Binding Effect of Common Ions to Human Serum Albumin in the Presence of Norfloxacin: Investigation with Spectroscopic and Zeta Potential Approaches

In this work, the toxic influence of metallic ions (Al³⁺, Cu²⁺, Mg²⁺, Pb²⁺) on human serum albumin (HSA) in the absence and presence of norfloxacin (NRF) was studied by spectroscopic approaches [fluorescence quenching, synchronous fluorescence, three-dimensional fluorescence, circular dichroism, resonance light scattering (RLS) and zeta potential techniques] under simulated physiological conditions. Fluorescence spectroscopy revealed that these metallic ions and NRF can quench the HSA fluorescence, and this quenching effect became more significant when both ion and drug are present together. The binding constants and binding sites of metal ions with HSA in the absence and presence of NRF were determined, based on the fluorescence quenching results. Ion aggregation gives rise to an enhancement of the RLS intensity for HSA and the critical induced aggregation concentration (C _CIAC) of the ions, causing HSA aggregation for binary and ternary systems. The zeta potential measurements indicate a combination of electrostatic and hydrophobic interactions between ion, NRF and HSA and the formed micelle-like clusters. These data illustrated that NRF has an effect on the interaction between HSA and metal ions, with relevance for various toxicological and therapeutic processes.     

A comparison of the inclusion behavior of human serum albumin and holo transferrin with fluoxymesterone in the presence of three different cyclodextrins

This article describes the interaction of fluoxymesterone (Flu) with HSA and HTF in the absence and presence of cyclodextrins (CDs) (α, β and γ). According to fluorescence data, the binding of Flu to the proteins caused strong static quenching in the binary and ternary systems. The fluorescence quenching results demonstrated that HSA and HTF had two and one class of apparent binding sites with a distinct binding constant in the presence of the CDs, respectively. The effects of Flu on the structure of HSA and HTF were analyzed using synchronous fluorescence spectroscopy, which showed that the interaction of Flu with both proteins in the binary and ternary systems altered the microenvironment around the Trp and Tyr residues. The distance, r, between Flu and the proteins was obtained according to FRET which pointed at a successful formation of a drug-protein complex. Far-UV CD spectra indicated that the binding of the drug to both proteins induced changes in the secondary structure of HSA and HTF in the binary and ternary systems. Finally, molecular modeling provided possible binding sites of Flu within the proteins for the binary and ternary systems and also confirmed the experimental results. The obtained data can be useful for determining usage drug doses in drug delivery.     

Interaction of tetrandrine with human serum albumin: a fluorescence quenching study.

The interaction of tetrandrine with human serum albumin (HSA) was studied by measuring fluorescence quenching spectra, synchronous fluorescence spectra and ultra-violet spectra. The fluorescence quenching spectra of HSA in the presence of tetrandrine showed that tetrandrine quenched the fluorescence of HSA. The quenching constants of tetrandrine on HSA were determined using the Stern-Volmer equation. Static quenching and non-radiation energy transfer were the two main reasons leading to the fluorescence quenching of HSA by tetrandrine. According to the F?rster theory of non-radiation energy transfer, the binding distances (r) and the binding constants (K(A)) were obtained. The thermodynamic parameters obtained in this study revealed that the interaction between tetrandrine and HSA was mainly driven by a hydrophobic force. The conformational changes of HSA were investigated by synchronous spectrum studies.     

Studies of the Interaction Between Ronidazole and Human Serum Albumin by Spectroscopic and Molecular Docking Methods

Ronidazole (RNZ) is widely used for the therapeutic treatment of farmed animals and is suspected of being a human carcinogen and mutagen. The interaction between RNZ and human serum albumin (HSA) was investigated systematically by fluorescence spectroscopy, synchronous fluorescence, three-dimensional fluorescence, CD spectroscopy, UV–vis absorption spectroscopy and a molecular docking study. The results indicate that the probable quenching mechanism of HSA by RNZ is dynamic quenching. The corresponding thermodynamic parameters, such as ΔH, ΔS and ΔG, etc., were calculated according to the van’t Hoff equation. The results indicate that the forces acting between RNZ and HSA are mainly hydrogen bonds and van der Waals forces. The conformational changes in the interaction were studied by synchronous fluorescence, CD spectroscopy and three-dimensional fluorescence spectra. The results reveal that the microenvironment and conformation of HSA has been changed. A molecular modeling study further confirmed the binding mode obtained by the experimental studies.     

利福布汀与人血清白蛋白相互作用的光谱研究

用荧光光谱法和圆二色谱法研究了利福布汀(RB)与人血清白蛋白(HSA)的相互作用. 结果表明, RB与HSA之间的相互作用主要是疏水作用, 作用机制是静态猝灭与动态猝灭的结合. 其结合常数(Ka)在106数量级, 说明RB和HSA有很强的结合. 此外, 探讨了金属离子(Cu2+, Zn2+, Mg2+ 和Ca2+)对RB与HSA结合常数的影响. 同步荧光光谱和圆二色谱数据表明, RB可导致HSA的构象改变.     

Studies on the Antagonistic Behavior Between Cyclophosphamide Hydrochloride and Aspirin with Human Serum Albumin: Time-Resolved Fluorescence Spectroscopy and Isothermal Titration Calorimetry

The interactions between cyclophosphamide hydrochloride (CYC) and aspirin (ASA) with human serum albumin (HSA) were investigated by measuring fluorescence anisotropy, poly-dispersity index, and time-resolved fluorescence. Also, isothermal titration calorimetry (ITC) was performed. The fluorescence spectra of the drugs exhibited an appreciable hypsochromic shift along with an enhancement in the fluorescence intensity. The gradual addition of HSA led to a marked increase in fluorescence anisotropy (r), and from this value it is argued that the drugs were located in a restricted environment of the protein. The binding constants for the ASA–HSA and CYC–HSA complexes were found to be 1.27 × 10⁸ and 4.23 × 10⁸ mol·L^?1, respectively, as calculated from the relevant fluorescence data. The polydispersity index and size distribution of the protein–drug complex were measured at several concentrations of the drugs by the zeta potential technique, which confirmed the already obtained experimental results. From the analysis of the steady-state and time-resolved fluorescence quenching of the drugs in aqueous solutions in the presence of HSA, it was found that the quenching is static in nature. ITC experiments revealed that, in the absence of drugs, the dominant forces are electrostatic, whereas hydrophobic and weak electrostatic forces became significant in the presence of the drug. The primary binding pattern between the drugs and HSA was interpreted as a combined effect of hydrophobic association and electrostatic interactions.     

分子内电荷转移荧光探针1-酮-2-（对二甲氨基苯亚甲基）四氢萘与人血清白蛋白作用

采用荧光及紫外光谱研究了1-酮-2-（对二甲氨基苯亚甲基）-四氢萘（KDTN）与人血清白蛋白（HSA）相互作用的光谱特性。 结果表明,静态猝灭和非辐射能量转移是导致KDTN对HSA荧光猝灭的主要原因。 测得17、27和37 ℃ 3个温度下的结合常数K_A分别为1.633×10⁸、0.7998×10⁸和0.347×10⁸ L/mol,结合位点数n分别为1.7、1.6和1.7;据Forster偶极 偶极非辐射能量转移理论,计算得到KDTN与HSA在3个温度下的作用距离r分别为2.64、2.59和2.64 nm;能量转移效率E分别为0.5100、0.4797和0.4210。 热力学参数表明,二者主要以范德华力或氢键结合;用同步荧光技术研究了KDTN对HSA构象的影响,结果表明,KDTN的加入对HSA构象影响不大。     

Study of caffeine binding to human serum albumin using optical spectroscopic methods

The binding of caffeine to human serum albumin (HSA) under physiological conditions has been studied by the methods of fluorescence, UV-vis absorbance and circular dichroism (CD) spectroscopy. The mechanism of quenching of HSA fluorescence by caffeine was shown to involve a dynamic quenching procedure. The number of binding sites n and apparent binding constant K _b were measured by the fluorescence quenching method and the thermodynamic parameters ΔH, ΔG, ΔS were calculated. The results indicate that the binding is mainly enthalpy-driven, with van der Waals interactions and hydrogen bonding playing major roles in the reaction. The distance r between donor (HSA) and acceptor (caffeine) was obtained according to the Förster theory of non-radiative energy transfer. Synchronous fluorescence, CD and three-dimensional fluorescence spectroscopy showed that the microenvironment and conformation of HSA were altered during the reaction.     

光谱法测定伊曲康唑与牛血清和人血清白蛋白相互作用

用荧光光谱和紫外吸收光谱法, 在pH=7.4±0.1的0.1 mol·L-1磷酸缓冲溶液中, 研究了伊曲康唑与牛血清白蛋白(BSA)和人血清白蛋白(HSA)的相互作用. 实验结果表明, 伊曲康唑与牛血清白蛋白和人血清白蛋白作用的猝灭常数均随着温度的升高而降低, 伊曲康唑可以有规律地使血清白蛋白内源荧光猝灭, 其猝灭机理可认为是伊曲康唑与白蛋白形成复合物的静态猝灭. 获得了在不同温度下, 伊曲康唑与血清白蛋白作用的结合常数以及△G、△H和△S等热力学参数. 根据所得结果可推断伊曲康唑与白蛋白的作用力主要为疏水作用力, 同时, 利用荧光共振能量转移理论(FRET)计算得出了伊曲康唑与白蛋白结合位置的距离d. 而且, 利用同步荧光光谱和紫外光谱揭示了该反应中蛋白的结构和其微环境的变化.     

Study of the interaction of aglycon of daunorubicin with human serum albumin by spectroscopy and modeling

The interaction between aglycon of daunorubicin (DNR-A) and human serum albumin (HSA) was investigated using fluorescence quenching and modeling. Results shown that fluorescence quenching of HSA by DNR-A resulted from the formation of DNR-A-HSA complex. The quenching constants were determined via measurement of the binding affinity between DNR-A and HSA using the Stern-Volmer equation. The thermodynamic parameters DeltaG, DeltaH, DeltaS and the binding distance r were calculated. Furthermore, SFS and UV spectra suggested that the complex changed the conformation of HSA and that hydrophobic interactions played a major role in DNR-A-HSA association, which was in good agreement with the results of the modeling study. Moreover, the SFS technique was successfully applied to determine the total proteins in biology samples with satisfactory results.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法,表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息.同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境;二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光,使其构象发生变化.荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息,揭示KA的存在使HSA的流动性和微粘度发生变化.定量求得不同温度下(298、308和318 K)考拉维酸与HSA作用的键合参数和热力学参数.分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内,并与赖氨酸Lys195和天冬氨酸Asp451形成三个氢键,与HSA的键合模式主要是疏水作用;位点竞争实验证明考拉维酸在HSA亚结构域的位点II位发生作用.另外,获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制.结果表明,HSA对考拉维酸有较强的结合能力,提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

考拉维酸对人血清白蛋白结构的影响

利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法, 表征了模拟生理条件下一种植物药活性组分考拉维酸(KA)影响人血清白蛋白(HSA)的结构信息. 同步荧光及紫外光谱证实考拉维酸的存在影响了HSA的微环境; 二维及三维荧光光谱表明考拉维酸可以猝灭HSA的内源荧光, 使其构象发生变化. 荧光偏振的测定提供了考拉维酸与HSA作用后生成的配合物弛豫时间与聚集特性的信息, 揭示KA的存在使HSA的流动性和微粘度发生变化. 定量求得不同温度下(298、308 和318 K)考拉维酸与HSA作用的键合参数和热力学参数. 分子模拟表明考拉维酸键合位点于HSA分子的疏水腔内, 并与赖氨酸Lys195 和天冬氨酸Asp451 形成三个氢键, 与HSA的键合模式主要是疏水作用; 位点竞争实验证明考拉维酸在HSA亚结构域的位点Ⅱ位发生作用. 另外, 获得的相关物理化学参数从分子水平上揭示了考拉维酸与HSA相互作用的机制. 结果表明, HSA对考拉维酸有较强的结合能力, 提示人血清白蛋白对考拉维酸可起到储存和转运的作用.     

Synthesis and interaction studies of benzimidazole derivative with human serum albumin

A benzimidazole derivative, 1-(2-picolyl)-3-(2-picolyl) benzimidazole iodide (PPB), was synthesized. Fourier transform infrared spectroscopy (FT-IR), UV–visible, three-dimensional (3D) fluorescence, synchronous fluorescence (SF) and fluorescence spectroscopic methods were used to determine the PPB binding mode and the effects of PPB on protein stability and secondary structure. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of PPB to human serum albumin (HSA). The binding constants between PPB and HSA were obtained according to Scatchard equation. The number of binding sites, the binding constants and the thermodynamic parameters were measured. The results showed a spontaneous binding of PPB to HSA through hydrogen bonds and van der Waals forces. In addition, the distance between PPB and the Trp 214 was estimated via employing the Förster's non-radiative energy transfer theory, and was found to be 3.49 nm, which indicated that PPB can bind to HSA with high probability. Site marker competitive experiments indicated that the binding of PPB to HSA primarily took place in subdomain IIA.     

Fluorescence study on the interaction of human serum albumin with bromsulphalein

The binding of bromsulphalein (BSP) with human serum albumin was investigated at different temperatures, 298 and 308 K, by the fluorescence spectroscopy at pH 7.24. The binding constant was determined by Stern-Volmer equation based on the quenching of the fluorescence HSA in the presence of bromsulphalein. The effect of various metal ions on the binding constants of BSP with HSA was investigated. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: DeltaH and DeltaS possess small negative (9.3 kJ mol(-1)) and positive values (22.3 J K(-l)mol(-l)), respectively. The experimental results revealed that BSP has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The binding constants between BSP to HSA were remarkable and independent on temperature. The binding constants between HSA and BSP decreased in the presence of various ions, commonly decreased by 30-55%. The hydrophobic force played a major role in the interaction of BSP with HSA. All these experimental results and theoretical data clarified that BSP could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design.     

Molecular mechanism of interaction between norfloxacin and trypsin studied by molecular spectroscopy and modeling

The molecular mechanism of the binding of norfloxacin (NRF) to trypsin was investigated by fluorescence, synchronous fluorescence and UV–vis absorbance spectroscopy and molecular modeling at physiological conditions. The quenching mechanism and the binding mode were investigated in terms of the association constants and basic thermodynamic parameters. The results of spectroscopic measurements suggested that NRF have a strong ability to quench the intrinsic fluorescence of trypsin through static quenching procedure. Moreover, fluorescence experiments were also performed at different values of pH to elucidate the effect of pH on the binding. The NRF–trypsin complex was stabilized by hydrophobic forces and hydrogen bonding, via tryptophan residue as indicated from the thermodynamic parameters, which was consistent with the results of molecular docking and accessible surface area calculations.     

Study of the interactions between fluoroquinolones and human serum albumin by affinity capillary electrophoresis and fluorescence method

The interactions between fluoroquinolones and human serum albumin (HSA) were investigated by affinity capillary electrophoresis (ACE) and fluorescence quenching technique. Based on the efficient separation of several fluoroquinolones using a simple phosphate buffer, the binding constants of fluoroquinolones with HSA were determined simultaneously during one set of electrophoresis by ACE method. The thermodynamic parameters were obtained from data at different temperatures, and the negative ΔH and ΔS values showed that both hydrogen bonds and van der Waals interaction played major roles in the binding of fluoroquinolones to HSA. The interactions were also studied by fluorescence quenching technique. The results of fluorescence titration revealed that fluoroquinolones had the strong ability to quenching the intrinsic fluorescence of HSA through the static quenching procedure. The binding site number n, apparent binding constant K_b and the Stern-Volmer quenching constant K_sv were determined. The thermodynamic parameters were also studied by fluorescence method, and the results were consonant with that of ACE.     

Binding of Dioxopromethazine Hydrochloride with Human Serum Albumin and Its Effect on the Conformation of the Protein

The interaction between dioxopromethazine hydrochloride (DPZ) and human serum albumin (HSA), in vitro, was investigated by means of fluorescence, absorption and circular dichroism (CD) spectroscopy. The results obtained from analysis of the fluorescence spectra indicate that DPZ has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. The HSA?CDPZ binding distance was determined to be less than 7?nm, suggesting that energy transfer from HSA to DPZ may occur. The thermodynamic parameters of the interaction between DPZ and HSA were measured according to the van??t Hoff equation. The negative enthalpy change (??H ⁰) and positive entropy change (??S ⁰) values indicate that hydrophobic and electrostatic interactions play major roles in the binding between DPZ and HSA. The binding process was a favorable process in which the Gibbs energy change (??G ⁰) is negative. The results of displacement experiments suggested that DPZ is located in site I of HSA within subdomain IIA. In addition, the results of absorption, synchronous fluorescence and CD spectra show that binding of DPZ with HSA can induce conformational changes in HSA.     

Spectroscopic and Molecular Docking Approaches for Investigation of Interaction of Phellopterin with Human Serum Albumin

The mechanism of interaction between human serum albumin (HSA) and natural product phellopterin (PL) from Angelica dahurica was investigated by spectroscopic techniques with molecular docking under simulated physiological conditions. The experimental results showed that the fluorescence of HSA was regularly quenched by PL, and the quenching constants (K_SV) decreased with increasing temperature, which indicated that the quenching mechanism was a static quenching procedure. The binding constants (K_A) were larger than 10^?5 M^?1 and the number of binding sites (n) was approximate to 1 at different temperatures, which indicated that the binding affinity was hige and there was just one main binding site in HSA for PL. According to thermodynamic parameters from Van't Hoff equation, the binding process of PL with HSA was spontaneous and exothermic process due to ΔG < 0, and the electrostatic force played major role in the binding between PL and HSA according to ΔH < 0 and ΔS > 0. The binding distance (r) was calculated to be about 3.35 nm, which implied that the energy transfer from HSA to PL occurred with high possibility according to the theory of Förster's non-radiation energy transfer. The microenvironment and conformation of HSA changed with the addition of PL based on the results of synchronous and three-dimensional fluorescence methods. The molecular docking analysis revealed the binding locus of PL to HSA in subdomain IIIA (Sudlow's site II).     

具抗凝血作用的三种钕配合物与人血清白蛋白的相互作用

采用荧光光谱法,紫外光谱法以及圆二色谱法研究了具抗凝血作用的水杨酸钕((NdL′3.2H2O,L′=水杨酸离子))、华法灵钕(NdL3.2H2O,L=华法灵离子)和华法灵水杨酸钕(NdL2L′.2H2O)3种配合物与人血清白蛋白的相互作用。结果表明:配合物对人血清白蛋白(HSA)的荧光产生猝灭现象;配合物的存在使得HSA紫外吸收光谱的强度增加;配合物的存在也对HSA的构象产生影响。水杨酸钕的猝灭方式为动态与静态猝灭,而华法灵钕和华法灵水杨酸钕的猝灭方式属于两者之间生成了不发荧光的复合物而导致的静态猝灭。并分别确定了它们的结合力类型:华法灵钕与HSA之间主要作用力是静电作用力;水杨酸钕与HSA之间主要作用力为典型的疏水作用力;华法灵水杨酸钕与HSA之间为氢键和范德华力。计算了配合物与人血清白蛋白的结合常数K和结合位点数n。     

光谱法结合分子模拟技术研究磺胺甲恶唑与左氧氟沙星的协同作用

在模拟人体生理酸度(pH=7.4)条件下,运用荧光光谱、紫外光谱和分子模拟技术,研究了磺胺甲恶唑(SMZ)和左氧氟沙星(LVFX)的协同作用.实验结果表明,SMZ、LVFX主要通过氢键和疏水作用与人血清白蛋白(HSA)发生相互作用形成复合物,导致HSA的内源荧光发生静态猝灭.SMZ、LVFX在HSA上有相同的结合位点,即Site I位.在HSA-SMZ(或HSA-LVFX)体系中分别加入LVFX(或SMZ),其结合常数均明显减小,表明LVFX(或SMZ)的存在削弱了HSA-SMZ(或HSA-LVFX)体系的结合能力,使得LVFX(或SMZ)更多被释放,血液中游离的LVFX(或SMZ)浓度增大,SMZ与LVFX共存能够协同增强药效.