植物活性成分与人血清白蛋白及核酸相互作用的光谱特性研究

发布时间：2018-05-04 00:27

  本文选题：荧光光谱 + 表面增强拉曼光谱　； 参考：《长春理工大学》2017年博士论文

【摘要】：近年来,天然植物活性成分所具有的抗肿瘤、抗菌等多种生理活性已逐渐为人所知,其来源丰富、结构多样以及副作用小等特点,引起了多个学科的普遍关注。蛋白质和核酸都是构成生命体重要的生物大分子,血清白蛋白(SA)承担了多种内源和外源性化合物的运载体作用,脱氧核糖核酸(DNA)也是许多抗癌和抗菌药物的主要靶向分子。因此,从分子水平上研究具有药理活性的植物成分与生物大分子的超分子作用机制,这对于超分子组装、分子识别、新药设计与开发等领域提供了必要的理论指导。文中利用多种光谱技术对七种具有抗癌等活性的植物成分与人血清白蛋白(HSA)和小牛胸腺DNA(ctDNA)之间的相互作用机制进行了研究。主要研究内容如下:1.紫檀芪与人血清白蛋白相互作用的光谱特性研究采用荧光光谱、紫外吸收光谱和表面增强拉曼光谱法,研究了在模拟生理条件下紫檀芪(PTE)与人血清白蛋白(HSA)之间相互作用机制。结果表明:HSA的荧光能被紫檀芪静态猝灭,并伴随有非辐射能量转移作用,两者间形成了1:1复合物,其作用距离为1.495 nm,结合常数为1.12×104(298 K)、4.07×104(304 K)和2.45×105 L/mol(310K)。表面增强拉曼光谱研究揭示,紫檀芪分子通过甲氧基与HSA进行结合。热力学数据表明,二者间的作用主要为疏水作用。标记竞争实验指出,紫檀芪优先结合HSA上的位点Ⅲ。三维荧光光谱、紫外吸收光谱、同步荧光光谱和表面增强拉曼光谱显示,与紫檀芪作用使HSA构象发生变化,导致色氨酸残基周围疏水性降低,但对紫檀芪分子构象影响不大。2.对-香豆酸与人血清白蛋白相互作用的光谱特性研究在模拟生理条件下,应用荧光光谱、紫外吸收光谱和表面增强拉曼光谱法对对-香豆酸(p-CA)与人血清白蛋白(HSA)的结合机理进行研究。结果表明,对-香豆酸对HSA的荧光猝灭机制为静态猝灭,其伴有非辐射能量转移。荧光光谱显示在298、304和310K下,对-香豆酸与HSA的结合常数(KA)分别为3.41×104、2.09×104和1.38×104 L/mol,结合位点数(n)近似为1。表面增强拉曼光谱研究揭示,对-香豆酸的酚基与HSA有效结合。标记竞争实验指出,对-香豆酸在HSA上的结合位点主要在SiteⅠ。反应过程热力学参数表明,二者间的作用主要为静电引力。根据F?rster非辐射能量转移理论求得对-香豆酸与HSA间的距离为5.11 nm。同步荧光光谱、三维荧光光谱显示,p-CA的结合没有导致HSA构象发生明显变化。3.柚皮素、柯里拉京和胡桃醌与人血清白蛋白相互作用的光谱特性研究基于荧光猝灭光谱、紫外吸收光谱法、同步荧光光谱、三维荧光光谱等技术及位点竞争实验,考察了柚皮素(NG)、柯里拉京(Cor)、胡桃醌(Jug)与HSA相互作用机制。结果表明,被分析分子均对HSA的内源荧光具有猝灭作用,而猝灭机制有所差别;其中柚皮素为静态猝灭伴有非辐射能量转移、柯里拉京为静态猝灭而胡桃醌是动、静态猝灭并伴有非辐射能量转移过程。计算了NG-HSA、Cor-HSA和Jug-HSA体系的结合常数(KA)、结合位点数(n)及反应的热力学参数(ΔG、ΔH和ΔS),以此判断了药物与HSA结合过程的非共价作用方式。位点竞争结果显示,柚皮素分子的结合位点主要是SiteⅡ,柯里拉京和胡桃醌均为SiteⅢ。由F?rster非辐射能量转移理论求得被分析分子与HSA间的结合距离。同步荧光光谱和三维荧光光谱指出,HSA与三种植物活性分子结合对其二级结构和构象有所改变但不显著。4.肉桂酸与小牛胸腺DNA相互作用光谱特性研究在模拟生理条件下,以溴化乙锭(EB)为荧光探针,采用荧光光谱、吸收光谱、共振散射光谱及表面增强拉曼光谱方法并结合离子强度和熔点实验,对肉桂酸(CA)与小牛胸腺DNA(ctDNA)间的作用机制进行了研究。结果表明,肉桂酸与ctDNA作用时吸收光谱产生减色效应,肉桂酸的加入能猝灭EB-ctDNA体系的荧光,其猝灭方式为静态猝灭,结合常数为1.63×103(293K)和4.97×103 L/mol(310 K)。共振散射光谱和ctDNA熔点结果指出,肉桂酸可在ctDNA结合处进行聚集,形成了超分子体系,使Tm值升高6℃。热力学参数结果显示,肉桂酸与ctDNA碱基间主要作用力为疏水作用。表面增强拉曼光谱揭示,肉桂酸在ctDNA的嵌入位置与碱基A或G相邻,形成了碱基堆积。上述结果均证明,在该实验条件下肉桂酸与ctDNA之间通过嵌入方式进行结合,其过程是熵驱动的自发、吸热过程。5.柯里拉京、胡桃醌和查尔酮与小牛胸腺DNA相互作用的光谱特性研究在pH7.4 Tris缓冲溶液中,采用吸收光谱、荧光光谱和共振散射光谱方法,结合离子强度影响、ctDNA熔点实验,对柯里拉京(Cor)、胡桃醌(Jug)和查尔酮(Cha)三种具有抗癌活性的药物小分子与小牛胸腺DNA(ctDNA)之间的作用机制进行了研究。结果表明,三种被分析分子与ctDNA作用后均出现了减色效应,且能有效地猝灭EB-ctDNA体系的荧光,猝灭方式有所差别,计算了结合常数。共振散射光谱和ctDNA熔点结果指出,三种化合物均可在ctDNA结合处进行聚集,形成了超分子体系,使Tm值升高5~6℃;三种分子均以嵌入方式与ctDNA进行结合,其强度为查尔酮柯里拉京胡桃醌,离子强度的改变对其结合影响不大。另外,根据热力学参数结果获得了三种化合物与ctDNA间非共价作用力类型。
[Abstract]:In recent years, many physiological activities, such as anti-tumor and antibacterial activities, have been gradually known by natural plant active ingredients. Their rich sources, diversity of structures and small side effects have caused widespread concern in many disciplines. Protein and nucleic acids are important biological molecules of life body, and serum albumin (SA) bears a variety of internal factors. Deoxyribonucleic acid (DNA) is also a major target molecule for many anticancer and antimicrobial agents. Therefore, the mechanism of supramolecular action of plant components with pharmacological activity and biological macromolecules is studied at the molecular level. This is a kind of field for supersub assembly, molecular recognition, new drug design and development. A variety of spectral techniques were used to study the interaction mechanism between seven kinds of plant components with anti-cancer activity and human serum albumin (HSA) and calf thymus DNA (ctDNA). The main contents are as follows: 1. the spectroscopic properties of the interaction between human serum albumin and human serum albumin The interaction mechanism between PTE and human serum albumin (HSA) under simulated physiological conditions was studied by spectral, UV absorption and surface enhanced Raman spectroscopy. The results showed that the fluorescence energy of HSA was quenched by statically and with the transfer of non radiation energy, and the 1:1 complex was formed between the two, and the range of action was 1.4. 95 nm, with a binding constant of 1.12 x 104 (298 K), 4.07 * 104 (304 K) and 2.45 x 105 L/mol (310K). The surface enhanced Raman spectroscopy revealed that the alseqi molecules were combined with HSA by the methoxy. The thermodynamic data showed that the role of the two was mainly hydrophobic. The labelled competition experiment indicated that the sandalwood Astragalus was first combined with the HSA site III. The fluorescence spectrum, UV absorption spectrum, synchronous fluorescence spectrum and surface enhanced Raman spectrum showed that the conformation of HSA was changed and the hydrophobicity of tryptophan residue was reduced, but the effect of the conformation on the molecular conformation of the sandalwood Astragalus was little.2.. The spectral characteristics of the interaction between the coumaric acid and the human serum white egg white were studied in the simulated physiological strips. The binding mechanism of p-CA and human serum albumin (HSA) was studied by fluorescence spectrum, UV absorption spectrum and surface enhanced Raman spectroscopy. The results showed that the fluorescence quenching mechanism of HSA was static quenching with non radiation energy transfer. The fluorescence spectra showed that at 298304 and 310K. The binding constant (KA) of acid and HSA was 3.41 x 104,2.09 x 104 and 1.38 x 104 L/mol respectively. The binding site number (n) approximated to 1. surface enhanced Raman spectroscopy, and the phenol group of - coumaric acid was effectively combined with HSA. The labeling competition experiment indicated that the binding site of para coumaric acid on HSA was mainly in Site I. The thermodynamic parameters of the reaction process showed that two The interaction between the people is mainly electrostatic force. According to the F? Rster non radiation energy transfer theory, the distance between the pair of coumaric acid and HSA is 5.11 nm. synchronous fluorescence spectra, and the three-dimensional fluorescence spectrum shows that the binding of p-CA does not lead to the obvious changes in the conformation of HSA, and the interaction between the.3. naringin, and the interaction between the cricket and walnut quinone and human serum albumin. Based on fluorescence quenching spectra, UV absorption spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy and site competition experiments, the interaction mechanism of naringenin (NG), Currie La Gin (Cor) and walnut quinone (Jug) with HSA was investigated. The results showed that the molecules were quenched by the endogenous fluorescence of HSA, and the quenching mechanism was found. It was judged by the static quenching with non radiation energy transfer, the static quenching of Hu Taokun, static quenching and non radiation energy transfer process. The binding constant (KA) of NG-HSA, Cor-HSA and Jug-HSA system, the number of bits (n) and the thermodynamic parameters of the reaction (delta G, Delta H and Delta S) were calculated. The non covalent action of drug and HSA binding process. The site competition results show that the binding site of naringenin is mainly Site II, Currie La Gin and Hu Taokun are Site III. The combination distance between the analys and HSA is obtained by the F rster non radiation energy transfer theory. The synchronous fluorescence spectrum and the three-dimensional fluorescence spectrum indicate that HSA and three The interaction of plant active molecules to the secondary structure and conformation has changed, but the spectral characteristics of the interaction between.4. cinnamic acid and calf thymus DNA are not significant. Under simulated physiological conditions, the fluorescence spectrum, absorption spectrum, resonance scattering and surface enhanced Raman spectroscopy combined with the strong ion intensity are combined with the fluorescence probe of ethidium bromide (EB). The effect mechanism of cinnamic acid (CA) and calf thymus DNA (ctDNA) was studied. The results showed that the absorption spectrum of cinnamic acid and ctDNA produced a color reduction effect. The addition of cinnamic acid could quench the fluorescence of EB-ctDNA system. The quenching method was static quenching, and the binding constant was 1.63 * 103 (293K) and 4.97 * 103 L/mol (310). K). The results of resonance scattering and ctDNA melting point indicate that cinnamic acid can be aggregated at the junction of ctDNA, forming a supramolecular system and increasing the value of Tm by 6. The thermodynamic parameters show that the main force between cinnamic acid and ctDNA bases is hydrophobic. The surface enhanced Raman spectrum reveals that cinnamic acid is embedded in ctDNA and the base A or G phase. The results show that the interaction between cinnamic acid and ctDNA is an entropy driven spontaneity, and the spectral specificity of the interaction of.5. Cox, walnut quinone and chalcone and calf thymus DNA is studied in the pH7.4 Tris buffer solution, using absorption light. The mechanism of action between small molecules of Currie La Gin (Cor), nutcrone quinone (Jug) and chalcone (Cha), three kinds of drugs with anticancer activity and calf thymus DNA (ctDNA), were studied by spectrum, fluorescence spectrum and resonance scattering spectroscopy. The results showed that three kinds of molecules were treated with ctDNA. The color reduction effect appeared, and the fluorescence of EB-ctDNA system could be quenched effectively. The quenching methods were different. The binding constants were calculated. The resonance scattering spectra and the results of ctDNA melting point indicated that the three compounds could be assembled at the ctDNA binding, forming a supramolecular system and increasing the Tm value at 5~6, and the three molecules were embedded with ctDNA. The strength of the combination was chalcone crunchone quinone, and the change of ionic strength had little effect on the binding. In addition, the non covalent forces between the three compounds and ctDNA were obtained according to the thermodynamic parameters.

【学位授予单位】：长春理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285;O657.3
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本文编号：1840781