【作者】 孔玲；

【导师】 刘绍璞；

【作者基本信息】 西南大学 ， 分析化学， 2011， 博士

【摘要】 共振瑞利散射(RRS)光谱(或共振光散射)和共振非线性散射(RNLS)是近年来发展起来的一种新分析技术。由于它的简易性和高灵敏度引起了人们的关注,在生化和药物分析、有机物和金属离子的测定以及纳米微粒的研究中得到越来越多的应用。但是对于蛋白质和核酸等生物大分子,这种方法主要用于研究它们与染料、药物等小分子以及纳米微粒的相互作用,尚很少用于蛋白质和核酸等生物大分子之间相互作用的研究。鉴于RRS光谱对于分子间静电引力,疏水相互作用,氢键作用等弱结合力的结合作用以及分子大小、形貌和构象变化较吸收光谱、荧光光谱等常见的分子光谱分析法更为锐敏,这就为RRS光谱技术用于研究蛋白质、核酸等生物大分子相互作用提供了有利条件,方法具有高灵敏度和简易性,它们能够为生物大分子间的相互作用提供独特的新信息。而且从研究的生物大分子相互作用和分子识别的体系,还可发现和筛选出对于核酸、蛋白质及多糖类物质的高灵敏度和某些高选择性的分析方法。本文的主要研究内容及结论如下：1.蛋白质与DNA相互作用的RRS和RNLS光谱及其分析应用(1)木瓜蛋白酶与DNA相互作用的RRS和RNLS光谱及其分析应用在pH1.0-2.8的酸性介质中,带正电荷的木瓜蛋白酶能与带负电荷的生物大阴离子DNA发生相互作用形成结合产物。此时不仅能引起吸收光谱的变化和木瓜蛋白酶荧光的猝灭,还将导致RRS和RNLS的显著增强并出现新的散射光谱。RRS对于两者的相互作用显示了更高的灵敏度。本文根据吸收、荧光和RRS光谱的变化并结合圆二色光谱和热力学数据讨论了木瓜蛋白酶与DNA之间相互作用的主要结合力、两者的结合位点和结合模式。认为两者反应的主要作用力是静电引力、氢键作用力、疏水作用和蛋白质芳香族氨基酸残基(特别是色氨酸残基)与DNA碱基之间的芳基堆集作用。木瓜蛋白酶的芳香族氨基酸残基在DNA大沟槽中与它的碱基之间的芳基堆积作用是引起吸收光谱变化和荧光猝灭的主要原因。而结合产物体积增大,两者电荷被大量中和导致表面疏水性增强以致疏水界面的形成,产生一种表面增强的散射效应。散射位于分子的吸收光谱带中产生的共振增强作用以及蛋白质和DNA反应前后构象变化均可能是引起散射增强的重要原因。由于RRS法具有高灵敏度,对DNA和木瓜蛋白酶的检出限(3σ)分别为4.5～15.3 ng·mL-1和5.6 ng·mL-1。因此木瓜蛋白酶与DNA反应体系为用RRS快速定量测定木瓜蛋白酶和DNA创造了条件。(2)胰岛素与ctDNA的相互作用的RRS和RNLS光谱及其分析应用研究在pH 7.4 Tirs-HCl缓冲溶液中,胰岛素与ctDNA相互作用生成复合物,此时将引起吸收光谱、圆二色光谱的变化,胰岛素的荧光发生猝灭,共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射的显著增强,并出现新的散射光谱。本文研究了胰岛素与ctDNA反应的适宜条件、影响因素及相关的分析化学性质,并结合RRS、吸收、荧光、圆二色光谱及其原子力显微镜讨论了胰岛素与DNA的相互作用,认为主要结合力包括氢键作用、静电引力、疏水作用力和芳环堆积作用力。并认为胰岛素进入B型构象的DNA大沟槽中,其Try-16、Tyr-26以及phe-24和phe-25四个芳香族氨基酸残基受到与DNA结合的影响。而反应产物体积增大、疏水性增强以及吸收-散射共振能量转移作用和DNA构象变化可能是散射增强的主要原因。此外,该反应体系还可用两者作探针RRS法对DNA和胰岛素进行相互测定。当用ctDNA作探针测定胰岛素时,检出限(36)为6.0ng.mL-1。当用胰岛素作探针测定ctDNA时,检出限(3σ)为7.2 ng.mL-1。两者均具有较高灵敏度和较好地选择性,而且方法简便快速,因此这一反应体系也对发展高灵敏度测定胰岛素类药物以及DNA的新方法有重要意义。(3)血红蛋白与sDNA相互作用的共振瑞利散射及荧光光谱研究在弱酸性介质中,血红蛋白与sDNA相互作用生成复合物,此时将引起共振瑞利散射(RRS)的显著增强,并出现新的散射光谱,同时血红蛋白的荧光发生明显猝灭。本文研究了血红蛋白与sDNA反应的适宜条件、影响因素及相关的分析化学性质。猝灭常数随温度的升高而增大,且加入sDNA后荧光寿命变短并结合荧光光谱及荧光寿命讨论了荧光猝灭的类型是动态猝灭。反应产物体积增大、疏水性增强、吸收-散射共振能量转移作用以及构象变化是散射增强的主要原因。此外,该反应体系还可用血红蛋白作探针RRS法和荧光法对DNA进行测定。方法具有较高的灵敏度,其检出限(3σ)分别为5.5 ng·mL-1和202.3 ng·mL-1。RRS法用于合成样品中DNA含量的测定,获得满意的效果。2.多肽与DNA相互作用的RRS和RNSL光谱及分析应用研究(1)多粘菌素B与DNA相互作用的RRS和RNSL光谱及分析应用研究在适宜的酸性介质中,多粘菌素B(PMB)能与DNA发生相互作用并形成复合物,此时将引起吸收光谱发生一定程度的变化,最大吸收波长略有红移并且吸光度有所提高。与此同时可观察到共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射的显著增强,并出现新的散射光谱。本文研究了PMB与DNA反应的适宜条件、影响因素及相关的分析化学性质,并以RRS增强和吸收以及圆二色光谱的变化讨论了PMB与DNA的主要结合力以及可能的结合模式。认为其主要结合力是静电引力、疏水作用力、氢键作用力以及芳基堆积作用等非共价键结合作用。此时PMB可能进入DNA的小沟槽并与磷酸链和相应的碱基结合而形成复合物。并认为反应产物体积增大、疏水性增强以及吸收-散射共振能量转移作用和DNA构象变化可能是散射增强的主要原因。此反应体系可用于RRS法测定DNA和PMB。当用ctDNA作探针测定PMB时,检出限(36)为9.8ng·mL-1。当用PMB作探针测定3中不同的DNA时,检出限(3σ)在3.8～9.0 ng·mL-1之间。两者均具有较高灵敏度而且方法简便快速,因此这一反应体系也对发展高灵敏度测定多肽类药物以及DNA的新方法有重要意义。(2)盐酸万古霉素与DNA相互作用的RRS和RNLS光谱及分析应用研究在适宜的酸性介质中,盐酸万古霉素(VCM)能与DNA发生相互作用并形成复合物,此时将引起共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射的显著增强,并出现新的散射光谱。本文研究了VCM与DNA反应的适宜条件、影响因素及相关的分析化学性质,并结合RRS、圆二色光谱、吸收光谱的变化和原子力显微镜讨论了VCM与DNA的相互作用。认为其主要结合力是静电引力、疏水作用力、氢键作用力以及芳基堆积作用等非共价键结合作用。此时VCM与DNA发生嵌入结合。并认为反应产物体积增大、疏水性增强以及吸收-散射共振能量转移作用和DNA构象变化可能是散射增强的主要原因。当用VCM作探针RRS法测定3种不同的DNA时,检出限(3σ)在6.8～11.1 ng.mL-1之间。方法具有较高灵敏度且简便快速,对发展高灵敏度测定DNA的新方法有重要意义。3.同多钨酸根RRS和RNLS光谱法测定硫酸多粘菌素B在pH 1.3～1.6的HCl-NaAc缓冲介质中,多粘菌素B(PMB)以及钨酸钠溶液本身的RRS和RNLS强度均十分微弱,但是当两者相互反应时,RRS和RNLS急剧增强。本文研究了PMB与钨酸根反应的适宜条件、影响因素及相关的分析化学性质,并以RRS增强、吸收和圆二色光谱的变化以及原子力显微镜照片讨论了PMB与同多钨酸的相互作用机理。并认为反应产物体积增大、疏水性增强以及吸收-散射共振能量转移作用可能是散射增强的主要原因。此反应体系可用于RRS、SOS和FDS法测定PMB。三种方法均具有较高的灵敏度,其中RRS法灵敏度最高,其检出限(3σ)为5.5 ng·mL-1。且方法具有较好的选择性,用于药物、人血清和尿液中多粘菌素B含量的测定,取得了满意的结果。更多还原

【Abstract】 Resonance Rayleigh scattering (RRS, or resonance light scattering, RLS) and resonance non-linear scattering (RNLS) are new techniques developed in recent years. They have received much attention due to their simplicity and high sensitivity. They have been extensively used to study and determination of biological macromolecules, pharmaceuticals, organic compounds and nanoparticles. However, for biological macromolecules such as proteins and nucleic acids, RRS method has been extensively used to research interactions between them and some small molecules such as dyes and medicines, and seldom used to study interactions between themselves. RRS is more sensitive to weak intermolecular forces (for instance, electrostatic force, hydrophobic force, hydrogen bonding and aromatic stacking interaction) and changes of molecular sizes, images and conformations than other molecular spectral methods such as absorption spectrum, fluorescence spectrum etc., this creates conditions for studying interactions of proteins with nucleic acids using RRS spectrum. When combining with common molecular spectral methods such as UV-Vis absorption spectrum, fluorescence spectrum, circular dichroism spectrum etc., it will provide much richer information for interactions between biological macromolecules. In addition, some high sensitive and selective analytical methods can be developed for determination of nucleic acids, proteins and polypeptides.The main contents and some conclusions of the dissertation are as follows:1. Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Proteins with DNA and Their Analytical Applications(1) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Papain with DNA and Their Analytical ApplicationsIn weak acidic medium, interaction between papain and DNA resulted in absorption spectral change, fluorescence quenching of papain and remarkable enhancement of RRS. The interaction types and binding modes were discussed by characteristics of RRS, absorption, fluorescence and circular dichroism spectra combining thermodynamic data. There are four interaction types including electrostatic attraction, hydrophobic force, hydrogen bonding and aromatic stacking interaction. Papain interacted with the major groove of ctDNA. Aromatic stacking interaction is the main reason of change of absorption spectrum and fluorescence quenching of papain. Surface enhanced scattering effect, resonance energy transfer effect, increase of molecular volume and conformational change make contribution to RRS enhancement. The enhanced RRS intensity (△I) is directly proportional to the concentration of ctDNA or papain. The detection limit (3σ) is 4.5-15.3 ng-mL-1 for DNA and 5.6 ng-mL-1 for papain. This creates conditions for determination of papain and DNA.(2) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Insulin with ctDNA and Their Analytical ApplicationsIn pH 7.4 Tris-HCl buffer solution, insulin interacted with ctDNA to form a complex, resulting in the change of absorption and circular dichroism spectra, the fluorescence quenching, enhancement of RRS, SOS and FDS as well as new scattering spectra. The optimum reaction conditions, influencing factors and relative analytical chemistry properties were investigated. The interaction mechanism was discussed combining scattering enhancement with absorption, fluorescence and circular dichroism spectra, as well as atomic force microscopy. There are four interaction types including electrostatic attraction, hydrophobic force, hydrogen bonding and aromatic stacking interaction. It was speculated that insulin entered the major groove of ctDNA, four aromatic residues including Try 16, Try 26, phe 24 and phe 25 were affected. The increase of molecular volume and hydrophobicity, resonance energy transfer and DNA conformation change are the main reasons of scattering enhancement. In addition, a RRS method based on this interaction was proposed to determination of insulin or DNA. When ctDNA is used as a probe to determine PMB, the detection limit (3σ) is 6.0 ng-mL-1. When insulin is used as a probe to determine ctDNA, the detection limit (3σ) is 7.2 ng-mL-1. The RRS method is very simple and sensitive. Therefore, it is significant of this interaction for developing a high sensitive method for determination of insulin or DNA.(3) Resonance Rayleigh Scattering and Fluorescence Rayleigh Scattering Spectra of Interaction of Hemoglobin with sDNA and Their Analytical ApplicationsIn weak acidic medium, interaction between hemoglobin and sDNA resulted in fluorescence quenching and remarkable enhancement of RRS of hemoglobin. And a new RRS spectrum appeared. The optimum reaction conditions, influencing factors and relative property of analytical chemistry were investigated. The quenching constant increased with increasing temperature, and the fluorescence life time decreased after adding sDNA, showing that the fluorescence quenching belongs to dynamic quenching. Surface enhanced scattering effect, resonance energy transfer effect, increase of molecular volume and conformational change make contribution to RRS enhancement. The enhanced RRS intensity (△I) or the quenched fluorescence intensity (△F) is directly proportional to the concentration of sDNA. The detection limits (3σ) of RRS and fluorescence quenching method are 5.5 ng mL-1 and 202.3 ng mL-1, respectively. RRS method was used to determine DNA in synthetic samples with satisfactory.2. Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Polypeptides with DNA and Their Analytical Applications(1) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Polymyxin B with DNA and Their Analytical ApplicationsA novel assay of DNA or polymyxin B (PMB) with a sensitivity at the nanogram level is proposed based on the measurement of enhanced resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (RNLS) including second order scattering (SOS) and frequency doubling scattering (FDS) resulting from interaction of PMB with DNA. The minor groove binding mechanism was suggested from RRS, absorption and circular dichroism spectra. The optimum reaction conditions, influencing factors and related analytical chemistry properties were tested. The interaction types and reasons of RRS enhancement were discussed. Linear relationships can be established between enhanced scattering intensity and DNA or PMB concentration. When ctDNA is used as a probe to determine PMB, the detection limit (3σ) is 9.8 ng mL-1. When PMB is used as a probe to determine DNA, the detection limit (3a) is in the range of 3.8-9.0 ng mL-1. Samples were analyzed satisfactorily. (2) Resonance Rayleigh Scattering and Resonance Non-linear Scattering Spectra of Interaction of Vancomycin with DNA and Their Analytical ApplicationsAssays were developed for DNA based on enhanced resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (including second order scattering and frequency doubling scattering) that result from the interaction of vancomycin with DNA. Vancomycin possesses the unusual property of promoting the aggregation of DNA which was concluded from the results obtained with RRS, absorption and circular dichroism spectroscopy and from atomic force microscope. The types of interaction and reasons of RRS enhancement are discussed. Linear relationships do exist over a wide range between the intensity of enhanced scattering and the concentrations of DNA. The detection limit (3σ) is in the range from 6.8 to 11.1 ng mL3. Resonance Rayleigh Scattering and Resonance Non-linear Scattering Methods for Determination of Polymyxin B Using Isopoly-tungstic Acid as a ProbeIn pH 1.3-1.6 HCl-NaAc buffer medium, RRS and RNLS of polymyxin B sulfate or isopoly-tungstic acid are very weak. However, when they react with each other, RRS and RNLS will be enhanced significantly. The optimum reaction conditions, influencing factors and relative property of analytical chemistry were investigated. The interaction mechanism is discussed combing RRS enhancement with atomic force microscopy, change of absorption and circular dichroism spectra. Increase of molecular volume, surface enhanced scattering and resonance energy transfer effect are the main reasons of scattering enhancement. RRS、SOS and FDS method for determination of PMB have been established. The three methods all have high sensitivity. The RRS has the highest sensitivity and the detection limit (3σ) for PMB is 5.5 ng-mL-1. The method has better selectivity. It was used to determination of PMB in practical samples with satisfactory results.更多还原