Study on interaction between a new fluorescent probe 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one and BSA

A new fluorescence reagent, 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one (mBPO), synthesized in our laboratory was used as the probe for protein and its interaction with Bovine Serum Albumin (BSA) was investigated in detail in this paper. It was found that BSA had the ability to quench the fluorescence of mBPO at 411 nm (λ(ex) = 286 nm), and the quenched intensity of fluorescence was proportional to the concentration of BSA. Based on this fact, mBPO has been used as a fluorescence probe for the detection of BSA. Under the optimal conditions, the calibration graph is linear up to 0.5 mg L(-1) for BSA and the limit of detection (LOD) was 0.06 mg L(-1). The regression equation is y = 1048.8x + 7.2093 with R(2) = 0.9913. The mechanism for the interaction of mBPO with BSA was also studied, while the binding constant and the number of binding sites were calculated. According to the thermodynamics parameter, the binding mode between mBPO and BSA was deduced. The results suggested the interaction between mBPO and BSA to be hydrophobic force in nature. It also proved that the fluorescence quenching reaction was affected by the tryptophan residue of BSA. For there are two tryptophan (Trp) residues, in site 134 and site 212 of BSA, and mBPO maybe has interaction with them respectively.     

Heterotropic binding of alclofenac and dansylsarcosine to bovine serum albumin

The binding data for the interaction of alclofenac (AF) and dansylsarcosine (DS) to bovine serum albumin (BSA) have respectively yielded nonlinear Scatchard plots. The plots have been subjected to Rosenthal’s method of analysis and thus the ligands have been found to possess two different kinds of sites in BSA. The binding capacities of these sites have been evaluated. The fluorescence competition studies have revealed that the binding of DS to BSA is noncompetitively inhibited by AF. Therefore, the presence of distinct binding sites for AF and DS in BSA could be inferred. The fluorescence quenching studies have also been able to demonstrate this aforesaid fact. The analysis of the quenching data by the modified Stern-Volmer plot has indicated that both the tryptophan (Trp) residues of BSA are accessible to DS for the quenching in absence of AF, but only one of them is accessible in presence of AF. This has led to suggest that the binding site of DS has been in the vicinity of loop 3–4, involving Trp-134 and Trp-212. The binding of AF at a distinct site from that of DS has exerted heterotropic interactions at the DS binding site and thereby inhibited the binding of DS to BSA.     

Effects of synthetic food colorants on the interaction between norfloxacin and bovine serum albumin by fluorescence spectroscopy

Abstract  

The effects of synthetic food colorants like tartrazine, sunset yellow, and erythrosine on the binding reaction between norfloxacin and bovine serum albumin (BSA) were investigated by fluorescence spectroscopy. Results showed that food colorants bound to BSA by van der Waals force and hydrogen bonding formation and norfloxacin by electrostatic interaction. In addition, marker competitive experiments suggested that the primary binding site for both norfloxacin and food colorants was located at subdomain IIA of BSA (site I). The presence of food colorants could alter the binding constant and distance between BSA and norfloxacin. The effects of colorants were dependent on their concentrations and binding affinity to BSA. The interaction could result in the change of the free, biologically active fraction of norfloxacin in blood.     

Investigation of the Interaction between Nitrite Ion and Bovine Serum Albumin Using Spectroscopic and Molecular Docking Techniques

The interaction between nitrite ion and bovine serum albumin (BSA), in an aqueous environment, was studied using spectroscopic methods, including fluorescence quenching technique, synchronous fluorescence, UV? Vis spectrophotometry and Resonance Rayleigh Scattering (RRS), and molecular docking technique. The experimental results showed that nitrite ion effectively quenched the intrinsic fluorescence of BSA with the static quenching. The ion‐BSA binding constant was determined to be 3.69×10³ L mol^?1. As the results showed the stoichiometry of binding nitrite ion to BSA was 1 : 1. Furthermore the thermodynamic parameters and nature of the binding force were calculated. The negative ΔH^o and ΔS^o values of reaction between nitrite ion and BSA indicated the predominant forces in the ion‐BSA interactions are hydrogen bonding interactions. Based on the Förster’s theory of non‐radiative energy transfer, the binding distance between nitrite ion and the inner tyrosine and tryptophan residue of BSA were determined to be 2.16 nm. Furthermore binding site of this ion on BSA was carried out by molecular docking technique.     

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.     

Study of the conjugation reaction between bovine serum albumin and gentamicin with Ponceau S as fluorescence probe

Abstract  

Ponceau S (PS) can quench the fluorescence of bovine serum albumin (BSA) in aqueous solution of pH 7.40. The static fluorescence-quenching process between BSA and PS was confirmed and the binding constant, the number of binding sites, and thermodynamic data for the interaction between BSA and PS were obtained. The results showed that the number of binding sites was 1 and that electrostatic attraction was important in the binding of BSA to PS. On the basis of the theory of F?rster resonance energy transfer, the binding distance (r < 7 nm) between PS and BSA was obtained. Site marker competitive experiments indicated that binding of PS to BSA primarily occurred in sub-domain IIA (site I). There was no obvious fluorescence intensity change on combining BSA and gentamicin (GM), so the conjugation reaction between BSA and GM cannot be studied by spectroscopy. It was observed that when GM was added to the BSA–PS system, the relative fluorescence intensity of the system recovered gradually with increasing concentration of GM, which showed there was a conjugation reaction between GM and BSA and that binding of GM to BSA primarily occurred in sub-domain IIA (site I).     

Interaction Study between ESIPT Fluorescent Lipophile-Based Benzazoles and BSA

In this study, the interactions of ESIPT fluorescent lipophile-based benzazoles with bovine serum albumin (BSA) were studied and their binding affinity was evaluated. In phosphate-buffered saline (PBS) solution these compounds produce absorption maxima in the UV region and a main fluorescence emission with a large Stokes shift in the blue–green regions due to a proton transfer process in the excited state. The interactions of the benzazoles with BSA were studied using UV-Vis absorption and steady-state fluorescence spectroscopy. The observed spectral quenching of BSA indicates that these compounds could bind to BSA through a strong binding affinity afforded by a static quenching mechanism (K_q~10¹² L·mol⁻¹·s⁻¹). The docking simulations indicate that compounds 13 and 16 bind closely to Trp134 in domain I, adopting similar binding poses and interactions. On the other hand, compounds 12, 14, 15, and 17 were bound between domains I and III and did not directly interact with Trp134.     

Study of the interaction of carbamazepine with bovine serum albumin by fluorescence quenching method.

The interaction between carbamazepine (CBZ) and bovine serum albumin (BSA) was studied using fluorescence spectroscopy (FS) and ultraviolet spectroscopy (UV). The experimental results showed that the CBZ could insert into the BSA and quench the inner fluorescence of BSA by forming the CBZ-BSA complex. It was found that both static quenching and non-radiation energy transfer were the main reasons leading to the fluorescence quenching. The apparent binding constants (K) between CBZ and BSA were found to be 1.8 x 10(4) (27 degrees C) and 2.8 x 10(4) (37 degrees C) and the binding site values (n) were 0.97 (27 degrees C) and 1.01 (37 degrees C), respectively. According to the Forster theory of non-radiation energy transfer, the binding distances (r) between CBZ and BSA were 3.6 nm and 3.4 nm at 27 degrees C and 37 degrees C, respectively. The process of the binding was a spontaneous molecular interaction in which entropy increased and Gibbs free energy decreased, indicating that the interaction between CBZ and BSA was mainly driven by the hydrophobic force.     

Study on the Interaction Between Bovine Serum Albumin and Potassium Perfluoro Octane Sulfonate

The interactions between potassium perfluorooctanesulfonate (PFOS) and bovine serum albumin (BSA) were studied by fluorescence spectroscopy. The association constants between PFOS and BSA were obtained by fluorescence enhancing and fluorescence quenching respectively. Furthermore, fluorescence quenching was studied at different temperatures, and the binding constant was also determined by the method of fluorescence quenching. According to the thermodynamic parameters, the main binding force could be judged. The experimental results revealed that BSA and PFOS had strong interactions. The mechanism of quenching belonged to dynamic quenching and the main sort of binding force was hydrophobic force. IR-spectra proved the interaction changed the conformation of BSA.     

三种香豆素类中药小分子与牛血清白蛋白的相互作用

运用荧光光谱(FS)、紫外光谱(UV)法研究了三种香豆素中药小分子与牛血清白蛋白(BSA)的相互作用.实验结果表明,香豆素类小分子能够插入BSA分子内部与BSA形成基态复合物导致BSA内源荧光猝灭,猝灭机理主要为静态猝灭和非辐射能量转移.药物分子极性及体积增大对BSA内源性荧光猝灭效应增强,与BSA中荧光性氨基酸残基之间的空间距离r增大,表观结合常数K_A增大且结合位点数n减少.结合过程的热力学参数变化表明上述相互作用过程是一个熵增加、Gibbs自由能降低的自发分子间作用过程,其中香豆素与BSA之间以疏水作用为主,而伞形花内酯、七叶内酯与BSA之间则还存在偶极-偶极作用,表明药物分子极性同样影响其与BSA间相互作用力的类型.     

Analysis of binding interaction between pegylated puerarin and bovine serum albumin by spectroscopic methods and dynamic light scattering

The interaction between bovine serum albumin (BSA) and pegylated puerarin (Pur) in aqueous solution was investigated by UV-Vis spectroscopy, fluorescence spectroscopy and circular dichroism spectra (CD), as well as dynamic light scattering (DLS). The fluorescence of BSA was strongly quenched by the binding of pegylated Pur to BSA. The binding constants and the number of binding sites of mPEG(5000)-Pur with BSA were 2.67±0.12 and 1.37±0.05 folds larger after pegylating, which were calculated from the data obtained from fluorescence quenching experiments. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be 4.09 kJ mol(-1) and 20.01 J mol(-1) K(-1), respectively, according to Van't Hoff equation, indicating that the hydrophobic force plays a main role in the binding interaction between pegylated Pur and BSA. In addition, the negative sign for Gibbs free energy change (ΔG) implies that the interaction process is spontaneous. Moreover, the results of synchronous fluorescence and CD spectra demonstrated that the microenvironment and the secondary conformation of BSA were changed. Comparing with Pur, all our data collected indicated that pegylated Pur interacted with BSA in the same way as that of Pur, but docked into the hydrophobic pocket of BSA with more accessibility and stronger binding force. DLS measurements showed monomethoxy polyethylene glycol (mPEG) have an effect on BSA conformation, and revealed that changes in BSA size might be due to increases in binding constant and the absolute values of ΔG after Pur pegylation.     

金属离子对次野鸢尾黄素与牛血清白蛋白相互作用的影响

应用荧光光度法研究了金属离子Fe3 、Ca2 、Cu2 或Mn2 对次野鸢尾黄素(IFR)与牛血清白蛋白(BSA)相互作用的影响.实验结果表明,不存在金属离子时,IFR对BSA的荧光猝灭过程为动态猝灭,其结合过程的表观结合常数KA值为104～105数量级,结合位点数n约等于1.由热力学参数得出IFR与BSA结合过程是一个熵增加、Gibbs自由能降低的自发过程,分子间相互作用力以疏水作用力为主.在Fe3 或Ca2 抖的存在下,IFR对BSA的荧光猝灭类型由动态猝灭转变为静态猝灭,作用力类型也由以疏水作用力为主转变为以氢键与范德华力为主或以静电引力为主.Cu2 或Mn3 存在下,IFR对BSA的荧光猝灭类型及分子间作用力类型均没有发生改变.四种金属离子的参与都使得IFR与BSA结合作用的袁观结合常数发生了明显的变化,但结合位点数仍维持在1左右.     

Binding equilibrium study of phosphotungstic acid and HSA or BSA with UV spectrum, fluorescence spectrum and equilibrium dialysis

The binding equilibrium between phosphotungstic acid (H7[P(W2O7)6] · XH2O;PTA) and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by UV-Vis, fluorescence spectroscopies and equilibrium dialysis. It has been observed that UV absorption enhanced and the fluorescence quenched as the PTA binding to HSA or BSA at physiological pH 7.43(?.02). The Scatchard analysis indicated that there exists a strong binding site of PTA in both HSA and BSA, and the successive stability constants of these two systems are obtained by nonlinear least-squares methods fitting Bjerrum formula.     

Binding of the bioactive component isothipendyl hydrochloride with bovine serum albumin

The binding of isothipendyl hydrochloride (IPH) to bovine serum albumin (BSA) was investigated by fluorescence spectroscopy combined with UV-visible absorption and circular dichroism (CD) techniques under simulative physiological conditions for the first time. The quenching mechanism of fluorescence BSA by IPH was discussed. The binding parameters have been evaluated by fluorescence quenching method. The thermodynamic parameters, ΔH°, ΔS° and ΔG° calculated at different temperatures indicated that the hydrophobic force played a major role in the interaction of IPH to BSA. The distance, r between donor (BSA) and acceptor (IPH) was obtained according to the Förster's theory of non-radiation energy transfer and was found to be 2.21 nm. Experimental results showed that the α-helicity of BSA decreased from 66.4% (in free BSA) to 39.1% (in bound BSA). The effect of common ions on the binding constant was also investigated.     

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.     

全硫取代三苯甲基自由基酯基衍生物与牛血清白蛋白的相互作用

采用荧光光谱、电子顺磁共振（EPR）波谱、紫外-可见吸收光谱和分子对接等技术研究了全硫取代三苯甲基（TAM）自由基酯基衍生物ET-03与牛血清白蛋白（BSA）的相互作用,发现ET--03与BSA能自发发生结合作用;主要以疏水作用力结合在BSA亚结构域ⅡA（位点Ⅰ）和亚结构域ⅢA（位点Ⅱ）上;ET-03对BSA的荧光猝灭效应为动态、静态混合猝灭机制,且可能存在非辐射能量转移.研究结果表明,酯基衍生化TAM自由基与白蛋白能自发结合,有望用于蛋白构效关系研究;同时也提示将TAM自由基酯基衍生物用于活体成像或自旋标记物时应考虑其与蛋白相互作用的影响.     

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。     

荧光光谱法研究茶碱与牛血清白蛋白的相互作用

荧光光谱法研究茶碱与牛血清白蛋白的相互作用;茶碱;牛血清白蛋白;荧光猝灭;能量转移     

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.     

二价铜、锌离子对盐酸巴马亭与牛血清白蛋白结合反应光谱的影响研究

The binding of Palmatine hydrochloride to bovine serum albumin (BSA) was studied, in the presence of Cu²⁺ and Zn²⁺, with fluorescence spectrum and ultra-violet spectrum. The results show that Cu²⁺ and Zn²⁺ don′t influence the first binding constant of Palmatine to BSA and the binding site of it to BSA. But Cu²⁺ has the fluorescence quenching effect on sensitive fluorescence of medicine binding to BSA.