药物分子与生物大分子相互作用的电化学及光谱研究
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摘要
蛋白质是生物体中必不可少的基本物质,在生物体的生命活动中起着重要的作用。建立药物-蛋白结合的体外模型,了解结合的紧密程度、结合部位、结合力、结合数等问题,有助于在分子层次的水平上认知蛋白质与小分子的作用机理和规律,为生命科学研究提供有用的信息和数据。
本文采用紫外-可见吸收光谱、荧光发射光谱并结合电化学方法,对3,3',3'',3'''-亚乙四基-4-羟基香豆素与牛血清白蛋白的相互作用进行了详细研究；采用多种电分析方法研究了肌红蛋白、血红蛋白的直接电化学及它们对过氧化氢还原的电催化作用,并采用差分脉冲伏安法及i-t曲线法研究了血红蛋白与3,4-二(4-羟基-3-香豆素)-2,5-己二醇的相互作用。这些研究可为抗艾滋病药物的研制开发及人类最终认识生命过程的本质起到重要作用。本文的主要研究内容如下：
1.采用紫外-可见吸收光谱法、荧光光谱法详细研究了3,3',3'',3'''-亚乙四基-4-羟基香豆素(EHC)与牛血清白蛋白(BSA)在不同缓冲体系、不同pH条件下的结合作用及EHC对蛋白质构象的影响,计算了二者不同条件下的结合数、结合常数；根据计算得到的热力学参数确定了不同条件下的作用力类型,推测出EHC-BSA的结合模式；并通过电化学方法讨论了酸性条件下BSA对EHC电还原行为的影响。
2.采用滴涂法将肌红蛋白(Mb)固定于二醋酸纤维素及1-丁基-3-甲基咪唑四氟硼酸盐离子液体形成的复合膜中,采用电化学及光谱手段对Mb进行了表征,并研究了Mb在修饰玻碳电极上的直接电化学及其对H202还原的电催化行为。为氧化还原蛋白的固定化研究以及电化学传感器的构建提供了一种新的生物相容性材料。
3.研究了固定于壳聚糖／1-丁基-3-甲基咪唑四氟硼酸盐离子液体复合膜中的血红蛋白(Hb)在玻碳电极上的直接电化学,实现了Hb对H2O2还原的电催化行为；采用i-t曲线法及差分脉冲伏安法探讨了Hb与HIV-1蛋白酶和整合酶的口服非肽抑制剂3,4-二(4-羟基-3-香豆素)-2,5-己二醇(HCH)的相互作用,探索了一种新的研究相互作用的方法i-t曲线法,构建了H2O2及HCH的电化学传感器。
Protein is an essential material of organisms, it plays an important role in life activities. The establishment of drug-protein binding model in vitro, understanding of binding tightness, binding sites, binding force and binding constant contribute to cognitive the interaction mechanism and rule, which will provide available information and data in the life science research.
In this thesis, the interaction of 3,3',3",3"'-ethylenetetrakis-4-hydroxycoumarin (EHC) with bovine serum albumin (BSA) was studied detailedly by UV-vis absorption, fluorescence spectrometry and electrochemical technique. Besides, the direct electrochemical behavior of both hemoglobin (Hb) and myoglobin (Mb), as well as their electrocatalysis to the reduction of hydrogen peroxide were investigated by means of a variety of electrochemical methods. In addition, the interaction of Hb with (3,4)-bis-(4-hydroxyl-3-coumarin)-2,5-hexylene glycol (HCH) was investigated by differential pulse voltammetry and amperometry. These studies will play the important role in the development of anti-AIDS drugs and understanding of the nature of life processes.
This thesis consists of five chapters and the main contents are summarized as follows:
1. The interaction of 3,3',3",3'"-ethylenetetrakis-4-hydroxycoumarin (EHC) with bovine serum albumin (BSA) was studied by UV-vis absorption spectrometry and fluorescence spectrometry both in acetate (pH 4.70) and phosphate (pH 7.40) buffer solutions, then the effect of EHC on the conformation of BSA was investigated by UV-vis absorption spectrometry. The binding ration and binding constant were calculated. The interaction forces at different pHs were elucidated according to the thermody parameters, the binding site of EHC in BSA was concluded. Lastly the effect of BSA on the electrochemical reduction behavior of EHC was discussed by electrochemical method.
2. Myoglobin (Mb) was immobilized into the cellulose diacetate/ 1-butyl-3-methyl-imidazolium tetrafluoroborate (CDA-[bmim]BF4) film by the simple casting method, and Mb was characterized by UV-vis spectroscopy and electrochemical impedance spectroscopy. The direct electron transfer of Mb on the modified glassy carbon electrode and electrocatalysis of Mb towards H2O2 were investigated, based on which the hydrogen peroxide biosensor was constructed. This study provided an efficient matrix for the immobilization of redox protein and development of electrochemical biosensors.
3. The direct electron transfer of hemoglobin (Hb) immobilized in biocompatible chitosan (Chi) and ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim]BF4) on the modified glassy carbon electrode was investigated, which realized electrocatalysis of Hb towards H2O2. The interaction of Hb with the inhibitor of HIV-1 protease and integrase (3,4)-bis-(4-hydroxyl-3-coumarin)-2,5-hexylene glycol (HCH) was investigated by differential pulse voltammetry and amperometry which was a novel method in the field of binding studies. The biosensor exhibited an attractive ability to measure H2O2 or HCH.
本文采用紫外-可见吸收光谱、荧光发射光谱并结合电化学方法,对3,3',3'',3'''-亚乙四基-4-羟基香豆素与牛血清白蛋白的相互作用进行了详细研究；采用多种电分析方法研究了肌红蛋白、血红蛋白的直接电化学及它们对过氧化氢还原的电催化作用,并采用差分脉冲伏安法及i-t曲线法研究了血红蛋白与3,4-二(4-羟基-3-香豆素)-2,5-己二醇的相互作用。这些研究可为抗艾滋病药物的研制开发及人类最终认识生命过程的本质起到重要作用。本文的主要研究内容如下：
1.采用紫外-可见吸收光谱法、荧光光谱法详细研究了3,3',3'',3'''-亚乙四基-4-羟基香豆素(EHC)与牛血清白蛋白(BSA)在不同缓冲体系、不同pH条件下的结合作用及EHC对蛋白质构象的影响,计算了二者不同条件下的结合数、结合常数；根据计算得到的热力学参数确定了不同条件下的作用力类型,推测出EHC-BSA的结合模式；并通过电化学方法讨论了酸性条件下BSA对EHC电还原行为的影响。
2.采用滴涂法将肌红蛋白(Mb)固定于二醋酸纤维素及1-丁基-3-甲基咪唑四氟硼酸盐离子液体形成的复合膜中,采用电化学及光谱手段对Mb进行了表征,并研究了Mb在修饰玻碳电极上的直接电化学及其对H202还原的电催化行为。为氧化还原蛋白的固定化研究以及电化学传感器的构建提供了一种新的生物相容性材料。
3.研究了固定于壳聚糖／1-丁基-3-甲基咪唑四氟硼酸盐离子液体复合膜中的血红蛋白(Hb)在玻碳电极上的直接电化学,实现了Hb对H2O2还原的电催化行为；采用i-t曲线法及差分脉冲伏安法探讨了Hb与HIV-1蛋白酶和整合酶的口服非肽抑制剂3,4-二(4-羟基-3-香豆素)-2,5-己二醇(HCH)的相互作用,探索了一种新的研究相互作用的方法i-t曲线法,构建了H2O2及HCH的电化学传感器。
Protein is an essential material of organisms, it plays an important role in life activities. The establishment of drug-protein binding model in vitro, understanding of binding tightness, binding sites, binding force and binding constant contribute to cognitive the interaction mechanism and rule, which will provide available information and data in the life science research.
In this thesis, the interaction of 3,3',3",3"'-ethylenetetrakis-4-hydroxycoumarin (EHC) with bovine serum albumin (BSA) was studied detailedly by UV-vis absorption, fluorescence spectrometry and electrochemical technique. Besides, the direct electrochemical behavior of both hemoglobin (Hb) and myoglobin (Mb), as well as their electrocatalysis to the reduction of hydrogen peroxide were investigated by means of a variety of electrochemical methods. In addition, the interaction of Hb with (3,4)-bis-(4-hydroxyl-3-coumarin)-2,5-hexylene glycol (HCH) was investigated by differential pulse voltammetry and amperometry. These studies will play the important role in the development of anti-AIDS drugs and understanding of the nature of life processes.
This thesis consists of five chapters and the main contents are summarized as follows:
1. The interaction of 3,3',3",3'"-ethylenetetrakis-4-hydroxycoumarin (EHC) with bovine serum albumin (BSA) was studied by UV-vis absorption spectrometry and fluorescence spectrometry both in acetate (pH 4.70) and phosphate (pH 7.40) buffer solutions, then the effect of EHC on the conformation of BSA was investigated by UV-vis absorption spectrometry. The binding ration and binding constant were calculated. The interaction forces at different pHs were elucidated according to the thermody parameters, the binding site of EHC in BSA was concluded. Lastly the effect of BSA on the electrochemical reduction behavior of EHC was discussed by electrochemical method.
2. Myoglobin (Mb) was immobilized into the cellulose diacetate/ 1-butyl-3-methyl-imidazolium tetrafluoroborate (CDA-[bmim]BF4) film by the simple casting method, and Mb was characterized by UV-vis spectroscopy and electrochemical impedance spectroscopy. The direct electron transfer of Mb on the modified glassy carbon electrode and electrocatalysis of Mb towards H2O2 were investigated, based on which the hydrogen peroxide biosensor was constructed. This study provided an efficient matrix for the immobilization of redox protein and development of electrochemical biosensors.
3. The direct electron transfer of hemoglobin (Hb) immobilized in biocompatible chitosan (Chi) and ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim]BF4) on the modified glassy carbon electrode was investigated, which realized electrocatalysis of Hb towards H2O2. The interaction of Hb with the inhibitor of HIV-1 protease and integrase (3,4)-bis-(4-hydroxyl-3-coumarin)-2,5-hexylene glycol (HCH) was investigated by differential pulse voltammetry and amperometry which was a novel method in the field of binding studies. The biosensor exhibited an attractive ability to measure H2O2 or HCH.
引文
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