三种β-内酰胺类抗生素与牛血清白蛋白的分子识别和相互作用研究

发布时间：2018-04-24 23:05

  本文选题：牛血清白蛋白 + β-内酰胺类抗生素　； 参考：《西北大学》2014年硕士论文

【摘要】：目的：阐明p-内酰胺类抗生素与牛血清白蛋白之间的分子识别和相互作用。 方法：以牛血清白蛋白为模型蛋白,选择青霉素G、青霉素V和头孢氨苄三种代表性抗生素,以荧光光谱法为主,同时辅以亲和色谱法和分子对接技术进行研究。通过不同温度下抗生素对BSA的荧光猝灭光谱,获得了猝灭常数(Ksv)、结合常数(Ka)和结合位点数(n)等参数,依据焓变(ΔH)、熵变(AS)和自由能变(ΔG)等热力学参数判断作用力类型,按照能量转移理论求得结合距离,通过抗生素与位点标志物的竞争来确定结合位置,采用同步荧光光谱以及红边激发荧光位移(REES)获得的信息讨论相互作用时BSA构象的改变,同时利用亲和色谱法计算了两者相互作用的结合位点数(n)和结合常数(Ka),最后,辅以分子对接技术,展现了精确的分子识别和相互作用过程。 结果：青霉素G、青霉素V和头孢氨苄等三种p-内酰胺类抗生素均能与BSA相互作用,并且猝灭BSA的内源性荧光光谱。猝灭常数(Ksv)随温度升高而降低,说明猝灭机制为静态猝灭过程。相互作用均为单一的结合位点,与标志物的竞争实验则证明了site Ⅱ位点为三种抗生素的共同结合位置。热力学参数计算出作用力类型为氢键或范德华力,而能量转移理论则求得它们与BSA中色氨酸残基的结合距离十分相似,且均小于7nm。同步荧光光谱以及REES则显示BSA的构象发生了不同程度的变化。荧光猝灭光谱所得到的猝灭常数(Ksv)、结合常数(Ka),热力学求出的吉布斯自由能变(ΔG),同步荧光产生的红移以及REES现象等结果共同验证了一个相同的结论,即此三种抗生素与BSA相互作用强弱由大到小的顺序为头孢氨苄、青霉素V和青霉素G。同时,分子对接给出的结合位置、结合距离、作用力大小和类型、模拟相互作用高级结构图,亲和色谱法测定的结合位点数n和结合常数Ka等信息,均与荧光光谱法所得结果基本一致。 结论：荧光光谱法、亲和色谱法和分子对接技术组合用于研究p-内酰胺类抗生素与牛血清白蛋白之间的分子识别和相互作用。这些研究结果有助于了解p-内酰胺类抗生素通过血清白蛋白在体内的运输和分布情况,对阐明抗生素的作用机制、药代动力学、药物不良反应以及药物设计和新药研发具有重要意义。
[Abstract]:Objective: to elucidate the molecular recognition and interaction between p- lactam antibiotics and bovine serum albumin (BSA). Methods: bovine serum albumin (BSA) was used as model protein. Three representative antibiotics, penicillin G, penicillin V and cefalexin, were selected and studied by fluorescence spectrometry, affinity chromatography and molecular docking technique. Based on the fluorescence quenching spectra of BSA by antibiotics at different temperatures, the parameters such as the quenching constant (Ksvn), binding constant (K) and binding site number (n) were obtained. The type of force was determined by thermodynamic parameters such as enthalpy change (螖 H ~ (2)), entropy change (BSA) and free energy variation (螖 G). According to the energy transfer theory, the binding distance was determined by the competition between antibiotics and site markers. The conformation changes of BSA were discussed by synchronous fluorescence spectrum and red edge excited fluorescence shift (REESs). At the same time, affinity chromatography was used to calculate the number of binding sites (n) and the binding constant (KA). Finally, the molecular docking technique was used to show the precise molecular recognition and interaction process. Results: penicillin G, penicillin V and cefalexin could interact with BSA and quench the endogenous fluorescence spectra of BSA. The quenching constant Ksv) decreases with the increase of temperature indicating that the quenching mechanism is a static quenching process. The interaction was a single binding site, and the competitive experiment with the marker showed that the site 鈪，

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