香兰素及金属离子与溶菌酶相互作用的研究
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摘要
蛋白质是最重要的生物大分子之一,是生物性状的直接表达者,在生物体内担负着各种生理功能。在分子水平上研究小分子与蛋白质的相互作用以及金属离子存在时对这种作用的影响,是当前生命科学、化学和医药学等众多领域所共同关注的课题之一。因此,深入研究小分子物质(特别是药物小分子)与蛋白质的作用机理和规律,不仅对于阐明生命的奥秘,了解小分子在体内的分配、运输、代谢、功效以及蛋白质的构象有重要的意义,而且对药物的设计和筛选、疾病的诊断具有指导意义。
本文采用光谱法,分别对小分子香兰素和金属离子与溶菌酶的相互作用进行了研究。论文包括以下几个部分:
第一部分简要介绍了香兰素的特性,溶菌酶的结构和功能性质,并概括了当今研究小分子与蛋白质相互作用的主要方法。
第二部分以紫外-可见吸收光谱法、荧光光谱法和圆二色谱法研究了香兰素与溶菌酶之间的相互作用。荧光光谱结果表明,香兰素有规律的猝灭溶菌酶的内源荧光,且是一种因相互作用形成复合物而引起的静态猝灭机制;并测定了不同温度下的猝灭常数和表观结合常数,求得1个香兰素分子约与1个溶菌酶分子结合,两者间的作用力类型主要是氢键和范德华力;基于F?rster非辐射能量转移理论测定了两者间的结合距离是3.01nm。同时,通过同步荧光光谱法和圆二色谱法对溶菌酶的构象进行了检测,结果表明,随着香兰素的加入,溶菌酶的二级结构发生改变,并且α-螺旋的含量有增加的趋势。另外,考察了香兰素对溶菌酶酶活力的影响,结果变现为激活作用。
第三部分研究了金属离子与溶菌酶的相互作用。实验结果表明,金属离子均能使溶菌酶的特征紫外吸收峰增强和特征荧光峰猝灭。根据荧光强度变化和荧光猝灭公式求得铁离子、铜离子分别与溶菌酶作用的荧光猝灭常数、结合常数和结合位点数,推得其猝灭机制属于静态猝灭,并根据热力学参数确定了主要作用力类型。
第四部分研究了香兰素与金属离子共存时,对溶菌酶(如荧光猝灭机理、结合位点数、作用力类型、酶结构变化等)的影响情况。实验结果表明,香兰素与金属离子对溶菌酶的荧光具有双重猝灭效应,使相互作用增强,并且与二元体系相比,结合位点数发生了变化。
Protein is one of the most important biomacromolecules. It directly expresses biologic characters and performs a wide variety of physiological functions in the life. Exploring the interaction mechanisms between small molecules and proteins is of current interest in many research areas such as biology, chemistry, medicine and so on. Therefore, further investigating the binding mechanisms between small molecules (especially for those drug molecules) and proteins is worthy to be done. This work not only helps us to clarify the secret of life, understand the distribution, transportion, metabolism, efficacy of small molecules in the body and structural features of protein, but also provides valuable information in drug designing and screening, or disease diagnosis.
In this thesis, the small molecules such as vanillin (VAN) and metal ions (Men+) interaction with lysozyme (LYS) were studied using molecule spectroscopy. The paper was consisted of the following parts:
In the first part, the properties of VAN, the structures and functions of LYS were introduced briefly. The methods of the research on the interaction between small molecules and proteins were summarized.
In the second part, the interaction of VAN with LYS in near physiological buffer condition was investigated by UV-Vis absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The fluorescence analysis showed that VAN could regularly quench the endogenous fluorescence of LYS and the quenching was initiated from compound formation, it was a static quenching mechanism. The quenching constants and apparent binding constants were obtained at different temperature, and every VAN combines to one LYS molecule. It showed that the binding power between VAN and LYS was mainly the hydrogen bond and van der waals force. The binding distance was an area 3.01nm away from LYS based on F?rster’s non-radiation energy transfer theory. The conformation of LYS was analyzed by CD spectroscopy and synchronous fluorescence spectroscopy, the results indicated that conformation of LYS changed with the addition of VAN, and theα-helix was more tightly linked. The effect on the activity of LYS after treated by VAN was studied. We found that VAN can increase its enzymic activity.
In the third part, the interaction of different Men+ with LYS was investigated. It was found out that these Men+ all can increase the UV absorption and quench the fluorescence intensity of LYS. The quenching constants, binding constants and the number of binding sites were calculated according to the fluorescence effect and quenching equation, and the quenching mechanism belongs to static quenching between Fe~(3+) or Cu~(2+) and LYS. The main sorts of binding force can be known according to the thermodynamic parameters.
In the forth part, the effect of the coexistence of VAN and Men+ on the interaction characteristics among VAN, Men+ and LYS were investigated such as the change of quenching mechanism of fluorescence, the number of binding sites, the interaction forces, the conformation of enzyme. The experimental results showed that VAN and Men+ can further quench the fluorescence of LYS and enhance the interaction. The number of binding sites also changed compared with the binary system.
本文采用光谱法,分别对小分子香兰素和金属离子与溶菌酶的相互作用进行了研究。论文包括以下几个部分:
第一部分简要介绍了香兰素的特性,溶菌酶的结构和功能性质,并概括了当今研究小分子与蛋白质相互作用的主要方法。
第二部分以紫外-可见吸收光谱法、荧光光谱法和圆二色谱法研究了香兰素与溶菌酶之间的相互作用。荧光光谱结果表明,香兰素有规律的猝灭溶菌酶的内源荧光,且是一种因相互作用形成复合物而引起的静态猝灭机制;并测定了不同温度下的猝灭常数和表观结合常数,求得1个香兰素分子约与1个溶菌酶分子结合,两者间的作用力类型主要是氢键和范德华力;基于F?rster非辐射能量转移理论测定了两者间的结合距离是3.01nm。同时,通过同步荧光光谱法和圆二色谱法对溶菌酶的构象进行了检测,结果表明,随着香兰素的加入,溶菌酶的二级结构发生改变,并且α-螺旋的含量有增加的趋势。另外,考察了香兰素对溶菌酶酶活力的影响,结果变现为激活作用。
第三部分研究了金属离子与溶菌酶的相互作用。实验结果表明,金属离子均能使溶菌酶的特征紫外吸收峰增强和特征荧光峰猝灭。根据荧光强度变化和荧光猝灭公式求得铁离子、铜离子分别与溶菌酶作用的荧光猝灭常数、结合常数和结合位点数,推得其猝灭机制属于静态猝灭,并根据热力学参数确定了主要作用力类型。
第四部分研究了香兰素与金属离子共存时,对溶菌酶(如荧光猝灭机理、结合位点数、作用力类型、酶结构变化等)的影响情况。实验结果表明,香兰素与金属离子对溶菌酶的荧光具有双重猝灭效应,使相互作用增强,并且与二元体系相比,结合位点数发生了变化。
Protein is one of the most important biomacromolecules. It directly expresses biologic characters and performs a wide variety of physiological functions in the life. Exploring the interaction mechanisms between small molecules and proteins is of current interest in many research areas such as biology, chemistry, medicine and so on. Therefore, further investigating the binding mechanisms between small molecules (especially for those drug molecules) and proteins is worthy to be done. This work not only helps us to clarify the secret of life, understand the distribution, transportion, metabolism, efficacy of small molecules in the body and structural features of protein, but also provides valuable information in drug designing and screening, or disease diagnosis.
In this thesis, the small molecules such as vanillin (VAN) and metal ions (Men+) interaction with lysozyme (LYS) were studied using molecule spectroscopy. The paper was consisted of the following parts:
In the first part, the properties of VAN, the structures and functions of LYS were introduced briefly. The methods of the research on the interaction between small molecules and proteins were summarized.
In the second part, the interaction of VAN with LYS in near physiological buffer condition was investigated by UV-Vis absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The fluorescence analysis showed that VAN could regularly quench the endogenous fluorescence of LYS and the quenching was initiated from compound formation, it was a static quenching mechanism. The quenching constants and apparent binding constants were obtained at different temperature, and every VAN combines to one LYS molecule. It showed that the binding power between VAN and LYS was mainly the hydrogen bond and van der waals force. The binding distance was an area 3.01nm away from LYS based on F?rster’s non-radiation energy transfer theory. The conformation of LYS was analyzed by CD spectroscopy and synchronous fluorescence spectroscopy, the results indicated that conformation of LYS changed with the addition of VAN, and theα-helix was more tightly linked. The effect on the activity of LYS after treated by VAN was studied. We found that VAN can increase its enzymic activity.
In the third part, the interaction of different Men+ with LYS was investigated. It was found out that these Men+ all can increase the UV absorption and quench the fluorescence intensity of LYS. The quenching constants, binding constants and the number of binding sites were calculated according to the fluorescence effect and quenching equation, and the quenching mechanism belongs to static quenching between Fe~(3+) or Cu~(2+) and LYS. The main sorts of binding force can be known according to the thermodynamic parameters.
In the forth part, the effect of the coexistence of VAN and Men+ on the interaction characteristics among VAN, Men+ and LYS were investigated such as the change of quenching mechanism of fluorescence, the number of binding sites, the interaction forces, the conformation of enzyme. The experimental results showed that VAN and Men+ can further quench the fluorescence of LYS and enhance the interaction. The number of binding sites also changed compared with the binary system.
引文
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