BSA) and acceptor (hesperidin) was obtained according to fluorescence resonance energy transfer. The effect of Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ on the binding constants between hesperidin and BSA were studied. The effect of hesperidin on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy and UV/Vis absorption spectroscopy.     

Study on the interaction between methyl blue and human serum albumin by fluorescence spectrometry

The interaction of methyl blue (MB) with human serum albumin (HSA) was studied by fluorescence and absorption spectroscopy. The intrinsic fluorescence of HSA was quenched by MB, which was rationalized in terms of the static quenching mechanism. The number of binding sites and the apparent binding constants at different temperatures were obtained from the Stern-Volmer analysis of the fluorescence quenching data. The thermodynamic parameters determined by the van’t Hoff analysis of the binding constants (ΔH°=39.8 kJ mol⁻¹ and ΔS°=239 J mol⁻¹ K⁻¹) clearly indicate that binding is absolutely entropy-driven and enthalpically disfavored The efficiency of energy transfer and the distance between the donor (HSA) and the acceptor (MB) were calculated as 60% and 2.06 nm from the Förster theory of non-radiation energy transfer.     

Binding studies of costunolide and dehydrocostuslactone with HSA by spectroscopy and atomic force microscopy

Human serum albumin (HSA), a major plasma protein and plasma-derived therapeutic, interacts with a wide variety of drugs and native plasma metabolites. In this study the interactions of costunolide (CE) and dehydrocostuslactone (DE) with HSA were investigated by molecule modeling, atomic force microscopy (AFM), and different optical techniques. In the mechanism discussion, it was proved that fluorescence quenching of HSA by both of the drugs is a result of the formation of drug-HSA complexes. Binding parameters for the reactions were determined according to the Stern-Volmer equation and static quenching. The results of thermodynamic parameters ΔG⁰, ΔH⁰, and ΔS⁰ at different temperatures indicated that hydrogen bonding interactions play a major role in the drug-HSA associations process. The binding properties were further studied by quantitative analysis of CD, FTIR, and Raman spectra. Furthermore, AFM results showed that the dimension of HSA molecules became more swollen after binding with the drugs.     

Determining the binding affinity and binding site of bensulfuron-methyl to human serum albumin by quenching of the intrinsic tryptophan fluorescence

Bensulfuron-methyl (BM) is a highly active sulfonylurea herbicide for use on paddy rice. Steady state fluorescence, UV/vis absorption, circular dichroism (CD), time-resolved fluorescence and molecular modeling methods have been exploited to determine the binding affinity and binding site of BM to human serum albumin (HSA). From the synchronous fluorescence, UV/vis, CD and three-dimensional fluorescence spectra, it was evident that the interaction between BM and HSA induced a conformational change in the protein. Steady state and time-resolved fluorescence data illustrates that the fluorescence quenching of HSA by BM was the formation of HSA-BM complex at 1:1 molar ratio. Site marker competitive experiments demonstrated that the binding of BM to HSA primarily took place in subdomain IIIA (Sudlow’s site II), this corroborates the hydrophobic probe ANS displacement and molecular modeling results. Thermodynamic analysis displays hydrophobic, electrostatic and hydrogen bonds interactions are the major acting forces in stabilizing the HSA-BM complex.     

Fluorescence spectrometric studies on the binding of puerarin to human serum albumin using warfarin, ibuprofen and digitoxin as site markers with the aid of chemometrics

The interaction of puerarin with human serum albumin (HSA) in pH 7.4 Tris-HCl buffer has been investigated by fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. The results revealed the presence of static type of quenching mechanism in the binding of puerarin to HSA. The association constants (K_a) between puerarin and HSA were obtained according to Modified Stern-Volmer equation. The calculated thermodynamic parameters indicated that the binding of puerarin to HSA was driven mainly by hydrophobic interaction. The competitive experiments of site markers suggested that the binding site of puerarin to HSA was located in the region of subdomain IIA (sudlow site I). Further, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way synchronous fluorescence spectra data of the competitive interaction between puerarin and warfarin with HSA. The concentration information for the three reaction components, warfarin, puerarin and puerarin−HSA, in the system at equilibrium was obtained simultaneously. The PARAFAC analysis indicated that puerarin in the puerarin-HSA complex was displaced by warfarin, which confirmed the binding site of puerarin to HSA was located in site I. Moreover, the results of CD and FT-IR spectra demonstrated that the secondary structure of HSA was changed in the presence of puerarin.     

Interaction of fisetin with human serum albumin by fluorescence, circular dichroism spectroscopy and DFT calculations: binding parameters and conformational changes

The interaction between fisetin, an antioxidant and neuroprotective flavonoid, and human serum albumin (HSA) is investigated by means of fluorescence (steady-state, synchronous, time-resolved) and circular dichroism (CD) spectroscopy. The formation of a 1:1 complex with a constant of about 10⁵ M⁻¹ was evidenced. Förster's resonance energy transfer and competitive binding with site markers warfarin and ibuprofen were considered and discussed. Changes in the CD band of HSA indicate a decrease in the α-helix content upon binding. An induced CD signal for bound fisetin was observed and rationalized in terms of density functional theory calculations.     

Interactions between 4-(2-dimethylaminoethyloxy)-N-octadecyl-1,8-naphthalimide and serum albumins: Investigation by spectroscopic approach

A novel 4-(2-dimethylaminoethyloxy)-N-octadecyl-1,8-naphthalimide (DON) has been synthesized as a spectrofluorimetric probe for the determination of proteins. Photophysics of DON in different solvents has been delineated in this paper. Progressive redshift with polarity of solvents in emission and absorption spectra hints at intramolecular charge transfer. The interactions of DON with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)) were studied by fluorescence and absorption spectroscopy. Fluorescence data revealed that the quenching of HSA/BSA by DON were static quenching and the DON–HSA/BSA complexes were formed. The binding constant (K_b) for HSA and was found to be 8.44×10^?4 and 60.26×10^?4 M^?1 and the number of binding sites (n) were 1.00 and 1.40, respectively. The thermodynamic parameters, ΔH and ΔS, for the DON–HSA system was calculated to be ?14.83 kJ mol^?1 and 23.61 J mol^?1 K^?1, indicating the hydrogen bonds and hydrophobic interactions were the dominant intermolecular force. ΔH and ΔS for the binding of DON with BSA was ?60.08 kJ mol^?1 and ?90.7441 mol^?1 K^?1, suggesting the hydrogen bonds and van der Waals force played the main role in the interaction. The results of displacement experiments showed that DON bound HSA/BSA occurred at the Trp-214 proximity, located in subdomain IIA of the serum albumin structure (the warfarin binding pocket). The effect of DON on the conformation of HSA was also analyzed by synchronous and three-dimensional fluorescence spectra. The fluorescence of DON could be quenched by HSA, based on which, a fluorometric method for the determination of microamount protein using DON in the medium of HCl?Tris buffer solution (pH=7.4) was developed. The linear range of the calibration curves was 0.1–10.0 μM for HSA, 0.1–11.2 μM for BSA and 0.2–9.7 μM for egg albumin (EA). The detection limit (3σ) for HSA was 1.12×10^?10 M, for BSA it was 0.92×10^?10 M and for EA it was 4.33×10^?10 M. The effect of metal cations on the fluorescence spectra of DON in ethanol was also investigated. The method has been applied to detect the total proteins in human serum samples and the results were in good agreement with those reported by the hospital.