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.     

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.     

光谱法和分子对接模拟技术研究异烟肼对人血清白蛋白和过氧化氢酶的特异性结合及抑制作用

异烟肼(Isoniazid, INH)是最常用的一线抗结核药物之一。据报道,人体内高浓度的异烟肼可导致癫痫, 肝功能衰竭, 甚至死亡。因此,研究异烟肼对人血清白蛋白(HSA)和过氧化氢酶(CAT)的结构和活性的潜在结合影响有利于评估其毒性和副作用。在模拟生理条件下,利用多种荧光光谱和分子对接技术研究INH与HSA和CAT之间的相互作用。所有荧光数据均进行了内滤光校正以获得更准确的结合参数。结果表明,INH-HSA和INH-CAT体系的猝灭常数(K_sv)随着温度的升高而降低,表明INH对HSA及CAT的荧光猝灭机理为静态猝灭。利用紫外-可见吸收光谱、同步荧光光谱、和圆二色(CD)光谱法研究了INH对HSA和CAT构象的影响。结果发现,INH可改变色氨酸残基的微环境并降低HSA和CAT中α-螺旋结构,导致蛋白质结构发生伸展,进而可能影响其生理功能。分子对接结果表明,INH与HSA的结合位点位于HSA的site Ⅰ,ⅡA子域。INH可以进入CAT中β-折叠的桶状空腔,从而抑制CAT的活性。Hill系数结果表明,INH与左氧氟沙星(LVFX,一种安全有效的二线抗结核药物,与其他抗结核药物联合使用可以提高抗结核疗效)之间存在药物协同性,促进INH与HSA的相互作用。另外,CD光谱测定表明INH与LVFX的协同作用改变HSA的二级结构,使α-螺旋结构降低约7.9%。该研究探讨了INH与HSA和CAT之间的结合作用和毒性机制,为INH的安全使用提供重要依据。     

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.     

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.     

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.     

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.     

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.     

Mn（Ⅱ）,Co（Ⅱ）与HSA相互作用的荧光光谱研究

用荧光光谱法研究了生理ｐＨ和等离子点（ｐＨ＝５．３０）时Ｍｎ（Ⅱ）、Ｃｏ（Ⅱ）与ＨＳＡ的相互作用。根据Ｆｏｒｓｔｅ非辐射能量转移理论,得到了不同ｐＨ时Ｍｎ（Ⅱ）、Ｃｏ（Ⅱ）在ＨＳＡ中的第一强结合位置与Ｔｒｐ－２１４残基间的距离。这一结果远大于文献报道值,根据Ｍｎ（Ⅱ）、Ｃｏ（Ⅱ）在ＨＳＡ中的结合部位及ＨＳＡ的畴结构对这一显著差异进行了讨论。     

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.     

Study of the interaction between fluoxetine hydrochloride and bovine serum albumin in the imitated physiological conditions by multi-spectroscopic methods

The mechanism of interaction of an antidepressant, fluoxetine hydrochloride (FLX) with bovine serum albumin (BSA) has been studied by different spectroscopic techniques under physiological conditions. FLX was found to quench the intrinsic fluorescence of protein by static quenching mechanism. The binding constant ‘K’ was found to be 7.06×10³ M⁻¹ at 296 K. The value of ‘n’ close to unity revealed that the BSA has a single class of binding site for FLX. Based on thermodynamic parameters, hydrogen bonding and van der Waals forces were proposed to operate between BSA and FLX. The change in conformation of protein was noticed upon its interaction with the drug. From displacement studies it was concluded that the FLX bound to protein at site I. The effects of various common metals ions on the binding were also investigated.     

Study on the interaction of La with bovine serum albumin at molecular level

The interaction of La³⁺ to bovine serum albumin (BSA) has been investigated mainly by fluorescence spectra, UV-vis absorption spectra, and circular dichroism (CD) under simulative physiological conditions. Fluorescence data revealed that the quenching mechanism of BSA by La³⁺ was a static quenching process and the binding constant is 1.75×10⁴ L mol⁻¹ and the number of binding sites is 1 at 289 K. The thermodynamic parameters (ΔH=−20.055 kJ mol⁻¹, ΔG=−23.474 kJ mol⁻¹, and ΔS=11.831 J mol⁻¹ K⁻¹) indicate that electrostatic effect between the protein and the La³⁺ is the main binding force. In addition, UV-vis, CD, and synchronous fluorescence results showed that the addition of La³⁺ changed the conformation of BSA.     

光谱法联合分子对接和等温滴定微量热法研究全氟壬酸与人血清白蛋白的相互作用

全氟壬酸(PFNA)是在血清中检测到第三多的全氟烷酸类(PFAAs)新型有毒环境污染物。目前PFNA对人血清白蛋白(HSA)结构甚至是功能的影响还处于起步阶段,借助于多光谱、分子对接和等温滴定微量热(ITC)技术研究了PFNA和HSA相互作用的结合机理。所有荧光数据均进行了内滤光校正以获得更准确的结合参数。荧光结果表明PFNA通过动静态猝灭方式可以猝灭HSA的内源荧光。取代实验和分子对接结果表明,PFNA主要通过极性键、疏水力和卤素键键合在HSA亚域ⅡA疏水腔中,最佳对接自由能为-26.54 kJ·mol-1,表明PFNA分子与HSA有较大的结合亲和力。ITC表明两者的结合属于两类结合位点模型并给出了相应的热力学参数：第一类结合位点有较大的亲和力,属于焓驱动,静电力和卤键作为主要驱动力;第二类结合位点亲和力较小,主要驱动力是疏水力。三维荧光光谱揭示PFNA与HSA生成复合物后,可以改变HSA的构象,引起Trp和Tyr残基微环境疏水性增强。圆二色谱(CD)定量测定了HSA与PFNA作用前后的二级结构含量：α-螺旋、β-折叠和β-转角含量分别降低14.3%,5.3%和3.5%,无规卷曲含量从14.4%增加到37.5%。以上结果表明,PFNA与HSA的结合可以改变HSA的二级结构,进而可能影响HSA的生理功能。结果阐述了PFNA与HSA相互作用机理,并且为PFNA在体内的运输和分配提供了可靠的生物物理和生物化学的相关依据。     

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.     

Luminescence studies of o-hydroxy p-amino and p-dimethylamino methyl benzoate

Steady-state and time-resolved spectroscopic studies of two bisubstituted derivatives of methyl benzoate dissolved in methylcyclohexane (McH), tetrahydrofuran (THF), and ethanol (EtOH) have been performed at 293 and 77 K. The compounds under study are capable of showing multimode fluorescence emission at room temperature. The kind of emission, i.e., locally excited state emission Fl(LE), proton-transfer emission Fl(PT) and twisted intramolecular charge-transfer emission Fl(CT) dependent on the solvent. Low temperature measurements show that for those molecules a phosphorescence enhancement via a proton-transfer tunneling is observed. For the molecules under study compositeness of the emission process is explained on an energy scheme, which is based on experimentally determined and theoretically calculated data. Stated dynamic Stokes shift and phosphorescence red-edge effect indicates that both molecules form an inhomogeneous emitting system in solvents used. The experiments are supported with quantum chemical semiempirical calculations.     

Interaction between titanium dioxide nanoparticles and human serum albumin revealed by fluorescence spectroscopy in the absence of photoactivation

Titanium dioxide (TiO₂) nanoparticles (NPs) are widely used as an important kind of biomaterials. The interaction between TiO₂ (P25) at 20 nm in diameter and human serum albumin (HSA) was studied by fluorescence spectroscopy in this work. Under the simulative physiological conditions, fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K_a) were 2.18±0.04×10⁴, 0.87±0.05×10⁴, 0.68±0.06×10⁴ M⁻¹ at 298, 304 and 310 K, respectively. In addition, according to the Van’t Hoff equation, the thermodynamic functions standard enthalpy (ΔH⁰) and standard entropy (ΔS⁰) for the reaction were calculated to be −75.18±0.15 kJ mol⁻¹ and −170.11±0.38 J mol⁻¹ K⁻¹. These results indicated that TiO₂ NPs bond to HSA mainly by van der Waals force and hydrogen bonding formation in low dielectric media, and the electrostatic interactions cannot be excluded. Furthermore, the effects of common ions on the binding constant of TiO₂ NPs-HSA complex were discussed.     

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。     

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.     

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.     

Realization of p-ZnO thin films by GaP codoping

An attempt has been made to realize p-ZnO by directly doping (codoping) GaP into ZnO thin films. GaP codoped ZnO thin films of different concentrations (1, 2 and 4 mol%) have been grown by RF magnetron sputtering. The grown films on sapphire substrate have been characterized by X-ray diffraction (XRD), Hall measurement, Photoluminescence (PL) and Energy dispersive spectroscopy (EDS) to validate the p-type conduction. XRD result shows that all the films have been preferentially oriented along (0 0 2) orientation. The decrease of full-width at half maximum (FWHM) with increase in GaP doping depicts the decrease in native donor defects. Hall measurement shows that among the three films, 2 and 4 mol% GaP doped ZnO shows p-conductivity due to the sufficient amount of phosphorous incorporation. It has been found that low resistivity (2.17 Ωcm) and high hole concentration (1.8×10¹⁸ cm⁻³) for 2% GaP codoped ZnO films due to best codoping. The red shift in near-band-edge (NBE) emission and donar-acceptor-pair (DAP) and neutral acceptor bound recombination (A°X) observed by room temperature and low temperature (10 K) PL, respectively, well acknowledged the formation of p-ZnO. The incorporated phosphorous in the film has been also confirmed by EDS analysis.