本文選題：牛血清白蛋白 + β-內(nèi)酰胺類(lèi)抗生素�。� 參考：《西北大學(xué)》2014年碩士論文

【摘要】：目的：闡明p-內(nèi)酰胺類(lèi)抗生素與牛血清白蛋白之間的分子識(shí)別和相互作用。 方法：以牛血清白蛋白為模型蛋白,選擇青霉素G、青霉素V和頭孢氨芐三種代表性抗生素,以熒光光譜法為主,同時(shí)輔以親和色譜法和分子對(duì)接技術(shù)進(jìn)行研究。通過(guò)不同溫度下抗生素對(duì)BSA的熒光猝滅光譜,獲得了猝滅常數(shù)(Ksv)、結(jié)合常數(shù)(Ka)和結(jié)合位點(diǎn)數(shù)(n)等參數(shù),依據(jù)焓變(ΔH)、熵變(AS)和自由能變(ΔG)等熱力學(xué)參數(shù)判斷作用力類(lèi)型,按照能量轉(zhuǎn)移理論求得結(jié)合距離,通過(guò)抗生素與位點(diǎn)標(biāo)志物的競(jìng)爭(zhēng)來(lái)確定結(jié)合位置,采用同步熒光光譜以及紅邊激發(fā)熒光位移(REES)獲得的信息討論相互作用時(shí)BSA構(gòu)象的改變,同時(shí)利用親和色譜法計(jì)算了兩者相互作用的結(jié)合位點(diǎn)數(shù)(n)和結(jié)合常數(shù)(Ka),最后,輔以分子對(duì)接技術(shù),展現(xiàn)了精確的分子識(shí)別和相互作用過(guò)程。 結(jié)果：青霉素G、青霉素V和頭孢氨芐等三種p-內(nèi)酰胺類(lèi)抗生素均能與BSA相互作用,并且猝滅BSA的內(nèi)源性熒光光譜。猝滅常數(shù)(Ksv)隨溫度升高而降低,說(shuō)明猝滅機(jī)制為靜態(tài)猝滅過(guò)程。相互作用均為單一的結(jié)合位點(diǎn),與標(biāo)志物的競(jìng)爭(zhēng)實(shí)驗(yàn)則證明了site Ⅱ位點(diǎn)為三種抗生素的共同結(jié)合位置。熱力學(xué)參數(shù)計(jì)算出作用力類(lèi)型為氫鍵或范德華力,而能量轉(zhuǎn)移理論則求得它們與BSA中色氨酸殘基的結(jié)合距離十分相似,且均小于7nm。同步熒光光譜以及REES則顯示BSA的構(gòu)象發(fā)生了不同程度的變化。熒光猝滅光譜所得到的猝滅常數(shù)(Ksv)、結(jié)合常數(shù)(Ka),熱力學(xué)求出的吉布斯自由能變(ΔG),同步熒光產(chǎn)生的紅移以及REES現(xiàn)象等結(jié)果共同驗(yàn)證了一個(gè)相同的結(jié)論,即此三種抗生素與BSA相互作用強(qiáng)弱由大到小的順序?yàn)轭^孢氨芐、青霉素V和青霉素G。同時(shí),分子對(duì)接給出的結(jié)合位置、結(jié)合距離、作用力大小和類(lèi)型、模擬相互作用高級(jí)結(jié)構(gòu)圖,親和色譜法測(cè)定的結(jié)合位點(diǎn)數(shù)n和結(jié)合常數(shù)Ka等信息,均與熒光光譜法所得結(jié)果基本一致。 結(jié)論：熒光光譜法、親和色譜法和分子對(duì)接技術(shù)組合用于研究p-內(nèi)酰胺類(lèi)抗生素與牛血清白蛋白之間的分子識(shí)別和相互作用。這些研究結(jié)果有助于了解p-內(nèi)酰胺類(lèi)抗生素通過(guò)血清白蛋白在體內(nèi)的運(yùn)輸和分布情況,對(duì)闡明抗生素的作用機(jī)制、藥代動(dòng)力學(xué)、藥物不良反應(yīng)以及藥物設(shè)計(jì)和新藥研發(fā)具有重要意義。
[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，

本文編號(hào)：1798644