本文選題：熒光光譜 + 表面增強(qiáng)拉曼光譜�。� 參考：《長(zhǎng)春理工大學(xué)》2017年博士論文

【摘要】：近年來(lái),天然植物活性成分所具有的抗腫瘤、抗菌等多種生理活性已逐漸為人所知,其來(lái)源豐富、結(jié)構(gòu)多樣以及副作用小等特點(diǎn),引起了多個(gè)學(xué)科的普遍關(guān)注。蛋白質(zhì)和核酸都是構(gòu)成生命體重要的生物大分子,血清白蛋白(SA)承擔(dān)了多種內(nèi)源和外源性化合物的運(yùn)載體作用,脫氧核糖核酸(DNA)也是許多抗癌和抗菌藥物的主要靶向分子。因此,從分子水平上研究具有藥理活性的植物成分與生物大分子的超分子作用機(jī)制,這對(duì)于超分子組裝、分子識(shí)別、新藥設(shè)計(jì)與開(kāi)發(fā)等領(lǐng)域提供了必要的理論指導(dǎo)。文中利用多種光譜技術(shù)對(duì)七種具有抗癌等活性的植物成分與人血清白蛋白(HSA)和小牛胸腺DNA(ctDNA)之間的相互作用機(jī)制進(jìn)行了研究。主要研究?jī)?nèi)容如下:1.紫檀芪與人血清白蛋白相互作用的光譜特性研究采用熒光光譜、紫外吸收光譜和表面增強(qiáng)拉曼光譜法,研究了在模擬生理?xiàng)l件下紫檀芪(PTE)與人血清白蛋白(HSA)之間相互作用機(jī)制。結(jié)果表明:HSA的熒光能被紫檀芪靜態(tài)猝滅,并伴隨有非輻射能量轉(zhuǎn)移作用,兩者間形成了1:1復(fù)合物,其作用距離為1.495 nm,結(jié)合常數(shù)為1.12×104(298 K)、4.07×104(304 K)和2.45×105 L/mol(310K)。表面增強(qiáng)拉曼光譜研究揭示,紫檀芪分子通過(guò)甲氧基與HSA進(jìn)行結(jié)合。熱力學(xué)數(shù)據(jù)表明,二者間的作用主要為疏水作用。標(biāo)記競(jìng)爭(zhēng)實(shí)驗(yàn)指出,紫檀芪優(yōu)先結(jié)合HSA上的位點(diǎn)Ⅲ。三維熒光光譜、紫外吸收光譜、同步熒光光譜和表面增強(qiáng)拉曼光譜顯示,與紫檀芪作用使HSA構(gòu)象發(fā)生變化,導(dǎo)致色氨酸殘基周?chē)杷越档?但對(duì)紫檀芪分子構(gòu)象影響不大。2.對(duì)-香豆酸與人血清白蛋白相互作用的光譜特性研究在模擬生理?xiàng)l件下,應(yīng)用熒光光譜、紫外吸收光譜和表面增強(qiáng)拉曼光譜法對(duì)對(duì)-香豆酸(p-CA)與人血清白蛋白(HSA)的結(jié)合機(jī)理進(jìn)行研究。結(jié)果表明,對(duì)-香豆酸對(duì)HSA的熒光猝滅機(jī)制為靜態(tài)猝滅,其伴有非輻射能量轉(zhuǎn)移。熒光光譜顯示在298、304和310K下,對(duì)-香豆酸與HSA的結(jié)合常數(shù)(KA)分別為3.41×104、2.09×104和1.38×104 L/mol,結(jié)合位點(diǎn)數(shù)(n)近似為1。表面增強(qiáng)拉曼光譜研究揭示,對(duì)-香豆酸的酚基與HSA有效結(jié)合。標(biāo)記競(jìng)爭(zhēng)實(shí)驗(yàn)指出,對(duì)-香豆酸在HSA上的結(jié)合位點(diǎn)主要在SiteⅠ。反應(yīng)過(guò)程熱力學(xué)參數(shù)表明,二者間的作用主要為靜電引力。根據(jù)F?rster非輻射能量轉(zhuǎn)移理論求得對(duì)-香豆酸與HSA間的距離為5.11 nm。同步熒光光譜、三維熒光光譜顯示,p-CA的結(jié)合沒(méi)有導(dǎo)致HSA構(gòu)象發(fā)生明顯變化。3.柚皮素、柯里拉京和胡桃醌與人血清白蛋白相互作用的光譜特性研究基于熒光猝滅光譜、紫外吸收光譜法、同步熒光光譜、三維熒光光譜等技術(shù)及位點(diǎn)競(jìng)爭(zhēng)實(shí)驗(yàn),考察了柚皮素(NG)、柯里拉京(Cor)、胡桃醌(Jug)與HSA相互作用機(jī)制。結(jié)果表明,被分析分子均對(duì)HSA的內(nèi)源熒光具有猝滅作用,而猝滅機(jī)制有所差別;其中柚皮素為靜態(tài)猝滅伴有非輻射能量轉(zhuǎn)移、柯里拉京為靜態(tài)猝滅而胡桃醌是動(dòng)、靜態(tài)猝滅并伴有非輻射能量轉(zhuǎn)移過(guò)程。計(jì)算了NG-HSA、Cor-HSA和Jug-HSA體系的結(jié)合常數(shù)(KA)、結(jié)合位點(diǎn)數(shù)(n)及反應(yīng)的熱力學(xué)參數(shù)(ΔG、ΔH和ΔS),以此判斷了藥物與HSA結(jié)合過(guò)程的非共價(jià)作用方式。位點(diǎn)競(jìng)爭(zhēng)結(jié)果顯示,柚皮素分子的結(jié)合位點(diǎn)主要是SiteⅡ,柯里拉京和胡桃醌均為SiteⅢ。由F?rster非輻射能量轉(zhuǎn)移理論求得被分析分子與HSA間的結(jié)合距離。同步熒光光譜和三維熒光光譜指出,HSA與三種植物活性分子結(jié)合對(duì)其二級(jí)結(jié)構(gòu)和構(gòu)象有所改變但不顯著。4.肉桂酸與小牛胸腺DNA相互作用光譜特性研究在模擬生理?xiàng)l件下,以溴化乙錠(EB)為熒光探針,采用熒光光譜、吸收光譜、共振散射光譜及表面增強(qiáng)拉曼光譜方法并結(jié)合離子強(qiáng)度和熔點(diǎn)實(shí)驗(yàn),對(duì)肉桂酸(CA)與小牛胸腺DNA(ctDNA)間的作用機(jī)制進(jìn)行了研究。結(jié)果表明,肉桂酸與ctDNA作用時(shí)吸收光譜產(chǎn)生減色效應(yīng),肉桂酸的加入能猝滅EB-ctDNA體系的熒光,其猝滅方式為靜態(tài)猝滅,結(jié)合常數(shù)為1.63×103(293K)和4.97×103 L/mol(310 K)。共振散射光譜和ctDNA熔點(diǎn)結(jié)果指出,肉桂酸可在ctDNA結(jié)合處進(jìn)行聚集,形成了超分子體系,使Tm值升高6℃。熱力學(xué)參數(shù)結(jié)果顯示,肉桂酸與ctDNA堿基間主要作用力為疏水作用。表面增強(qiáng)拉曼光譜揭示,肉桂酸在ctDNA的嵌入位置與堿基A或G相鄰,形成了堿基堆積。上述結(jié)果均證明,在該實(shí)驗(yàn)條件下肉桂酸與ctDNA之間通過(guò)嵌入方式進(jìn)行結(jié)合,其過(guò)程是熵驅(qū)動(dòng)的自發(fā)、吸熱過(guò)程。5.柯里拉京、胡桃醌和查爾酮與小牛胸腺DNA相互作用的光譜特性研究在pH7.4 Tris緩沖溶液中,采用吸收光譜、熒光光譜和共振散射光譜方法,結(jié)合離子強(qiáng)度影響、ctDNA熔點(diǎn)實(shí)驗(yàn),對(duì)柯里拉京(Cor)、胡桃醌(Jug)和查爾酮(Cha)三種具有抗癌活性的藥物小分子與小牛胸腺DNA(ctDNA)之間的作用機(jī)制進(jìn)行了研究。結(jié)果表明,三種被分析分子與ctDNA作用后均出現(xiàn)了減色效應(yīng),且能有效地猝滅EB-ctDNA體系的熒光,猝滅方式有所差別,計(jì)算了結(jié)合常數(shù)。共振散射光譜和ctDNA熔點(diǎn)結(jié)果指出,三種化合物均可在ctDNA結(jié)合處進(jìn)行聚集,形成了超分子體系,使Tm值升高5~6℃;三種分子均以嵌入方式與ctDNA進(jìn)行結(jié)合,其強(qiáng)度為查爾酮柯里拉京胡桃醌,離子強(qiáng)度的改變對(duì)其結(jié)合影響不大。另外,根據(jù)熱力學(xué)參數(shù)結(jié)果獲得了三種化合物與ctDNA間非共價(jià)作用力類(lèi)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.

【學(xué)位授予單位】：長(zhǎng)春理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R285;O657.3
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本文編號(hào)：1840781