金属离子及其配合物与生物大分子相互作用的研究及分析应用

【作者】 郭长英；

【导师】 吴霞；

【作者基本信息】 山东大学 ， 分析化学， 2006， 硕士

【摘要】 机体是由数以亿万计分子量大小不等的分子组成。蛋白质和核酸是体内主要的生物大分子。核酸是遗传信息的载体，是生物物种延续、物种进化的决定因素；蛋白质担负着各种生理功能，是生物性状的直接表达者。它在人体代谢中扮演极为重要的角色，从整体上维持生物体新陈代谢活动的进行，人类绝大多数疾病都是由蛋白质异常引起的。因此核酸与蛋白质的定量分析在生命科学、生化药物、食品分析及临床检验中都有着重要作用。深入研究小分子物质与核酸和蛋白的作用，建立核酸，蛋白快速简便的分析方法，对分子水平上阐明生命的奥秘、开发新型药物、攻克许多疑难病症以及促进信息科学的发展都具有重要的意义，是当前生物分析化学研究的前沿和热点。 本文致力于寻找新的核酸和蛋白质探针，以建立灵敏的定量检测的方法，以荧光和共振光散射技术为主要研究手段。同时应用吸收光谱技术，表面张力测定技术，电势电泳技术，透射电子显微技术等手段进行了机理研究和讨论。本文共五部分。第一部分综述了核酸和蛋白质发光探针的基本原理、研究进展、发展趋势。 在论文的第二部分，我们以Eu³⁺-BA的配合物为探针建立了灵敏的核酸分析方法。研究表明，在CTMAB存在下，核酸的加入能显著增强Eu³⁺-BA配合物中Eu³⁺的荧光，且荧光增强程度与核酸的浓度在一定范围内成正比。在最佳实验条件下，fsDNA、ctDNA和RNA的线性范围分别为1.0×10^-9-5.0×10^-6g/mL、3.0×10^-9-1.0×10^-6g/mL和8.0×10^-9-1.0×10^-6g/mL。它们的检出限分别为0.33、0.21和0.99ng／mL。该方法用于实际样品拟南芥中DNA含量的测定，结果令人满意。机理研究表明，CTMAB与DNA所形成的离子聚合物为Eu³⁺-BA配合物提供了疏水的微环境，从而使其荧光增强。另外，我们推测形成Eu³⁺-BA配合物之后多余的BA分子可以通过疏水作用进入到CTMAB与DNA所形成的离子缔合物中，并通过分子间作用力将能量转移给Eu³⁺离子。所以我们认为，体系的荧光增强源于疏水微环境的形成和分子间能量转移。 论文的第三部分中，Tb³⁺-BA配合物被用作蛋白质的荧光探针，研究了蛋白质与配合物间的作用机理。实验表明，在SDBS存在下，蛋白质的加入能显著增更多还原

【Abstract】 Organism is constituted by hundreds of millions of molecules with different molecular weight. Nucleic acids and proteins are the most important biomacromolecule. Nucleic acid is the carrier of genetic information. It is also the determination factor for species’ continuation and evolution. Proteins are the carrier of many physiological functions, and are also the direct expresser of physiological characters. Protein play an important role in the metabolism of human body, and the most disease of human being is caused by the abnormal of protein. So the quantitative analysis of nucleic acid and protein are important in life science, biochemical medicament, food analysis and clinic analysis. It is very important for researchers to explore the secret of life, to develop new functional medicine for curing many difficult diseases and promote the development of information science. What we should do is to further penetrate the interaction between small molecules and nucleic acids and protein, and to develop rapid and convenient assays for nucleic acids and protein. Therefore, it is a hot point in bio-analytical chemical research.This thesis focused on the development of new probes of nucleic acids and proteins and to establish sensitive methods for the quantitative determination of them, used the fluorescence and resonance light scattering （RLS） as the primary technique. The absorption, determination of surface tension, electrophoresis by TV, TEM techniques were also used to study the interaction mechanism. In the first part of this thesis, we commented on the basic theory and progress of luminescent probes for nucleic acid and protein. The developing tendency of this area was also included in this part. In the other parts, four systems for the determination of nucleic acids and proteins.In the second section, we developed Eu³⁺-benzoylacetone （BA） complex as fluorescent probe to detect micro amounts of nucleic acids. The study indicated that in the presence of cetyltrimethyl-ammonium bromide （CTMAB） the instrinc fluorescence intensity of Eu³⁺ in Eu³⁺-benzoylacetone （BA） complex can be greatly更多还原