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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题名:	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
作者:	許甯貽;Ning-Yi Hsu
贡献者:	化學工程與材料工程研究所
关键词:	鹼性抗生胜肽;微脂粒;indolicidin;peptide-membrane interaction;fluorescence spectroscopy
日期:	2009-07-02
上传时间:	2009-09-21 12:32:13 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	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。 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.
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