Competitive binding of small molecules with biopolymers: a fluorescence spectroscopy and chemometrics study of the interaction of aspirin and ibuprofen with BSA

The interaction of aspirin and ibuprofen with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both aspirin and ibuprofen quenched the intrinsic fluorescence of BSA and the binding ratios obtained were 2 : 1 for aspirin-BSA and 3 : 1 for ibuprofen-BSA interactions, respectively. The thermodynamic parameters (ΔH, ΔS and ΔG) obtained from the fluorescence spectroscopy data showed that the binding of aspirin to BSA involved van der Waals interactions and hydrogen bonds. Competitive experiments using warfarin and diazepam as site markers indicated that aspirin was mainly located in the hydrophobic pocket of site II of the protein as well as to a small extent in site I. Furthermore, the competitive interaction of the aspirin and ibuprofen with BSA, which was studied with the use of the three-way excitation-emission fluorescence spectra and a parallel factor analysis (PARAFAC) chemometrics method, showed that the competitive effect of ibuprofen was stronger than that of aspirin, i.e. the former molecule replaced the aspirin from the aspirin-BSA complex.     

Spectroscopic study on the inherent binding information of cationic perfluorinated surfactant with bovine serum albumin

UV-vis, FT-IR, fluorescence and synchronous fluorescence spectra are applied to discuss the inherent binding information of model protein bovine serum albumin (BSA) with perfluorinated surfactant trimethyl-1-propanaminium iodide (FC-134). According to the results analyzed from Stern-Volmer equation, FC-134 can quench the fluorescence intensity of BSA via a dynamic quenching mechanism with complex formation. The thermodynamic parameters are calculated, revealing that hydrophobic force is the main interaction driven force. The binding constants and number of binding sites are also obtained. With the aid of site markers—warfarin and ibuprofen, we first report that FC-134 primarily binds to tryptophan residue Trp-214 of BSA within site I (sub-domain IIA).     

Spectrometric and voltammetric studies of the interaction between quercetin and bovine serum albumin using warfarin as site marker with the aid of chemometrics

The interaction of quercetin, which is a bioflavonoid, with bovine serum albumin (BSA) was investigated under pseudo-physiological conditions by the application of UV-vis spectrometry, spectrofluorimetry and cyclic voltammetry (CV). These studies indicated a cooperative interaction between the quercetin-BSA complex and warfarin, which produced a ternary complex, quercetin-BSA-warfarin. It was found that both quercetin and warfarin were located in site I. However, the spectra of these three components overlapped and the chemometrics method - multivariate curve resolution-alternating least squares (MCR-ALS) was applied to resolve the spectra. The resolved spectra of quercetin-BSA and warfarin agreed well with their measured spectra, and importantly, the spectrum of the quercetin-BSA-warfarin complex was extracted. These results allowed the rationalization of the behaviour of the overlapping spectra. At lower concentrations ([warfarin]<1x10(-5) mol L(-1)), most of the site marker reacted with the quercetin-BSA, but free warfarin was present at higher concentrations. Interestingly, the ratio between quercetin-BSA and warfarin was found to be 1:2, suggesting a quercetin-BSA-(warfarin)(2) complex, and the estimated equilibrium constant was 1.4x10(11)M(-2). The results suggest that at low concentrations, warfarin binds at the high-affinity sites (HAS), while low-affinity binding sites (LAS) are occupied at higher concentrations.     

Spectrophotometric study of the interaction between chlorotetracycline and bovine serum albumin using Eosin Y as site marker with the aid of chemometrics

Interaction of chlorotetracycline (CTC) with bovine serum albumin (BSA) was investigated under simulated physiological conditions by spectroscopy with the aid of multivariate curve resolution-alternating least squares (MCR-ALS). Eosin Y was selected as an alternative site I marker on the BSA to study the above molecular interaction. The binding of Eosin Y and CTC to BSA showed that CTC was displaced from CTC-BSA complex by Eosin Y, and Eosin Y-BSA complex was formed. However, the recorded fluorescence spectra of Eosin Y and Eosin Y-BSA overlapped and MCR-ALS was applied to resolve the two-way fluorescence spectra. From the resolved equilibrium concentration profiles, it was observed that Eosin Y competed with CTC in the binding process with BSA; it was also shown that the binding site of CTC on BSA was site I, and this was further confirmed by the fluorescence polarization method. Compared with some common site I markers for BSA, the fluorescence and UV-vis spectral shapes of the Eosin Y-BSA complex were quite different from that of Eosin Y, and this feature facilitated the investigation of the small molecule-BSA interaction.     

光谱法研究11-羟基喜树碱与牛血清白蛋白的相互作用

在生理pH条件下用荧光光谱法和紫外光谱法结合化学计量学来研究10-羟基喜树碱(10-HCPT)与牛血清白蛋白(BSA)的相互作用,结果发现,10-HCPT对BSA的内源荧光有静态猝灭作用。以华法林作为标记药物,采用三维荧光技术,分别对竞争实验过程进行扫描,进而用平行因子法(PARAFAC)处理所得三维数据,并根据蛋白质结合位上药物的置换作用确定了10-HCPT的结合位置是BSA的site I。利用同步荧光光谱,考察了10-HCPT对BSA构象的影响,10-HCPT的加入使BSA构象发生变化,BSA内部残基所处环境的疏水性下降。     

Spectrophotofluorimetric Study of the Interaction between Trazodone Hydrochloride and Bovine Serum Albumin

Abstract

The binding of trazodone hydrochloride (TZH), an antidepressant drug, to bovine serum albumin (BSA) has been investigated by fluorescence spectroscopic analysis. The fluorescence emission of BSA (λ_em=350 nm) was quenched by TZH while that of this ligand was enhanced (λ_em=443 nm). The spectral behavior was consistent with the static quenching mechanism, and the apparent binding constant, K _a (1.05×10⁴ l mol^?1) as well as binding site number, n (～1), were estimated. Thermodynamic parameters obtained from the measured data at different temperatures showed that the binding of TZH to BSA involved predominantly hydrophobic interactions as well as smaller contributions from electrostatic forces. Phenylbutazone and ibuprofen were utilized as competitive markers for sites I and II, respectively, in the interaction of TZH with BSA. This competitive displacement procedure indicated that the likely binding was site I, i.e., subdomain IIA, and this was supported by the observation that up to 50% of this site marker, phenylbutazone, could be exchanged with TZH whilst only a few percent of ibuprofen were so affected.     

Fluorescence spectrometric study on the interactions of Isoprocarb and sodium 2-isopropylphenate with bovine serum albumin

The binding interaction of the pesticide Isoprocarb and its degradation product, sodium 2-isopropylphenate, with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both Isoprocarb and sodium 2-isopropylphenate quenched the intrinsic fluorescence of BSA. This quenching proceeded via a static mechanism. The thermodynamic parameters (DeltaH(o), DeltaS(o) and DeltaG(o)) obtained from the fluorescence data measured at two different temperatures showed that the binding of Isoprocarb to BSA involved hydrogen bonds and that of sodium 2-isopropylphenate to BSA involved hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy of the interaction of BSA with either Isoprocarb or sodium 2-isopropylphenate showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide, i.e., the protein is denatured. The sodium 2-isopropylphenate, was estimated to be about 4-5 times more toxic than its parent, Isoprocarb. Synchronous fluorescence spectroscopy and the resolution of the three-way excitation-emission fluorescence spectra by the PARAFAC method extracted the relative concentration profiles of BSA, Isoprocab and sodium 2-isopropylphenate as a function of the added sodium 2-isopropylphenate. These profiles showed that the degradation product, sodium 2-isopropylphenate, displaced the pesticide in a competitive reaction with the BSA protein.     

Fluorescence Spectrometric Study on the Interactions of Terazosin Hydrochloride and Prazosin Hydrochloride with Bovine Serum Albumin Using Warfarin and Diazepam as Site Markers

Abstract

The binding interaction of the terazosin hydrochloride and prazosin hydrochloride with bovine serum albumin (BSA) was studied by spectrofluorimetry. Both of these two compounds quenched the fluorescence of BSA. The thermodynamic parameters (ΔH ⁰, ΔS ⁰ and ΔG ⁰) obtained from the fluorescence data measured at two different temperatures showed that the binding of terazosin hydrochloride to BSA involved hydrogen bonds and that of prazosin hydrochloride to BSA involved hydrophobic and electrostatic interactions. In this work, the competitive interaction of the terazosin hydrochloride and prazosin hydrochloride with BSA was studied by three-way excitation-emission fluorescence with the aid of parallel factor analysis (PARAFAC).     

Biophysical Studies on the Site-Selective Binding of a Synthesized Selenium–Quercetin Complex on a Protein

A selenium?Cquercetin complex (SEQC) was synthesized and its structure was identified by IR, LC-MS and ¹H-NMR. Its biochemical effects on bovine serum albumin (BSA) were studied by measuring its molecular spectra including three-dimensional fluorescence, ultraviolet and circular dichroism (CD) spectra. The interaction of SEQC and BSA is discussed in terns of fluorescence quenching and F?rster??s non-radiation energy transfer theory. The thermodynamic parameters $\Delta H^{\uptheta}$ , $\Delta G^{\uptheta}$ , and $\Delta S^{\uptheta}$ were calculated at different temperatures and the results indicate that the interaction is an endothermic as well as an entropy generating process. The binding site was explored by the fluorescence probe technique using warfarin and ibuprofen as markers. It was found that around body temperature hydrophobic forces maintained the average distance from the tryptophan residue in domain?II of BSA (donor) to SEQC (acceptor) at 3.76 nm. The conformational changes of BSA were investigated by three-dimensional fluorescence and circular dichroism spectra.     

Interaction of epicatechin with bovine serum albumin using fluorescence quenching combined with chemometrics

The interaction between BSA and epicatechin was studied using fluorescence quenching titrations combined with trilinear decomposition method and excitation-emission matrix(EEM)fluorescence.The resolved spectra were highly similar with the actual ones which indicated that the resolved results were reliable.The relevant parameters of the binding process were obtained by quantifying each substance in the complicated mixtures in situ.The quenching was static quenching,epicatechin had a weak interaction with BSA and the binding site was one.The total concentration and the free concentration of quenchers had different effect on the system.The results demonstrated that the method exploited in this article is a useful tool to investigate complicated interactions,avoiding complicated pretreatment and simplify experimental procedure.     

Investigating the interaction of juglone (5-hydroxy-1, 4-naphthoquinone) with serum albumins using spectroscopic and in silico methods

The interaction between juglone at the concentration range of 10–110 µM and bovine serum albumin (BSA) or human serum albumin (HSA) at the constant concentration of 11 µM was investigated by fluorescence and UV absorption spectroscopy under physiological-like condition. Performing the experiments at different temperatures showed that the fluorescence intensity of BSA/HSA was decreased in the presence of juglone by a static quenching mechanism due to the formation of the juglone–protein complex. The binding constant for the interaction was in the order of 10³ M^?1, and the number of binding sites for juglone on serum albumins was determined to be equal to one. The thermodynamic parameters including enthalpy (ΔH), entropy (ΔS) and Gibb’s free energy (ΔG) changes were obtained by using the van’t Hoff equation. These results indicated that van der Waals force and hydrogen bonding were the main intermolecular forces stabilizing the complex in a spontaneous association reaction. Moreover, the interaction of BSA/HSA with juglone was verified by UV absorption spectra and molecular docking. The results of synchronous fluorescence, UV–visible and CD spectra demonstrated that the binding of juglone with BSA/HSA induces minimum conformational changes in the structure of albumins. The increased binding affinity of juglone to albumin observed in the presence of site markers (digoxin and ibuprofen) excludes IIA and IIIA sites as the binding site of juglone. This is partially in agreement with the results of molecular docking studies which suggests sub-domain IA of albumin as the binding site.     

光谱法研究氨基己酸与牛血清白蛋白的相互作用

利用荧光光谱、紫外-可见吸收光谱及圆二色（CD）光谱研究了模拟生理条件下的氨基己酸(ACA)与牛血清白蛋白(BSA)的相互作用。 实验结果分析表明,氨基己酸对BSA的内源性荧光具有猝灭作用,属于动态猝灭过程。 计算了2种温度下ACA-BSA体系的结合常数、结合位点数及反应的热力学参数ΔG、ΔH和ΔS分别约为-21.00 kJ/mol、-0.64 kJ/mol和-72.00 kJ/(mol·K),由此推出了二者主要通过氢键和范德华力形成摩尔比为1∶1的复合物。 依据Forster非辐射能量转移理论求得二者之间的结合距离为2.3 nm。 位点取代实验指出氨基己酸主要结合在位点Site I。 CD光谱表明,氨基己酸诱导了BSA分子二级结构微变。     

Graphene oxide as a nanocarrier for loading and delivery of medicinal drugs and as a biosensor for detection of serum albumin

The interaction of graphene oxide (GO), a medicinal drug (10-hydroxy camptothecin (HCPT)), and bovine serum albumin (BSA) was investigated with the aim of developing a method for the analysis of serum albumin proteins. It was demonstrated that HCPT could be readily loaded onto GO via the π–π stacking interaction, and the delivery of HCPT to BSA was improved in the presence of GO; this, in turn, facilitated the binding interaction of HCPT and BSA. Chemometrics methods, multivariate curve resolution-alternating least squares (MCR-ALS) and parallel factor analysis (PARAFAC), were applied to resolve spectral data, and this assisted in the elucidation of the above interaction. GO was found to enhance the fluorescence response of HCPT to BSA, and thus, a low cost fluorescence bio-sensing platform was developed for fluorescence-enhanced detection of BSA based on GO. The satisfactory analytical performance of this biosensor for BSA was attributed to the structure and electronic properties of GO.     

Mechanism and conformational studies of farrerol binding to bovine serum albumin by spectroscopic methods

The mechanism and conformational changes of farrerol binding to bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence quenching technique, UV–vis absorption, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that farrerol could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The thermodynamic parameters enthalpy change and entropy change for the binding were calculated to be −29.92 kJ mol⁻¹ and 5.06 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which suggested that the both hydrophobic interactions and hydrogen bonds play major role in the binding of farrerol to BSA. The binding distance r deduced from the efficiency of energy transfer was 3.11 nm for farrerol–BSA system. The displacement experiments of site markers and the results of fluorescence anisotropy showed that warfarin and farrerol shared a common binding site I corresponding to the subdomain IIA of BSA. Furthermore, the studies of synchronous fluorescence, CD and FT-IR spectroscopy showed that the binding of farrerol to BSA induced conformational changes in BSA.     

Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism

The binding of farrerol to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence spectra at pH 7.40. The results of fluorescence titration indicated that farrerol could quench the intrinsic fluorescence of BSA in a static quenching way. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed. The binding sites number n and binding constant K using fluorescence quenching equation at 310 K were calculated. The binding distance and the energy transfer efficiency between farrerol and BSA were also obtained according to the theory of F?rster's non-radiation energy transfer. The effect of some metal ions on the binding constant of farrerol with BSA was also studied. The effect of farrerol on the conformation of BSA was analyzed using CD, synchronous fluorescence spectra and three-dimensional (3D) fluorescence spectra under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that farrerol could bind to the site I of BSA.     

Independent component analysis as a pretreatment method for parallel factor analysis to eliminate artefacts from multiway data

Parallel factor analysis (PARAFAC) has successfully been used in many applications for the analysis of excitation-emission fluorescence data. However, some measurement “artefacts”, such as Rayleigh or Raman scattering, can pose a problem for the extraction of the PARAFAC components and their interpretation. Replacing the spectral zones corresponding to these signals by missing values in the data is not necessarily a method of choice in the cases where informative signals lie in the same wavelength regions. In this article, independent component analysis (ICA) is used on the unfolded cubic array, and the independent components related to the Rayleigh and Raman scattering are identified and removed prior to the reconstruction of the excitation-emission fluorescence data cube. PARAFAC is then applied on these data reconstructed after selective artefact removal, and satisfactory models can be obtained. This procedure, although particularly useful for 3D fluorescence data, may be applied to other types of data as well.     

三七总皂甙对牛血清白蛋白溶液构象的影响

应用衰减全反射傅立叶变换红外光谱结合荧光光谱和紫外光谱研究了中药三七 的有效成分三七总皂甙与牛血清白蛋白（BSA）的相互作用，采用对蛋白质红外光 谱酰氨Ⅰ带和酰氨Ⅲ带进行曲线拟合的方法，定量分析了不同浓度三七总皂甙对 BSA二级结构的影响，发现随着三七总皂甙浓度的增加，蛋白分子结构逐渐发生了 由螺旋向折叠的转化。a-螺旋结构减少了3%，β-折叠结构增加了约5%，其它二级 结构没有明显的变化，红外差谱和荧光光谱的结果为药物与蛋白质的作用引起牛血 清白蛋白溶液构象的变化提供了佐证，紫外光谱反映了单体皂甙与蛋白质的结合常 数的差异。     

Spectrometric study of the interaction between Alpinetin and bovine serum albumin using chemometrics approaches

The binding interaction of Alpinetin (APT) with bovine serum albumin (BSA) was studied by fluorescence, UV-visible and synchronous fluorescence spectroscopy (SFS) under simulated physiological conditions. The measured complex spectra were resolved by multivariate curve resolution-alternating least squares (MCR-ALS), yielding a host of data and information, which otherwise would have been impossible to obtain. The extracted profiles corresponded to the spectra of the single species in the APT/BSA mixture. In addition, the presence of the APT-BSA complex was demonstrated, and it was shown that the associated quenching of the fluorescence from the BSA protein resulted from the formation of APT-BSA complex via a static mechanism. The binding constant (K_a(ave) = 2.34 × 10⁶ L mol⁻¹) and the number of sites (n = 1) were obtained by fluorescence methods as were the thermodynamic parameters (ΔH⁰, ΔS⁰ and ΔG⁰). This work suggested that the principal binding between APT to BSA was facilitated by hydrophobic interactions. The thermodynamic parameters for APT were compared to those from the structurally similar Chrysin and Wogonin molecules. It appeared that the entropy parameters were relatively more affected by the small structural changes. SFS from the interaction of BSA and APT showed that the ligand affected the conformation of BSA. The competitive interaction of APT and site makers with BSA indicated site I as the binding area of APT in BSA.     

Studies on the interaction of cinnamic acid with bovine serum albumin

The interaction between cinnamic acid and bovine serum albumin (BSA) have been studied at three temperatures, 296, 303 and 310 K. Fluorescence quenching spectra in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to investigate the drug-binding mode, the binding constant and the protein structure changes in the presence of cinnamic acid in aqueous solution at pH 7.40. The fluorescence quenching constant K(q), K(sv) and the binding constant K were calculated according to Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of cinnamic acid. The thermodynamic parameters, the enthalpy (DeltaH) and the entropy change (DeltaS) were estimated to be -16.457 kJ mol(-1) and 38.028 J mol(-1) K(-1) according to the van't Hoff equation. The displacement experiment shows that cinnamic acid can bind to the subdomain IIA (corresponding to Sudlow's drug binding site I). The distance between the tryptophan residues in BSA and cinnamic acid bound to site I was estimated to be 1.63 nm using F?ster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of common ions indicates that common ions have effect on drug-BSA system.