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.     

Structural analysis and binding domain of albumin complexes with natural dietary supplement humic acid

Humic acid, a natural ionic molecule, is rapidly being recognized as one of the crucial elements in our modern diets of the new century. A biophysical protocol utilizing circular dichroism (CD), steady state and time-resolved fluorescence for the investigation of the complexation of the humic acid to the staple in vivo transporter, human serum albumin (HSA), as a model for protein-humic substances, is proclaimed. The alterations of CD and three-dimensional fluorescence suggest that the polypeptide chain of HSA partially folded after complexation with humic acid. The data of fluorescence emission displayed that the binding of humic acid to HSA is the formation of HSA-humic acid complex with an association constant of 10⁴ M⁻¹; this corroborates the fluorescence lifetime measurements that the static mechanism was operated. The precise binding domain of humic acid in HSA has been verified from the denaturation of albumin, hydrophobic ANS displacement, and site-specific ligands; subdomain IIA (Sudlow's site I) was earmarked to possess high-affinity for humic acid. The observations are relevant for other albumin-humic substance systems when the ligands have analogous configuration with humic acid.     

Complexation of fluoroquinolone antibiotics with human serum albumin: A fluorescence quenching study

Mechanism of interaction and detailed physico-chemical characterization of the binding of four fluoroquinolones: levofloxacin, sparfloxacin, ciprofloxacin HCl and enrofloxacin with human serum albumin has been studied at physiological pH (7.4) using fluorescence spectroscopic technique. The stoichiometry of interaction was found to be 1:1 for all the drugs used. The association constants for the interaction were of the order of 10⁴ in most cases. At low drug:protein ratios, a significant fraction of the added drug was bound. The predominant interactions involved are hydrogen bonding and Van der Waal’s interactions in the case of levofloxacin, hydrophobic interactions in the case of ciprofloxacin hydrochloride and enrofloxacin and hydrogen bonding, hydrophobic and electrostatic interactions in the case of sparfloxacin.The drug binding region did not coincide with that of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). From the displacement of site-specific probes and site-marker drugs, it was concluded that ciprofloxacin hydrochloride is site II-specific while enrofloxacin is a site I-specific drug. Levofloxacin binds at both site I and site II with equal affinity. Sparfloxacin had higher affinity for site II than site I. It is also possible that sparfloxacin binds at the interface between site I and site II. Stern-Volmer analysis of the data showed that the quenching mechanism is predominantly collisional for the binding of ciprofloxacin HCl and enrofloxacin while both static and collisional quenching mechanisms are operative in the case of levofloxacin and sparfloxacin. High magnitude of the rate constant for quenching showed that the process is not entirely diffusion controlled. Circular dichroism (CD) spectroscopic studies showed that the presence of drugs did not cause any major changes in the secondary structure of HSA.     

Interaction of nobiletin with human serum albumin studied using optical spectroscopy and molecular modeling methods

The binding of nobiletin to human serum albumin (HSA) was investigated by fluorescence, UV-vis, FT-IR, CD, and molecular modeling. Fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K) at four different temperatures (289, 296, 303 and 310 K) were 4.054, 4.769, 5.646 and 7.044×10⁴ M⁻¹, respectively. The enthalpy change (ΔH⁰) and the entropy changes (ΔS⁰) were calculated to be 1.938 kJ mol⁻¹ and 155.195 J mol⁻¹ K⁻¹ according to the Van’t Hoff equation. The binding average distance, r, between the donor (HSA) and the acceptor (nobiletin) was evaluated and found to be 2.33 nm according to the Förster's theory of non-radiation energy transfer. Changes in the CD and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. Computational mapping of the possible binding sites of nobiletin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.     

Interaction of the docetaxel with human serum albumin using optical spectroscopy methods

Docetaxel is a semi-synthetic product derived from the needles of the European yew. It is an antineoplastic agent belonging to the taxoid family. The interaction between docetaxel and human serum albumin (HSA) has been investigated systematically by the fluorescence quenching technique, synchronous fluorescence spectroscopy, ultraviolet (UV)-vis absorption spectroscopy, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) under physiological conditions. Our fluorescence data showed that HSA had only one docetaxel binding site and the binding process was a static quenching procedure. According to the Van’t Hoff equation, the thermodynamic parameters standard enthalpy (ΔH⁰) and standard entropy (ΔS⁰) were calculated to be −41.07 KJ mol⁻¹ and −49.72 J mol⁻¹ K⁻¹. These results suggested that hydrogen bond was the predominant intermolecular force stabling the docetaxel-HSA complex. The data from the CD, FT-IR and UV-vis spectroscopy supported the change in the secondary structure of protein caused by the interaction of docetaxel with HSA.     

Probing the binding of vitexin to human serum albumin by multispectroscopic techniques

The interaction between vitexin and human serum albumin (HSA) has been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of vitexin to HSA. The binding constants (K_a) between vitexin and HSA were obtained according to the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -57.29 kJ mol⁻¹ and -99.01 J mol⁻¹ K⁻¹ via the van't Hoff equation, which indicated that the interaction of vitexin with HSA was driven mainly by hydrogen bond and van der Waals forces. Fluorescence anisotropy data showed that warfarin and vitexin shared a common binding site I corresponding to the subdomain IIA of HSA. The binding distance (r) between the donor (HSA) and the acceptor (vitexin) was 4.16 nm based on the Förster theory of non-radioactive energy transfer. In addition, the results of synchronous fluorescence, CD and FT-IR spectra demonstrated that the microenvironment and the secondary structure of HSA were changed in the presence of vitexin.     

光谱及电化学方法研究大黄酸与牛血清白蛋白的相互作用

采用荧光光谱、紫外光谱和圆二色光谱法并结合电化学方法,研究了大黄酸与牛血清白蛋白之间的相互作用。结果表明:大黄酸对牛血清白蛋白有较强的荧光猝灭作用且为静态猝灭,并计算得出不同温度下其结合常数(KA)与结合位点数(n)分别为:3.67×105,0.95(298 K);2.60×104,0.83(309 K)。由热力学参数确定它们间的作用力主要是静电引力,并依据F rster能量转移理论求得其结合距离为3.28 nm,同步荧光光谱及圆二色谱表明大黄酸对牛血清白蛋白的构象产生影响。     

Binding of rare earth metal complexes with an ofloxacin derivative to bovine serum albumin and its effect on the conformation of protein

The water-soluble Pr (Ⅲ) and Nd (Ⅲ) complexes with an ofloxacin derivative have been prepared and characterized. The single-crystal X-ray diffraction showed that the Pr (III) and Nd (III) complexes have the similar molecular structure. Under physiological pH condition, the effects of [PrL(NO₃)₂(CH₃OH)](NO₃) and [NdL(NO₃)₂(CH₃OH)](NO₃) on bovine serum albumin (BSA) were examined using fluorescence spectroscopy in combination with UV-vis absorbance and circular dichroism (CD) spectra. The result reveals that the quenching mechanism of fluorescence of BSA by two complexes is a static quenching process and the number of binding sites is about 1 for both. The thermodynamic parameters (ΔH=−14.52 kJ mol⁻¹, ΔS=56.54 J mol⁻¹ K⁻¹ for [PrL(NO₃)₂(CH₃OH)](NO₃) and ΔH=−24.63 kJ mol⁻¹, ΔS=22.07 J mol⁻¹ K⁻¹ for [NdL(NO₃)₂(CH₃OH)](NO₃)) indicate that hydrophobic and electrostatic interactions are the main binding force in the complexes-BSA system. The binding average distance between complexes and BSA was obtained on the basis of Förster's theory. In addition, it was proved by the CD spectra that the BSA secondary structure was changed in the presence of complexes in an aqueous solution.     

多光谱和分子模拟技术研究全氟十二酸与人血清白蛋白的相互作用

全氟十二酸(PFDoA)是8～12个碳链的全氟烷酸(PFAAs)中毒性最强的新型环境污染物。已有大量研究表明PFAAs在环境中广泛积累,但对PFDoA与HSA的相互作用还处于起步阶段。本研究力争在模拟生理条件下,采用荧光猝灭法、分子模拟技术和圆二色谱确定HSA与PFDoA的相互作用机理。研究结果表明,PFDoA对HSA的猝灭是动态猝灭与形成PFDoA-HSA基态复合物引起的猝灭共同作用的结果。计算得到的结合距离(r=3.65 nm)表明,PFDoA(受体)与HSA(供体)之间的相互作用发生了非辐射能量转移。取代反应结果表明,PFDoA键合在HSA的site Ⅰ位点上。分子对接进一步研究了PFDoA与HSA作用的详细结合情况,表明PFDoA通过多种作用力结合在HSA的亚域IIA内,例如,PFDoA上的O 1原子主要通过极性键与HSA上的Arg 257和Ser 287残基结合。计算得到的最优对接能量为-25.87 kJ·mol-1,表明PFDoA对HSA有较大的结合亲和力。同步荧光光谱和三维荧光光谱研究了PFDoA对HSA构象的影响,结果显示,与PFDoA结合后,色氨酸的微环境疏水性增加,HSA的构象也发生改变。PFDoA与HSA作用前后圆二色谱二级结构的定量分析结果表明,PFDoA-HSA复合物的形成使螺旋稳定性降低。该研究结果为全氟烷酸与HSA的动力学研究提供了理论依据和可靠数据,并揭示了生物大分子与配体相互作用的化学本质。     

三种异黄酮类药物与不同异构体人血清白蛋白的作用机制研究

利用荧光光谱研究了三种异黄酮类化合物染料木素、鸡豆黄素A和3’,4’,7-三羟基异黄酮与不同异构体人血清白蛋白的相互作用机制。计算了异黄酮与蛋白质形成复合物的各种结合参数(猝灭速率常数、结合常数及结合位点数),结果表明三种异黄酮类化合物在人血清白蛋白上只有一个结合位点,位于结合位点siteⅠ,结合常数在0.17×105～1.20×105 L.mol-1之间。荧光增强光谱结果显示,与蛋白质作用后药物的荧光强度明显增加,说明了药物与人血清白蛋白发生了结合,在此基础上讨论了三种异黄酮与人血清白蛋白的结合机理。     

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.     

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.     

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).     

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.     

Study on the interaction between theasinesin and human serum albumin by fluorescence spectroscopy

The binding properties on theasinesin to human serum albumin (HSA) have been studied for the first time using fluorescence spectroscopy in combination with UV–vis absorbance spectroscopy. The results showed that theasinesin strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and non-radiation energy transfer happened within molecules. The number of binding site was 1, and the efficiency of Förster energy transfer provided a distance of 4.64 nm between tryptophan and theasinesin binding site. At 298, 310 and 323 K, the quenching constants of HSA–theasinesin system were 2.55×10³, 2.16×10³ and 1.75×10³ mol L⁻¹. ΔH^θ, ΔS^θ and ΔG^θ were obtained based on the quenching constants and thermodynamic theory (ΔH^θ<0, ΔS^θ>0 and ΔG^θ<0). These results indicated that hydrophobic and electrostatic interactions are the mainly binding forces in the theasinesin–HSA system. In addition, the results obtained from synchronous fluorescence spectra showed that the binding of theasinesin with HSA could induce conformational changes in HSA.     

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.     

光谱法和分子对接研究红斑红曲胺与牛血清白蛋白相互作用

近年来,随着对红曲色素的深入研究,其越来越多的功能活性被发现,但其某些致毒作用也使红曲色素的安全性受到了质疑。因此,阐明红曲色素在人体中与大分子的相互作用对深入研究其转运代谢及毒副作用具有重要作用。光谱法是研究溶液中小分子与蛋白质相互作用的一种有效方法,其具有灵敏度高、选择性强、用样量少、方法简单等优点,在研究中得到越来越广泛的应用。为探究红曲色素在体内的转运机制和血液中与转运蛋白的相互作用,本研究首次用红斑红曲胺(Rubropunctamine,Rub）作为红曲色素的典型代表与牛血清白蛋白（bovine serum albumin, BSA）相互作用。利用内源荧光光谱、同步荧光光谱探究不同浓度的Rub对BSA的荧光猝灭作用,采用Stern-Volmer方程、Lineweaver-Burk函数和Van’t-Hoff方程对不同温度下BSA与Rub作用后在λ_EX/λ_EM(280.0 nm/340.0 nm)（λ_EX/λ_EM表示荧光的激发波长和发射波长）的内源荧光强度值确定二者作用类型、结合位点数及相互作用机理,进一步利用圆二色谱定量测定了Rub的结合对BSA二级结构影响,最后运用软件Discovery Studio2.5对Rub与BSA的相互结合进行分子对接模拟。结果显示：(1) Rub对BSA具有较强的内源荧光猝灭效果,在λ_EX/λ_EM(280.0 nm/340.0 nm)的荧光强度下降306.1,发射波长由338.6 nm蓝移到331.8 nm,同步荧光显示荧光猝灭主要发生在色氨酸残基上。(2)Stern-Volmer方程计算得到动态猝灭速率常数K_q为2.335×1012 L·(mol·s)-1,远大于此类型允许的最大扩散碰撞常数2.0×1010 L·(mol·s)-1,判定该猝灭是单纯的静态猝灭过程。利用Lineweaver-Burk函数计算得到静态猝灭速率常数K_q随温度升高而减小,即该复合物在温度升高时变得不稳定。(3)利用等式lg[(F₀-F)/F]=lgK₀＋nlgc_Q得到两者结合常数可达103 L·mol-1以上,结合位点数近似为1,且随着温度增加表观结合常数变小。(4)不同温度下Van’t-Hoff方程计算得到ΔH,ΔS,ΔG都小于0,则该相互作用能自发进行且氢键和范德华力是其主要的相互作用力。(5)圆二色谱测得BSA与Rub结合后二级结构中α-螺旋含量由29.4%降至20.2%;β-折叠由39.9%上升到50.7%;β-转角由6.5%下降到3.5%;无规则卷曲由24.2%上升到25.6%。(6)分子对接发现Rub结合点位于 BSA中由Arg458,Asp108,Glu424和Ser428等氨基酸形成的口袋内,与Arg458有范德华力作用,与Arg144形成分子内氢键,影响到Trp213微环境。     

Spectroscopic studies on the binding of barbital to bovine serum albumin

In this paper, the interaction between barbital and bovine serum albumin (BSA) was investigated by the method of fluorescence spectroscopy under simulative physiological conditions. Fluorescence data revealed that the fluorescence quenching of BSA by barbital was the result of the formation of BSA-barbital complex, and the effective quenching constants (K_a) were 1.468×10⁴, 1.445×10⁴ and 1.403×10⁴ M⁻¹ at 297, 303 and 310 K, respectively. The thermodynamic parameters enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be −2.679 kJ mol⁻¹ and 70.76 J mol⁻¹ K⁻¹, respectively, according to the van’t Hoff equation. The results indicated that hydrophobic and electrostatic interactions were the dominant intermolecular force in stabilizing the complex. The results of synchronous fluorescence spectra showed that binding of barbital with BSA can induce conformational changes in BSA. In addition, the effects of Cu²⁺ and Zn²⁺ on the constants of BSA-barbital complex were also discussed.     

Extrinsic fluorescence probe study of human serum albumin using Nile red

Nile red bound to human serum albumin (HSA) shows an order of magnitude increase in the probe's fluorescence intensity. Here, we report on the fluorescence characteristics of the probe-protein complex in Trizma buffer (pH 7.1), urea, guanidine hydrochloride, and AOT/isooctane/buffer reverse micelles using both steady—state and time-resolved fluorescence techniques. With a view to illustrating the use of extrinsic probe fluorescence spectroscopy in protein research, we demonstrate that protein unfolding can be observed through measurements of the probe's time-resolved anisotropy and steady-state fluorescence spectrum. Moreover, this shows that thermal unfolding is fundamentally different from using denaturant, with respect to changes in both the nanosecond diffusional rotation of the probe at intermediate stages and in the denatured protein's structure. Also, the large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size to be easily identified in the steady-state fluorescence. This was not seen in earlier work exploiting the intrinsic tryptophan fluorescence of HSA and further demonstrates the complementary information that extrinsic fluorescence probe studies can offer protein science. We discuss the complex acrylamide quenching characteristics of Nile red bound to HSA in terms of the possibility of at least two binding sites for the probe and the effect of acrylamide on the probe-protein structure at very high quencher concentrations.     

Molecular spectroscopic insight into the binding of batatasin V isomers to human serum albumin

Fluorescence, ultraviolet-visible absorption spectroscopy, circular dichroism spectrum, and molecular docking methods were employed to study the interaction mechanisms of batatasin V and its isomer with human serum albumin. The two isomers both bond reactively to the hydrophobic activity in subdomain IIA, with an approximate binding affinity. Thermodynamic parameters and molecular modeling results manifested that hydrogen bonds and van der Waals force were the main contributors to the interaction. The secondary structure of human serum albumin was altered with the obvious decreased amount of α-helix. The results overall suggested similar binding mechanisms of batatasin V isomers with human serum albumin. This work will promote the further study of batatasins for pharmacological function. It could also help to provide some useful information for further drug design based on batatasins.