The Interaction Between Crystal Violet and Bovine Serum Albumin: Spectroscopic and Molecular Docking Investigations

The present work reported the investigations on the interaction between a triphenylmethane industrial dye—crystal violet (CV)—and bovine serum albumin (BSA) by spectroscopic methods and molecular docking calculation. The static quenching mechanism of the intrinsic fluorescence of BSA by CV was deduced by the fluorescence measurements and the ground-state complex formation was confirmed from the UV-vis spectra. The site maker competition binding experiments together with the molecular docking showed that the CV molecule specifically bound on the subdomain IIA of BSA. The obtained values of thermodynamic properties of binding suggested that the hydrophobic interaction was dominated as suggested by molecular docking results that the CV molecule was surrounded by hydrophobic amino acid residues. The conformation change of BSA in the binding process was detected by circular dichroism spectra and Fourier-transform infrared (FTIR) spectra and also reflected by the size change of BSA from the measurements by dynamic light scattering (DLS).     

A comparison study on the interaction of hyperoside and bovine serum albumin with Tachiya model and Stern–Volmer equation

The interaction between hyperoside and bovine serum albumin (BSA) was examined by fluorescence spectroscopy at 298, 304, and 310 K. The spectroscopic data were analyzed using Tachiya model and Stern–Volmer equation to determine the binding sites and apparent binding constant between hyperoside and BSA. For Tachiya model, both binding sites and apparent binding constants increased with the increasing of temperature, whereas for Stern–Volmer equation, the corresponding binding constants decreased as temperature increasing and the binding sites were independent of temperature. The positive sign of enthalpy change (ΔH) and entropy change (ΔS) suggested that hydrophobic forces played a major role in the interaction. Synchronous fluorescence spectra indicated that the conformation of protein was perturbed by the interaction of hyperoside with BSA. Moreover, the presence of metal ion affected the hyperoside-BSA binding.     

激光光散射与荧光光谱法研究大豆苷与牛血清白蛋白作用及聚集态

荧光光谱法和动态光散射法研究大豆苷与牛血清白蛋白在生理条件下的相互作用. 研究表明, 大豆苷与牛血清白蛋白能形成2∶l复合物, 荧光猝灭属于静态猝灭过程; 大豆苷与牛血清白蛋白分子间主要的结合作用力为疏水作用; 310 K下, 两者结合常数和结合位点数分别为7.4×l04 L•mol―1和1.75; 大豆苷使牛血清白蛋白的构象发生了变化; 动态光散射数据探讨了牛血清白蛋白与大豆苷分子产生聚集与之相互作用, 进一步证实了牛血清白蛋白在大豆苷水溶液中的构象变化. 实验结果为进一步研究大豆苷对心血管疾病的药理作用, 特别是对血浆蛋白构象的影响提供了重要依据.     

Spectroscopic Studies on the Interaction of Asiatic Acid with Bovine Serum Albumin

Fluorescence spectroscopy, Fourier transform infrared (FT‐IR) spectroscopy, circular dichroism (CD) and FT‐Raman spectroscopy were employed to analyze the binding of the asiatic acid (AA) to bovine serum albumin (BSA) under simulative physiological conditions. Fluorescence data revealed that the fluorescence quenching of BSA by AA was the result of the formation of BSA‐AA complex. The fluorescence quenching mechanism of BSA by AA was a static quenching procedure. According to the Van′t Hoff equation, the thermodynamic parameters enthalpy change (ΔH⁰) and entropy change (ΔS⁰) for the reaction were evaluated to be ?12.55 kJ·mol^?1 and 67.08 kJ·mol^?1, respectively, indicating that hydrophobic and electrostatic interactions played a major role in stabilizing the complex. The influence of AA on the conformation of BSA has also been analyzed on the basis of FT‐IR, CD and FT‐Raman spectra.     

Cr(Ⅵ)与牛血清白蛋白的相互作用

采用荧光光谱、紫外光谱、CD光谱法研究了K2Cr2O7与牛血清白蛋白(BSA)的相互作用。实验结果表明,铬(Ⅵ)使BSA的紫外吸收降低,峰位红移,表明铬(Ⅵ)与BSA发生较强的相互作用;铬(Ⅵ)酸根离子与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。测定了不同温度下该反应的热力学参数,ΔGθ0,ΔHθ和ΔSθ分别为-12.60kJ/mol和56.60J/(mol.k),表明上述作用过程是一个熵增加、自由能降低的自发分子间作用过程,铬(Ⅵ)酸根离子与BSA之间以静电作用力为主;非辐射能量转移机理确定了铬(Ⅵ)与牛血清白蛋白中色氨酸残基之间的距离r=2.85nm;同步荧光和CD光谱研究表明,铬(Ⅵ)使BSA的二级结构发生改变,α-螺旋含量降低,色氨酸残基所处微环境的极性减小。     

Characterization of Interaction Between Raltitrexed and Bovine Serum Albumin by Optical Spectroscopic Techniques

The interaction of raltitrexed(RTX) with bovine serum albumin(BSA) was investigated by steady state/lifetime fluorescence spectroscopy and circular dichroism(CD) spectroscopy under the simulative physiological conditions. The results of fluorescence titration reveal that RTX could strongly quench the intrinsic fluorescence of BSA via a static quenching procedure. The obtained binding constant K_A of RTX with BSA was 478630 and 44259 L/mol at 298 and 310 K, respectively. According to van’t Hoff equation, the thermodynamic parameters ΔH, ΔG and ΔS were calculated, indicating that hydrophobic forces were the predominant intermolecular forces in stabilizing the complex. The binding process was a spontaneous process, in which Gibbs free energy change was negative. According to Förster’s non-radioactive energy transfer theory, the distance r between donor(BSA) and acceptor(RTX) was 3.82 nm, suggesting that the energy transfer from BSA to RTX occurred with high probability. Displacement experiment and the number of binding sites calculation confirmed that RTX could bind to the site-I of BSA. Furthermore, the effects of pH and some metal ions on the interaction of RTX with BSA were also investigated. The results of synchronous fluorescence and CD spectra show that the RTX-BSA binding induced conformational changes in BSA.     

大黄酸铜(Ⅱ)配合物与血清白蛋白的相互作用

采用荧光光谱法和紫外光谱法研究了大黄酸铜配合物与牛血清白蛋白之间的相互作用.大黄酸铜配合物能显著猝灭牛血清白蛋白的内源荧光并以静态猝灭为主;计算了298 K和309 K温度下结合常数、结合位点,根据热力学参数判断大黄酸铜配合物与牛血清白蛋白之间具有较强的疏水作用力;依据F?rster的偶极-偶极非辐射能量转移理论,计算出大黄酸铜在蛋白质中结合位置与色氨酸残基间的距离为3.21 nm, 表明大黄酸铜的部分片段能够插入蛋白质分子内部;用同步荧光光谱和圆二色光谱技术探讨了大黄酸铜对牛血清白蛋白构象的影响.     

荧光光谱法研究橙皮甙与牛血清白蛋白的相互作用

在pH7.40的Tris-HCl 缓冲体系中,采用荧光光谱技术研究了橙皮甙与牛血清白蛋白(BSA)的相互作用.随着温度升高,橙皮甙与BSA 的猝灭常数逐渐增加.实验表明:橙皮甙对BSA的荧光猝灭为动态猝灭过程.由热力学参数焓变(△H=70.71 kJ/mol)大于零和熵变[△S=316.29J/(mol·K)]大于零,...     

Spectroscopic studies of the interaction between pirimicarb and calf thymus DNA

The interaction between pirimicarb and calf thymus DNA in physiological buffer (pH 7.4) was investigated with the use of Neutral Red (NR) dye as a spectral probe by UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopy, as well as viscosity measurements and DNA melting techniques. The results revealed that an intercalation binding should be the interaction mode of pirimicarb to DNA. CD spectra indicated that pirimicarb induced conformational changes of DNA. The binding constants of pirimicarb with DNA were obtained by the fluorescence quenching method. The thermodynamic parameters, enthalpy change (ΔHθ) and entropy change (ΔSθ) were calculated to be -52.13±2.04 kJ mol(-1) and -108.8±6.72 J mol(-1) K(-1) according to the van't Hoff equation, which suggested that hydrogen bonds and van der Waals forces might play a major role in the binding of pirimicarb to DNA. Further, the alternative least squares (ALS) method was applied to resolve a complex two-way array of the absorption spectra data, which provided simultaneously the concentration information for the three reaction components, pirimicarb, NR and DNA-NR. This ALS analysis indicated that the intercalation of pirimicarb into the DNA by substituting for NR in the DNA-NR complex.     

Interaction of Bovine Serum Albumin with N‐(4‐Ethoxyphenyl)‐N′‐(4‐Antipyrinyl)Thiourea (EPAT) Using Spectroscopies

The interaction between N‐(4‐ethoxyphenyl)‐N′‐(4‐antipyrinyl)thiourea (EPAT) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy in combination with UV absorption spectroscopy. The intrinsic fluorescence of bovine serum albumin was quenched by EPAT through a static quenching procedure. The binding constants of EPAT with BSA were estimated according to the fluorescence quenching results at different temperatures. The thermodynamic parameters: enthalpy change (ΔH) and entropy change (ΔS) were calculated to be ?10.69 kJ/mol and 42.64 J·mol^?1·K^?1 according to thermodynamic equations, respectively, and indicating that the binding force was suggested to be mainly a hydrophobic force. The effect of common ions on the binding constant was also investigated. A new fluorescence spectroscopy assay of the proteins was presented in this paper. The determination results of the proteins in bovine serum by means of this method were very close to those obtained using Coomassie Brilliant Blue G‐250 colorimetry.     

吡蚜酮与牛血清白蛋白的相互作用

利用紫外吸收、荧光、同步荧光光谱及圆二色谱研究了吡蚜酮与牛血清白蛋白(BSA)的相互作用. 结果发现, 吡蚜酮使BSA的紫外吸收峰强度降低, 峰位红移; BSA的特征荧光峰猝灭, 荧光猝灭常数KSV随着温度的升高而降低, 表明吡蚜酮与BSA发生了较强的相互作用, 且吡蚜酮对BSA的荧光猝灭机制属于静态猝灭. 计算了不同温度下的结合常数和结合位点数; 由van′t Hoff方程计算出体系的ΔH和ΔS值, 得出二者之间的作用力主要为氢键和范德华力; 根据非辐射能量转移理论确定了给体-受体间的结合距离r=2.4 nm. 采用同步荧光光谱和圆二色谱考察了吡蚜酮对牛血清白蛋白构象的影响.     

Spectroscopic studies on binding of 1-phenyl-3-(coumarin-6-yl)sulfonylurea to bovine serum albumin

The interaction of 1-phenyl-3-(coumarin-6-yl)sulfonylurea (SU22) with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectroscopy combined with UV-absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy techniques under simulative physiological conditions for the first time. Fluorescence data and UV-absorption spectra revealed that the quenching mechanism of fluorescence of BSA by SU22 was a static quenching process and the number of binding sites was about 0.8858; the thermodynamic parameters (DeltaG=-29.23 kJ mol(-1), DeltaH=-47.48 kJ mol(-1), and DeltaS=-61.24 J mol(-1)K(-1)) explained that hydrogen bond and Van der Waals interaction were the main binding force stabilizing the complex. The binding average distance between SU22 and BSA was obtained (3.20 nm) on the basis of the F?rster's theory. In addition, The CD spectra and FT-IR spectra have proved that BSA secondary structure changed in the presence of SU22 in aqueous solution.     

咪唑型离子液体与牛血清蛋白的缔合特性

通过紫外-可见光谱、荧光光谱、同步荧光光谱、圆二色谱、衰减全反射红外光谱、负染-透射电镜、等温滴定微量热等实验方法系统地探讨了咪唑型离子液体与牛血清蛋白(BSA)的缔合特性.结果发现,离子液体[Bmim]Cl的加入使得BSA的紫外吸收强度增加,同时也会导致其荧光猝灭,并且这种猝灭是静态猝灭.同步荧光的研究结果表明,[Bmim]Cl分子可与蛋白质中接近色氨酸残基的区域发生相互作用,使蛋白质的构象和内部的疏水结构发生改变;负染色法透射电镜直观地显示了加入离子液体后形成的蛋白质-离子液体复合物结构逐渐变大;圆二色谱和衰减全反射红外光谱表明:在离子液体与BSA缔合过程中,离子液体的加入使得BSA二级结构中的α-螺旋和β-折叠的含量降低,从而引起蛋白质二级结构的变化;表面张力法和等温滴定微量热法进一步证实上述缔合作用为静电作用和疏水作用共同作用的结果,但离子液体的烷基链与BSA疏水内腔之间的疏水作用是离子液体与BSA缔合的主要驱动力.     

光谱法与分子模拟技术研究杨梅素与牛血清白蛋白的相互作用

利用紫外光谱、荧光光谱、红边激发荧光位移(REES)法、圆二色谱(CD)结合分子模拟技术共同研究了模拟生理条件下杨梅素与牛血清白蛋白(BSA)的相互作用,阐述了相互作用机制.分子模拟结果表明,杨梅素与蛋白在亚结构域II A的疏水腔内结合,主要作用力为疏水作用力和氢键.依据荧光猝灭法判断猝灭机制为静态猝灭,并得到不同温度下药物与蛋白相互作用的结合常数(Ka)及结合位点数(n),根据热力学参数判断出作用力类型,并且计算出杨梅素与蛋白的结合距离,与分子模拟得到的判定结果基本一致.通过紫外光谱、同步荧光光谱以及REES法获得的信息讨论了相互作用时BSA中色氨酸(Trp)微环境的变化;并利用CD谱的测定结果定量计算了BSA二级结构中α-螺旋含量的变化.     

Study of the interaction of indirubin with bovine serum albumin

This study examined the interaction of indirubin with bovine serum albumin (BSA) at three temperatures (286, 297, 308 K) at pH 7.40. In the presence of indirubin, the drug-BSA binding mode, binding constant and the protein structure changes in aqueous solution were determined by fluorescence quenching methods including Fourier transform infrared (FT-IR) spectroscopy and UV-Vis spectroscopy. The FT-IR change indicates that indirubin binds to BSA. The change in protein secondary structure accompanying ligand binding has been proved by fluorescence spectra data. The thermodynamic parameters, the enthalpy change (DeltaH), and the entropy change (DeltaS) calculated by the van't Hoff equation possess small negative (-2.744 kJ.mol(-1)) and positive values (112.756 J.mol(-1).K(-1)), respectively, which indicated that hydrophobic interactions play the main role in the binding of indirubin to BSA. Furthermore, the displacement experiment shows that indirubin can bind to the subdomain IIA and the distance between the tryptophan residues in BSA and indirubin bound to site I was estimated to be 2.24 nm according to F?ster's equation on the basis of fluorescence energy transfer.     

溴百里酚蓝与牛血清白蛋白的相互作用研究

在模拟动物体生理条件和不同温度下,用荧光光谱和紫外-可见吸收光谱法研究了溴百里酚蓝(BTB)与牛血清白蛋白(BSA)结合反应的光谱行为。用Stern-Volmer和Lineweaver-Burk方程分别处理试验数据,发现BSA与BTB发生反应生成了新的复合物,属于静态荧光猝灭。求出了反应时复合物的形成常数KLB(2.792×105L.mol-1)、热力学参数(ΔHθ=(20.24 kJ.mol-1,ΔSθ=37.22J.K-1,ΔGθ=(31.25kJ.mol-1)与结合位点数(1.1578)。根据F rster偶极-偶极非辐射能量转移理论计算出结合位置距离212位色氨酸残基2.60nm,证明二者主要靠静电作用力结合。同时用同步荧光光谱和三维荧光光谱法探讨了BTB对BSA构象的影响,表明BTB使色氨酸残基所处微环境的极性减弱、疏水作用增强,为阐明BTB的染色机理、毒理效应和生物学效应提供重要信息。     

小檗碱与人免疫球蛋白在仿生膜中的相互作用

利用荧光光谱、圆二色谱(CD)和动态激光散射技术研究了小檗碱与人免疫球蛋白(HGG)在以双(2-乙基己酯)-磺酸基琥珀酸钠(AOT)/异辛烷/水微乳液为膜的模拟环境下的结合反应. 通过Scatchard方程分别计算了不同温度下的反应结合常数. CD光谱结果表明: 小檗碱与HGG键合改变了蛋白原有的构象. 同时由热力学结果可知, 小檗碱与HGG之间的相互作用主要以疏水作用为主, 同时存在静电作用力和氢键作用力. 动态激光散射结果进一步证明了药物与HGG在微乳液中的相互作用. 通过计算机模拟技术, 在不考虑环境影响的条件下从理论上探讨了HGG与小檗碱的结合区域和结合模式.     

Investigation of the association behaviors between biliverdin and bovine serum albumin by fluorescence spectroscopy

The interaction between biliverdin and bovine serum albumin (BSA) has been studied by steady fluorescence spectroscopy, synchronous fluorescence and resonance light scanning spectra. The binding of biliverdin to BSA quenches the tryptophan residue fluorescence and the results show that both static and dynamic quenching occur together with complex formation. The binding constant and binding sites of biliverdin to BSA at pH 7.1 are calculated to be 3.33 × 10⁸ L/mol and 1.54, respectively, according to the double logarithm regression curve. In addition, the distance between the biliverdin and BSA is estimated to be 1.25 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues has not obvious changes, which obeys the phase distribution model. Finally, the thermodynamic data show that biliverdin molecules enter the hydrophobic cavity of BSA via hydrophobic interaction.     

Interaction Between Surfactin and Bovine Serum Albumin

The interaction of surfactin, a typical biosurfactant, with bovine serum albumin (BSA) was investigated by surface tension, fluorescence, freeze-fractured transmission electron microscopy (FF-TEM) and circular dichroism (CD) measurements. The surface tension curves of pure surfactin solution and surfactin/BSA solutions have different phenomena, where two obvious inflections determined as the critical aggregation concentration (cac) and the critical micelle concentration (cmc) appear for surfactin/BSA solutions. The higher BSA concentration, the higher cac and cmc values for surfactin/BSA solution. Fluorescence spectra show that the structure change of BSA is dependent on both surfactin and BSA concentration. The micropolarity, FF-TEM and CD results further demonstrate the interaction between BSA and surfactin. The excess free energy (ΔG⁰) of surfactin/BSA interactions have been obtained as ?6.13 and 5.32 kJ/mol for 1.0 × 10^?6 and 3.8 × 10^?6 mol/L BSA concentration, respectively. The binding ratio (R) determined for surfactin/BSA systems are higher than that reported for dirhamnolipid to BSA. Above all, it can be concluded that the hydrophobic interaction and the hydrogen bonds between surfactin and BSA play the key role for the high binding ratio for surfactin to BAS.     

In Vitro Binding of Furadan to Bovine Serum Albumin

Under physiological conditions, the interaction between furadan (FRD) and bovine serum albumin (BSA) was investigated by spectroscopy including fluorescence emission, UV-visible absorption, scattering, circular dichroism (CD) spectra, synchronous and three-dimensional fluorescence spectra. The observed binding constant K _b and the number of binding sites n were determined by the fluorescence quenching method. The distance r between donor (BSA) and acceptor (FRD) was obtained according to the Förster theory of non-radiation energy transfer. The enthalpy change (ΔH ^θ ), Gibbs energy change (ΔG ^θ ) and entropy change (ΔS ^θ ) at four different temperatures were calculated. The process of binding was proposed to be a spontaneous process since the ΔG ^θ values were negative. The positive ΔS ^θ and ΔH ^θ values indicated that the interaction of FRD and BSA was driven mainly by hydrophobic interactions. The addition of FRD to BSA solutions leads to enhancement in scattering intensity, exhibiting the formation of an aggregate in solution. CD spectra, synchronous and three-dimensional fluorescence spectra were used to measure the structural change of BSA molecules with FRD present.