以螢光光譜探討Indolicidin及其類似物與微脂粒之交互作用

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許甯貽(Ning-Yi Hsu) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

以螢光光譜探討Indolicidin及其類似物與微脂粒之交互作用
(Investigation of the interactions of indolicidin analogs with SUVs by Fluorescence Spectroscopy Analysis)

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摘要(中)

鹼性抗生胜肽Indolicidin(IL)為13個胺基酸所組成之短鏈胜肽，對許多微生物具有抗生的活性，包括細菌、病毒、真菌，由於具有經濟效益及廣效的抗菌活性，因此IL被認為是具有潛力的抗生藥物。然而，Indolicidin對於人類紅血球的溶血活性限制其在醫療應用的進一步發展。在過去文獻中，Ahmad等人[1]曾提出IL之溶血行為可能跟其自身聚集(Self-aggregation)有關。以此作為基礎，在本研究的第一部分，主要為利用螢光光譜探討IL及其類似物之寡聚(Oligomerization)行為，發現這些胜肽在水相的螢光有自身淬滅(Self-quenching)現象，可能為寡聚體形成所造成。
　　更進一步地，IL及其類似物在乙醇中的螢光圖譜及丙烯醯胺螢光淬滅實驗也證實了寡聚體的存在，且寡聚程度由大到小為：IL > IL-K7 > IL-F89 > IL-K7F89。此外，藉由探討IL及其類似物與細胞膜的作用可了解影響生物活性的關鍵因素。因此，本研究利用POPG /POPC 微脂粒作為細菌細胞膜的模型，以及POPC微脂粒作為紅血球細胞膜的模型，探討IL及其類似物之寡聚體與仿細胞膜之間的作用。結合作用後螢光光譜及胜肽在乙醇中的螢光光譜，可了解IL及其類似物吸附於微脂粒的多寡及寡聚體分散程度，並以雙重螢光淬滅方法獲得插膜深度的資訊。在與負電性的POPG /POPC微脂粒作用中，發現這些胜肽幾乎全部插入微脂粒之疏水碳鏈區，且在低濃度時正電荷較多的IL-K7及IL-K7F89插膜較深，且兩者在微脂粒之疏水區較為散開；相反地，這些胜肽對於電中性之POPC微脂粒的吸附量較低，恰好與寡聚的程度正相關，且並未出現寡聚體散開的現象。綜合本研究，IL及其類似物對於抗菌的關鍵可能來自於胜肽寡聚體插膜對磷脂質的擾亂，而溶血活性可能主要與其吸附在紅血球上的量或在水相寡聚程度有關。

摘要(英)

Indolicidin(IL) is a tryptophan(Trp)-rich cationic peptide isolated from bovine neutrophils. In the past two decades, it’s found that its bactericidal activity toward many kinds of pathogens. However, its hemolytic activity limits its application. Ahmad et al. has suggested that the self-association of IL should be related to its hemolytic behavior. Based on this suggestion, the investigations of peptide oligomerization have been conducted with fluorescence spectroscopy. Both of intrinsic fluorescence spectroscopy and fluorescence quenching by acrylamide indicated that IL and its analogs show various degrees of oligomerization, and follow the order: IL > IL-K7 > IL-F89 > IL-K7F89. Furthermore, to investigate the interactions of peptide oligomers and phospholipids, the POPG/POPC(1:1) and POPC small unilamellar vesicles (SUVs) were used in this study, which are representatives of bacterial cell membrane and the cell membrane of erythrocytes, respectively. The adsorption amount and oligomer dispersion were identified by the fluorescence spectra of peptides both in SUV and in ethanol. Also, the dual quenching of Trp by acrylamide and 10-doxylnonadecane determined the insertion depth of peptides. It was found that IL and its analogs show high affinities to the POPG/POPC SUVs, and may disperse in the SUV, especially for IL-K7 and IL-K7F89. On the other hand, IL and its analogs have lower affinities to the POPC SUVs. Interestingly, the hemolytic activities followed the same order as that of their affinities and also the oligomerization degrees. As a result, the phospholipid perturbation caused by peptides’ insertion and dispersion may be crucial to their high antimicrobial activities. As for the various hemolytic activities of IL and its analogs, they could be derived from the adsorption amount or insertion depth.

關鍵字(中)

★ 螢光光譜
★ 鹼性抗生胜肽
★ 微脂粒

關鍵字(英)

★ indolicidin
★ peptide-membrane interaction
★ fluorescence spectroscopy

論文目次

摘要 i
Abstract ii
目錄 iii
圖目錄 v
表目錄 vi
一、緒論 1
1-1　　研究動機 1
1-2　　研究目的 2
二、文獻回顧 3
2-1　　鹼性抗生胜肽 3
2-1-1　鹼性抗生胜肽的發展 3
2-1-2　鹼性抗生胜肽的抗生機制 3
2-2　　鹼性抗生胜肽Indolicidin的生物活性及作用機制 8
2-2-1　鹼性抗生胜肽Indolicidin 8
2-2-2　Indolicidin的抗菌活性 9
2-2-3　Indolicidin的抗菌機制 10
2-2-4　Indolicidin的溶血活性及機制 11
2-3　　蛋白質或胜肽聚集之探討 12
2-3-1　探討蛋白質聚集之方法 12
2-3-2　以螢光光譜觀測蛋白質聚集行為 13
2-3-2-1螢光光譜儀量測原理 13
2-3-2-2色胺酸的螢光光譜 15
2-3-2-3螢光淬滅 17
2-4　　胜肽與微脂粒之交互作用 18
2-4-1　微脂粒簡介 18
2-4-2　Indolicidin與微脂粒作用的螢光光譜分析 19
2-4-2-1色胺酸的自身螢光光譜 19
2-4-2-2利用Dye leakage探討抗生胜肽對磷脂質膜的擾動 19
2-4-2-3利用螢光淬滅探討蛋白質在膜內的深度 19
2-5　　Indolicidin之類似物 21
2-5-1　Indolicidin類似物的設計 21
2-5-2　Indolicidin類似物的生物活性 22
三、實驗藥品、設備與方法 24
3-1　　實驗藥品 24
3-2　　實驗設備 25
3-3　　實驗方法 26
3-3-1　Indolicidin及其類似物在緩衝液中的聚集及結構探討 26
3-3-1-1　UV-VIS吸收值 26
3-3-1-2　螢光光譜測定 26
3-3-1-3　圓二色光譜測定 26
3-3-1-4　螢光淬滅實驗 26
3-3-2　Indolicidin及其類似物在微脂粒中的行為及結構探討 27
3-3-2-1　微脂粒之製備 27
3-3-2-2　IL及其類似物與微脂粒之交互作用 29
3-4　　計算方法 31
3-4-1　Indolicidin及其類似物之聚集行為 31
3-4-2　Indolicidin及其類似物與微脂粒之交互作用 31
四、結果與討論 35
4-1　　Indolicidin及其類似物的在緩衝液中聚集行為 35
4-1-1 不同濃度的Indolicidin及其類似物之UV280吸收值 35
4-1-2　不同濃度的Indolicidin及其類似物之螢光強度 36
4-1-3　Indolicidin及其類似物在PBS緩衝液中的單位螢光強度 38
4-1-4　Indolicidin及其類似物的結構探討 39
4-1-5　Indolicidin及其類似物在乙醇中的單位螢光強度 40
4-1-4　以丙烯醯胺進行螢光淬滅實驗 43
4-2　　Indolicidin及其類似物與POPG/POPC微脂粒之交互作用 45
4-2-1　以螢光光譜觀測IL及其類似物與POPG/POPC微脂粒之交互作用 45
4-2-2　以螢光光譜探討吸附在POPG/POPC SUV的胜肽寡聚體 47
4-2-2-1胜肽寡聚體吸附於POPG/POPC SUV之分散index 48
4-2-2-2胜肽寡聚體吸附於POPG/POPC SUV之10-DN淬滅率 49
4-2-2-3胜肽寡聚體吸附於POPG/POPC SUV之丙烯醯胺淬滅率 50
4-3　　Indolicidin及其類似物與POPC微脂粒之交互作用 52
4-3-1　以螢光光譜觀測Indolicidin及其類似物與POPC微脂粒之交互作用 52
4-3-2　以螢光光譜探討吸附於POPC SUV的胜肽寡聚體 54
4-3-2-1胜肽寡聚體在POPC SUV的吸附百分比 54
4-3-2-2胜肽寡聚體吸附於POPC SUV之分散index 55
4-3-2-3胜肽寡聚體吸附於POPC SUV之10-DN淬滅率 56
4-3-2-4胜肽寡聚體在POPC SUV中之丙烯醯胺淬滅率 56
五、結論 58
六、參考文獻 59

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指導教授

阮若屈(Ruoh-Chyu Ruaan)

審核日期

2009-7-16

推文