Indolicidin及其類似物與微脂粒交互作用之熱力學研究

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姓名

張立煒(Li-wei Chang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

Indolicidin及其類似物與微脂粒交互作用之熱力學研究
(Thermodynamics of the interaction of indolicidin analogs with SUVs)

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摘要(中)

鹼性抗生胜肽Indolicidin(IL)因其具有廣效且迅速的抗菌性，同時不易造成微生物的抗藥性，因此近年來被廣泛的研究以期能成為新一代的抗生藥物。然而，IL因對紅血球有溶血活性而限制了其進一步的發展。本實驗室利用分子動態模擬，設計了其低溶血性的類似物ILK7、ILF89和ILK7F89，而這些類似物也各自展現出不同強弱的抗菌性。本研究主要是從熱力學的角度，探討IL及其類似物與仿紅血球、仿細菌生物膜系統微脂粒(small unilamellar vesicle，SUV)間的交互作用，進而觀察其不同溶菌與溶血性的根源。首先，我們利用平衡透析的實驗方法，獲得作用達平衡時胜肽吸附在微脂粒上的吸附量，並由胜肽初始濃度的不同進一步獲得其恆溫吸附曲線。由於傳統用來描述蛋白質分子吸附於基質上的Langmuir adsorption model無法恰當的描述IL及其類似物與磷脂膜之吸附作用，於是本研究提出了一個新的吸附模式：Two-site adsorption model。此吸附模式主要將IL及其類似物自我聚集(self-aggregate)成寡聚體之現象考量進吸附反應當中，而此模式也精確的模擬了胜肽分子於兩種不同生物膜之吸附作用。
接下來本研究利用van’t Hoff方程式的二次式關係式來求得反應的熱力學參數，並進一步的由熱力學參數分析IL及其類似物與仿生物細胞膜之吸附作用。從熱力學參數的分析我們可以得知：(1) IL及其類似物於液相中的寡聚現象主要是疏水力作用的一種亂度趨動(Entropy driven)之反應。(2) 無論與細菌細胞膜或紅血球細胞膜作用，IL及其類似物都傾向於以聚集體的方式吸附於其上。(3) 無論以單體形式或以聚集體形式吸附，IL及其類似物對生物膜的作用力大小都與其生物活性強弱呈現相當大的相關性。(4)藉由比較單體與聚集體之熱力學參數，可得知聚集體因為其吸附於膜上後可能產生分散現象，而造成細胞膜較大的擾亂，所以其吸附焓及亂度都較單體來的大。
綜合來看，IL及其類似物無論以單體或聚集體吸附，其與細胞膜之親和性皆與生物活性呈現正相關，又吸附作用中其傾向以聚集體吸附的程度又大於單體吸附，所以可知IL及其類似物之寡聚現象對其生物活性的影響相當重要。

摘要(英)

Cationic antimicrobial peptide Indolicidin(IL) are beimg increasingly recognized as potential candidates for antibacterial drugs in the face of the rapidly emerging bacterial resistance to conventional antibiotics in recent years.However,its hemolytic activity limit its application. We design it’s anlogoues ILK7, ILF89 and ILK7F89 according to the results of molecular dynamics simulation in order to reduce the hemolytic activity but remain the antimicrobial activity. Understand the interaction between peptides and cell membrane may help us to reach our goal. In this study, we want to investigate thermodynamics of the interaction between IL-analogues and lipid bilayers. Because of the less accuracy of Langmuir adsorption model, we proposed a new adsorption model: Two-site sdsorption model to simulate the isothermal adsorption curve obtained from equlilibrium dialysis experiment . The difference between the two models is the self-aggregate phenomenom of IL-anlogues is taken into consideration. The results of using Two-site adsorption model simulate the isothermal adsorption curve is quite well.
Then we use van’t Hoff quadratic equation to get the thermodynamic parameters of the peptide-membrane interaction. Through analysis of the thermodynamic parameters we could understand that (1) the self-aggregation of IL-anlogues is driven by entropy. (2) IL and its analogues tend to adsorb to both bacterial and mammalian red blood cell membrane in the aggregated form.(3) The biological activities of IL-analogues are well correlated with its binding affinity to lipid bilayer.(4) Because of the membrane interruption and membrane dispersion of peptides, the enthalpy and entropy change of aggregate adsorption are more than monomer adsorption. As a result, self-aggregation phonomenom is a important factor to affect its biological activity.

關鍵字(中)

★ 熱力學
★ 鹼性抗生胜肽
★ 微脂粒

關鍵字(英)

★ indolicidin
★ SUV
★ equilibrium dialysis
★ thermodynamics
★ antimicrobial peptides

論文目次

中文摘要i
Abstractii
誌謝iii
目錄iv
圖目錄viii
表目錄x
第一章緒論1
1-1 研究動機1
1-2 研究目的2
第二章文獻回顧3
2-1 細胞膜3
2-1-1 細胞膜的基本性質3
2-1-2 細胞膜的組成與結構4
2-1-3 常見磷脂質的種類與特性5
2-1-4 原核生物與真核生物細胞膜之差異8
2-2 微脂粒介紹8
2-2-1 微脂粒的結構8
2-2-2 微脂粒的形成與機制9
2-2-2-1 脂質分子間與脂質分子聚集之力10
2-2-2-2 微脂粒之形成11
2-2-3 微脂粒的分類13
2-2-4 微脂粒之製備方法14
2-2-5 微脂粒的應用16
2-3 鹼性抗生胜肽簡介17
2-3-1 鹼性抗生胜肽的發展17
2-3-2 鹼性抗生胜肽的特色18
2-3-3 鹼性抗生胜肽的抗生作用機制19
2-4 鹼性抗生胜肽Indolicidin其生物活性與作用機制22
2-4-1 Indolicidin簡述22
2-4-2 Indolicidin的抗菌活性及機制23
2-4-3 Indolicidin的溶血活性及機制24
2-4-4 影響Indolicidin生物活性之因素24
2-4-4-1 胜肽電荷變化25
2-4-4-2 胜肽結構變化25
2-4-4-3 胜肽疏水性變化25
2-4-4-4 胜肽自身聚集的影響 26
2-5 Indolicidin之類似物27
2-5-1 Indolicidin類似物的設計27
2-5-2 Indolicidin類似物的生物活性28
2-6 Indolicidin類似物寡聚現象之探討 29
2-7 鹼性抗生胜肽與磷脂膜交互作用之熱力學探討33
2-7-1 探討胜肽與磷脂膜交互作用熱力學的方法33
2-7-2 以van’t Hoff 方程式探討熱力學34
2-7-3 等溫吸附曲線研究35
2-7-3-1 以平衡透析獲得恆溫吸附曲線35
2-7-3-2 吸附模式的探討37
2-7-3-3 吸附機制的探討38
第三章實驗藥品、儀器設備與方法40
3-1 實驗藥品40
3-2 實驗儀器設備41
3-3 實驗方法42
3-3-1 Peptide溶液配置及定量42
3-3-1-1 UV-VIS光譜測定42
3-3-1-2 螢光光譜測定42
3-3-2 微脂粒之製備43
3-3-3 以平衡透析實驗得到IL及其類似物與微脂粒之恆溫吸附曲線44
3-3-3-1 透析膜選用及前處理44
3-3-3-2 平衡透析實驗46
3-4 IL及其類似物與微脂粒作用之熱力學現象探討47
3-4-1 恆溫吸附曲線之吸附模式分析47
3-4-2 利用數據處理軟體Matlab最適化恆溫吸附曲線48
3-4-3 熱力學參數計算49
第四章結果與討論50
4-1 不同溫度下Indolicidin及其類似物與微脂粒作用之恆溫吸附曲線50
4-1-1 不同溫度下IL及其類似物與POPG/POPC SUV作用之恆溫吸附曲線探討51
4-1-1-1 以Langmuir adsorption吸附模式分析52
4-1-1-2 以Two-site adsorption 吸附模式分析53
4-1-1-3 由吸附模式分析所得的參數討論與POPG/POPC之吸附現象54
4-1-2 不同溫度下IL及其類似物與POPC SUV 作用之恆溫吸附曲線57
4-1-2-1 以Two-site adsorption 吸附模式分析58
4-1-2-2 由吸附模式分析所得的參數討論與POPC之吸附現象59
4-2 Indolicidn及其類似物與POPG/POPC SUV作用之熱力學參數探討62
4-2-1 液相胜肽分子寡聚反應之熱力學參數探討62
4-2-2 單體吸附之熱力學參數探討64
4-2-3 聚集體吸附之熱力學參數探討66
4-2-4 單體吸附與聚集體吸附之熱力學現象比較68
4-3 Indolicidn及其類似物與POPC SUV作用之熱力學參數探討71
4-3-1 液相胜肽分子寡聚反應之熱力學參數探討71
4-3-2 單體吸附之熱力學參數探討73
4-3-3 聚集體吸附之熱力學參數探討75
4-3-4 單體吸附與聚集體吸附之熱力學現象比較77
4-4 IL及其類似物與兩種不同SUV系統作用之熱力學參數比較79
第五章結論82
第六章參考文獻84
附錄89

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指導教授

阮若屈(Ruoh-chyu Ruaan)

審核日期

2010-7-27

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