蛋白质和核酸与小分子的相互作用及其分析应用的研究

【作者】 王飞；

【导师】 杨景和； 申大忠；

【作者基本信息】 山东大学 ， 分析化学， 2006， 博士

【摘要】 蛋白质和核酸是生命科学与生物化学中两类最重要的生物大分子。蛋白质担负着各种生理功能，从整体上维持生物体新陈代谢活动的进行，是生物性状的直接表达者，而核酸是揭示生命遗传与变异的主要物质。因此，深入研究小分子物质与蛋白质和核酸的作用机理，特别是建立蛋白质以及核酸快速、简便的分析方法，对分子水平上阐明生命的奥秘等方面具有重要的意义，是当前生物分析化学研究的前沿和热点。 本论文以分析化学，生物化学与生物物理为研究背景，利用荧光技术、光散射技术、吸收光谱技术、圆二色谱技术、小角散射技术、核磁共振技术、扫描电镜和压电传感器等手段研究了小分子与蛋白质、核酸的作用机理，建立了蛋白质以及核酸快速、准确、简便、灵敏而选择性的分析方法。论文共分五个部分。 论文的第一部分评述了蛋白质和核酸发光探针的基本原理、研究进展和发展趋势，以及小分子物质与生物大分子相互作用的研究手段等，共引用文献192篇。 论文的第二部分中，在两个激发波长下，研究了morin-Al³⁺-表面活性剂-蛋白质体系的荧光增强效应。研究发现在阴离子表面活性剂SDBS和阳离子表面活性剂CTAB存在下蛋白质都能增强morin-Al³⁺的荧光。同时实验了morin-Al³⁺-SDBS-BSA和morin-Al³⁺-CTAB-BSA体系的荧光增强效应的最佳实验条件，建立了BSA的浓度与体系的荧光强度之间的线性方程，并将该效应应用于蛋白质的检测中。在morin-Al³⁺-SDBS-蛋白质体系中，检测BSA、HSA和EA的线性范围分别为0.010-13.0μg／mL、0.050-12.0μg／mL、0.040-12.0μg／mL（420nm激发）和0.0070-12.0μg／mL、0.0080-15.0μg／mL、0.020-15.0μg／mL（280nm激发），检出限分别为5.0ng／mL、16ng／mL、18ng／mL（420nm激发）和2.0ng／mL、5.6ng／mL、12ng／mL（280nm激发）；在morin-Al³⁺-CTAB-蛋白质体系中，420nm和280nm激发波长下，检测BSA、HSA和EA的线性范围分别为0.0050-20.0μg／mL、0.020-15.0μg／mL、0.040-13.0μg／mL和0.0030-20.0μg／mL、0.0060-20.0μg／mL、0.010-18.0μg／mL，检出限分别为2.6ng／mL、14ng／mL、16ng／mL和0.70ng／mL、4.1ng／mL、5.6ng／mL，显然，280nm激发下的灵敏度较更多还原

【Abstract】 Proteins and nucleic acids are important biomolecules of life science and biochemistry. Proteins are the carrier of many physiological functions, and are also the direct expresser of physiological characters; whereas nucleic acids are the primary materials in showing the heredity and variation of life. It is very important for researchers to expound the secrets of life, to develop new functionalized medicine to surmount many difficult diseases. What we should do is to further penetrate the interaction mechanism between small molecules and proteins or nucleic acids, to develop rapid and convince assay for proteins and nucleic acids. This project is the forward position and hot point in biochemical and biophysical researches.Based on the research of analytical chemistry, biochemistry and biophysics, this thesis studied the interaction mechanism between protein and small molecules, and nucleic acid and small molecules using the research techniques including fluorescence, absorption, RLS, CD, SAXS, NMR, SEM, TEM, AFM and QCM. Some rapid, accurate assays with high sensitivity and selectivity were developed for protein and nucleic acid. The main conclusions are listed as below:In the first section, we summarize the basic theory and progress of luminescent probes for proteins and nucleic acids, and comment on the experimental techniques to investigate the interaction between biomacromolecules and small molecules. 192 references are cited here.In the second section, at two excitation wavelengths, fluorescence enhancement effect in morin-Al³⁺-surfactant-protein system was reported here. From the research, we found that protein could enhance the fluorescence of morin-Al³⁺ in the presence of anionic surfactant SDBS or cationic surfactant CTAB. Different effects were tested on the fluorescence intensity of the morin-Al³⁺-SDBS-BSA and morin-Al³⁺-CTAB-BSA systems, and under the optimum conditions, the calibration graph for proteins were obtained. In morin-Al³⁺-SDBS-protein system, the enhanced intensity of fluorescence is in proportion to the concentration of proteins in the range of 0.010-13.0μg/mL and 0.0070-12.0μg/mL for BSA, 0.050-12.0μg/mL and 0.0080-15.0μg/mL for HSA and 0.040- 12.0μg/mL and 0.020-15.0μg/mL for EA at 420m excitation wavelength and 280nm excitation wavelength, respectively. Their detection limits （S/N=3） are 5.0 ng/mL and 2.0 ng/mL for BSA, 16 ng/mL and 5.6 ng/mL for HSA and 18 ng/mL and 12 ng/mL for EA at 420nm excitation wavelength and 280nm excitation wavelength,更多还原