小分子与血清白蛋白相互作用的研究

近年来,药物普遍应用的势态尤为明显,在这一势态之下,对小分子与血清白蛋白之间的相互作用机理进行研究便显得极为重要。因此对其进行研究,能够对药物运输及代谢过程有一定程度的了解,并能够为临床用药及新药研发提供依据。本课题重点分析了药物小分子与人血清白蛋白之间的相互作用。     

人血清白蛋白与金属离子作用的荧光光谱研究

通过金属离子对人血清白蛋白内源荧光的猝灭,探讨了金属离子Cu²⁺,Mn²⁺,Ni²⁺,Co²⁺,Cr³⁺等与人血清白蛋白的结合;基于Förster无辐射能量转移机理,得出了人血清白蛋白第一类Cu²⁺结合部位与214位色氨酸残基间的距离。     

罗丹明类荧光分子探针与血清白蛋白之间相互作用的研究

本研究旨在对罗丹明类荧光探针ZM-6与人血清白蛋白(HSA)的相互作用进行研究。采用了荧光光谱法、三维荧光光谱法、同步荧光光谱法以及CD光谱法在模拟生理条件下对二者的相互作用以及HSA的构象进行了研究。研究结果表明,探针与ZM-6之间的猝灭机理主要是静态猝灭方式。根据热力学数据确定了二者之间的作用力,类型为范德华力和氢键。二者之间的结合距离为4.45 nm。同时得出,ZM-6对HSA的构象产生了影响。此研究对于探针分子的设计以及修饰提供有效的数据以及理论支持。     

气相色谱法测定食品中植物甾醇含量研究

目的：建立气相色谱法测定食品中植物甾醇含量的方法。方法：对常见食品中的菜油甾醇、豆甾醇、β-谷甾醇含量进行定性定量分析。食品样品经皂化后提取,浓缩后用正己烷定容,上机测定。结果：本方法可有效分离食品中植物甾醇组分,各组分在0.005~0.2 mg/mL范围内线性关系良好,相关系数R2均在0.99以上;检出限为0.3 mg/100 g,其回收率在84.9%~106.2%,方法的重现性、精密度和稳定性良好,RSD均小于5%。结论：与液相色谱法相比,本方法能够有效分离和测定植物甾醇组分,且具有样品前处理简单、方法重现性好、准确度和灵敏度高等优点,可用于测定食品中植物甾醇组分含量。     

白色念珠菌羊毛甾醇14α-去甲基化酶三维结构分子模型研究

基于四种原核细胞色素Ｐ４５０晶体蛋白Ｐ４５０ＢＭ３、Ｐ４５０ｃａｍ、Ｐ４５０ｔｅｒｐ、Ｐ４５０ｅｒｙＦ模建白色念珠菌羊毛甾醇１４α－去甲基化酶三维结构。序列匹配采用四种晶体结构比较结果基础之上提出的细胞色素Ｐ４５０超家族蛋白基于结构知识的序列匹配方法。以Ｐ４５０ＢＭ３晶体结构坐标模建目标蛋白结构保守区主链结构,结构保守区侧链构象来源于四种晶体蛋白与模建蛋白对应残基同源性得分最高残基构象。模建结果用分子力学和分子动力学进行结构优化,模建结果蛋白采用Ｐｒｏｆｉｌｅ－３Ｄ图、Ｒａｍａｃｈａｎｄｒａｎ图和疏水图分析确证结构的合理性。并根据模型推测与血红素辅基相互作用的残基、与氧化还原偶联蛋白作用和参与电子传递的残基、底物进出通道和活性位点的残基。这些研究结果为定点突变研究、抗多肽抗体结合实验等提供理论依据,为高效低毒抗真菌药物合理设计提供靶标。     

生长素与油菜素甾醇相互作用机制的研究进展

植物生长发育是一个复杂、精细的调控过程,涉及细胞、组织和器官间多层次的信息交流,激素在其间发挥了重要调控作用．生长素和油菜素甾醇（BR）都能促进植物伸长,随着对其作用机制研究的深入,人们发现它们协同调控很多生理过程,对二者作用机制和信号转导的相互作用研究也成为激素研究领域的热点之一．对生长素和BR转导途径的揭示及直接下游基因的大规模发掘为二者通过相互作用调控不同生理过程的机制研究提供了重要线索．生长素和BR的相互作用涉及到下游基因转录的调控、信号组分互作以及合成代谢和极性运输等多层次的调控．     

水稻中油菜素甾醇功能机制解析与分子设计利用

植物激素油菜素甾醇(brassinosteroids,BR)调控许多重要农艺性状,在农业生产上具有巨大的应用潜力.近年来,水稻中BR功能机制解析进展迅速,一些相关基因的克隆为利用BR进行作物设计改良奠定了基础.然而,由于激素功能的复杂性,所调控的不同性状之间相互关联,容易带来负效应,对BR分子的利用造成了障碍.本文对水...     

芦丁与人血清白蛋白相互作用的紫外可见光谱特性研究

本文通过测定芦丁与HSA相互作用前后的紫外可见吸收光谱、圆二色性及人血清白蛋白(HSA)的荧光特性,研究了芦丁与HSA结合作用。结果表明,芦丁在紫外区有三个特征的吸收峰(264.0、285.5及354.5nm)、在330～300 nm及300～230 nm处显示圆二色性,HSA引起芦丁紫外可见吸收光谱波峰红移;芦丁与HSA相互作用后,不引起HSA二级结构的改变,但对其三级结构有影响,同时对HSA荧光激发及发生光谱最大峰位及幅度有影响。     

胡椒碱分子键合人血清白蛋白位点的表征

利用荧光光谱法、紫外光谱法并结合计算机模拟技术在分子水平上研究了胡椒碱与人血清白蛋白(human serum albumin HSA)的键合作用.同步荧光及紫外光谱图表明,胡椒碱对HSA微环境有影响.位点竞争试验证明,胡椒碱分子键合在HSA的位点Ⅱ区.通过荧光光谱滴定数据求得不同温度下(300K 310K和318 K)药物与蛋白相互作用的结合常数及结合位点数.分子模拟的结果显示了胡椒碱与HSA的键合区域和键合模式,表明药物与蛋白有较强的键合作用;维持药物与蛋白质的相互作用力主要是疏水用,兼有氢键(位于氨基酸残基Arg 257,Arg 222及Arg218位).通过实验数据计算得到的热力学参数(ΔH0与ΔS0的值分别为原33.11 kJ·mol-1和原18.90 J·mol原1·K-1)确定了胡椒碱与HSA分子的相互作用力类型主要为氢键兼范德华力.     

笋篼黄酮的结构鉴定及其与人血清白蛋白结合的分子模拟

以竹笋加工剩余物笋篼为原料,采用红外光谱(FT-IR)、液质联用技术(HPLC-MS)对笋篼黄酮(BSDF)的主要组分进行鉴定,并利用分子模拟技术研究了笋篼黄酮类物质与人血清白蛋白(HSA)的结合作用。红外结果表明,BSDF具有黄酮苷类化合物的特征峰;液质研究结果表明,BSDF中主要有4种黄酮苷类物质,分别是夏佛塔苷、白杨素-6-C-阿拉伯糖-8-C-葡萄糖苷、芹菜素-6,8-C-二那阿拉伯糖苷、白杨素-6-C-β-D-葡萄糖苷-8-C-α-L-阿拉伯糖苷;分子模拟结果显示,BSDF苷类物质白杨素-6-C-β-D-葡萄糖苷-8-C-α-L-阿拉伯糖苷能够很好地结合HSA的子域ⅠA和ⅢA。两者之间的结合存在氢键作用力、范德华作用力及阳离子-π堆积。该研究为了解笋篼黄酮结构及小分子药物在体内的运输情况提供一定的理论基础。     

Synthesis,characterization, and binding assessment with human serum albumin of three bipyridine lanthanide(III) complexes

In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2′-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV–Vis and ¹H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV–Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln³⁺ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.     

Interaction of a tyrosine kinase inhibitor,vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking

Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB–HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92–6.89?×?10^3?M^?1 at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB–HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J?mol^?1 K^?1) and negative ΔH (?6.57?kJ?mol^?1) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB–HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow’s site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca²⁺, Zn²⁺, Cu²⁺, Ba²⁺, Mg²⁺, and Mn²⁺ in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.     

Exploring the interaction between tyrphostin 9 and human serum albumin using biophysical and computational methods

Abstract

Tyrphostin 9 (Tyr 9) is a potent platelet-derived growth factor receptor (PDGFR) inhibitor, which induces apoptosis in various cancer cell types. The binding of Tyr 9 to the major transport protein, human serum albumin (HSA) was investigated using several spectroscopic techniques and molecular docking method. Fluorescence quenching titration results showed progressive decrease in the protein fluorescence with increasing drug concentrations. A decreasing trend of the Stern-Volmer constant, K _sv with increasing temperature characterized the drug-induced quenching as static quenching, thus pointed towards the formation of Tyr 9–HSA complex. The binding constant of Tyr 9–HSA interaction was found to lie within the range 3.48–1.69?×?10⁵ M^?1 at three different temperatures, i.e. 15 °C, 25 °C and 35?°C, respectively and suggested intermediate binding affinity between Tyr 9 and HSA. The drug–HSA complex seems to be stabilized by hydrophobic forces, van der Waals forces and hydrogen bonds, as suggested from the thermodynamic data as well as molecular docking results. The far-UV and the near-UV CD spectral results showed slight alteration in the secondary and tertiary structures, respectively, of the protein upon Tyr 9 binding. Interaction of Tyr 9 with HSA also produced microenvironmental perturbations around protein fluorophores, as evident from the three-dimensional fluorescence spectral results but increased protein’s thermal stability. Both competitive drug binding results and molecular docking analysis suggested Sudlow’s Site I of HSA as the preferred Tyr 9 binding site.

Communicated by Ramaswamy H. Sarma     

Interaction and photo–induced cleavage studies of meropenem drug with human serum albumin using spectroscopic and molecular docking investigations

The interaction between Meropenem drug and human serum albumin (HSA) has been studied under physiological condition in Tris–HCl buffer solution at pH 7.4 by various spectroscopic (UV spectra, fluorescence spectra, CD spectra), Photo–induced HSA cleavage, and molecular docking techniques. The results of fluorescence titration revealed that the Meropenem strongly quench the intrinsic fluorescence of HSA through a static quenching procedure. Binding constants (K_b) and the number of binding sites (n ? 1) were calculated using modified Stern–Volmer equations. The thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were calculated which revealed that the electrostatic and hydrogen bonding interactions play a major role in HSA–Meropenem association. The distance r between donor (HSA) and acceptor (Meropenem) was obtained according to fluorescence resonance energy transfer (FRET) and the alterations of HSA secondary structure induced by Meropenem were confirmed by FT–IR and CD measurements. The molecular docking technique was utilized to ascertain the mechanism and mode of action towards the molecular target HSA indicating that Meropenem was located within the subdomain IIA of protein by electrostatic interactions and hydrogen bonds, consistent with the corresponding experimental results. Additionally, Meropenem shows efficient photo–induced HSA cleavage. Our results may provide valuable information to understand the mechanistic pathway of drug delivery and to pharmacological behavior of drug.

• Research Highlights

• The interaction of Meropenem with HSA was studied by spectroscopic, photo-induced cleavage and molecular docking techniques.

• The secondary structure of protein has been changed upon the interaction with Meropenem.

• Subdomain IIA of the HSA is found to be the main binding site for Meropenem.

Communicated by Ramaswamy H. Sarma     

Characterizing the interaction between pyrogallol and human serum albumin by spectroscopic and molecular docking methods

In the present study, the interaction of Pyrogallol (PG) with human serum albumin (HSA) was investigated by UV, fluorescence, Circular dichroism (CD), and molecular docking methods. The results of fluorescence experiments showed that the quenching of intrinsic fluorescence of HSA by PG was due to a static quenching. The calculated binding constants (K) for PG-HSA at different temperatures were in the order of 10⁴?M ^?1, and the corresponding numbers of binding sites, n were approximately equal to unity. The thermodynamic parameters, ΔH and ΔS were calculated to be negative, which indicated that the interaction of PG with HSA was driven mainly by van der Waals forces and hydrogen bonds. The negative value was obtained for ΔG showed that the reaction was spontaneous. In addition, the effect of PG on the secondary structure of HSA was analyzed by performing UV–vis, synchronous fluorescence, and CD experiments. The results indicated that PG induced conformational changes in the structure of HSA. According to Förster no-radiation energy transfer theory, the binding distance of HSA to PG was calculated to be 1.93?nm. The results of molecular docking calculations clarified the binding mode and the binding sites which were in good agreement with the results of experiments.

Communicated by Ramaswamy H. Sarma     

Deciphering the binding of carbendazim (fungicide) with human serum albumin: A multi-spectroscopic and molecular modelling studies

Carbendazim is a benzimidazole fungicide used to control the fungal invasion. However, its exposure might lead to potential health problems. The present study evaluates the interaction of carbendazim (CAR) with human serum albumin (HSA) which is an important drug carrier protein and plays a very crucial role in the transportation of small molecules. A number of biophysical techniques were employed to investigate the binding of CAR with HSA. The increased UV-absorption of HSA on titrating with CAR suggests the formation of HSA–CAR complex and it could be due to the exposure of aromatic residues. The fluorescence study confirmed that CAR quenches the fluorescence of HSA and showed the static mode of quenching. CAR (50 µM) quenches around 56.14% of the HSA fluorescence. The quenching constant, binding constant, number of binding site and free energy change was calculated by fluorescence quenching experiment. Competitive displacement assay showed Sudlow’s site I as the primary binding site of CAR on HSA. The synchronous fluorescence study revealed the perturbation in the microenvironment around tyrosine and tryptophan residues upon binding of CAR to HSA. The circular dichroism results suggested that the binding of CAR to HSA altered its secondary structure. Molecular docking experiment demonstrated the binding of CAR to Sudlow’s site I of HSA. Docking studies suggested that the hydrogen bonding, van der Waals and pi-alkyl are playing role in the interaction of CAR with HSA. The study confirmed the conformational changes within HSA upon binding of CAR.