探討多胜肽與蛋白質分子間之交互作用

Intercations between Polypeptide and Protein molecules

賴潤錕(Jun-Kun Lai)

指導教授 ： 王勝仕

國立臺灣大學/工學院/化學工程學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.03366

摘要

蛋白質是生物體內重要的組成元素，在各種生理活動扮演著不可或缺的角色。酵素是一種以蛋白質為構造主體的催化劑，用以促進生物體內各種特定生化反應之進行。具有正確構形的蛋白質，才能發揮其本身的功能，進行各種反應，酵素亦是如此，而摺疊錯誤的蛋白質不僅可能失去其正常之功能，更可進而產生聚集體，導致疾病的發生。
本論文中，吾人嘗試對於多胜肽和蛋白質分子間之作用進行探討，藉由不同組成、比例之共聚多胜肽對蛋白質進行包覆、吸附等方式，進而維持蛋白質構形。此外，並利用共聚多胜肽試驗其對於蛋白質折疊、結構變化和聚集、類澱粉纖維生成之影響。
本論文第一部份，以木瓜酵素為模型酵素，藉由嵌段共聚多胜肽Lys-b-Gly形成囊泡(vesicle)後，再結合與矽之礦化作用(mineralization)形成矽網絡結構完成固定化。相對於未固定化和只由囊泡包覆之酵素，本系統polypeptide mediated silica-immobilized papain提升木瓜酵素對於pH值變化和熱之穩定性以及重覆使用率(例如在25°C下放置48小時後，以0小時的活性為基準，固定化之系統保持約68.5%的活性、未固定化之組別約29.6%之活性)。藉由多胜肽所形成之矽網絡結構保護木瓜酵素，進而維持固定化酵素之活性。由動力學分析得知，矽/多胜肽形成網絡結構之特性和構形，對於酵素之功能有著重要的影響。
本論文第二部份，以牛胰島素和母雞蛋白溶菌酶為模型，探討兩種隨機共聚多胜肽D,L-lysine-co-glycine和D,L-lysine-co-L-phenylalanine對於在試管內之蛋白質形成類澱粉纖維的影響。由穿透式電子顯微和ThT螢光光譜分析之實驗結果觀察得知，對於抑制類澱粉纖維生成之效果，主要受到所加入多胜肽之數量和組成比例的影響。例如在添加1 mM隨機共聚多胜的組別中，母雞蛋白類澱粉纖維的形成受到約35 %的抑制，而添加2 mM之組別，則可達到約65 %之抑制效果。而對於牛胰島素，加入0.5mM或是1mM隨機共聚多胜則可達到約25 %或是80 %之抑制效果。隨著共聚多胜肽中glycine 或是phenylalanine之比例提高，對於抑制類澱粉纖維生成有更好的效果。此外，吾人藉由圓二色光譜、ANS 螢光光譜、自身螢光光譜和SDS-PAGE等分析方法探討兩種蛋白質的結構變化以及抑制機制。母雞蛋白溶菌酶和共聚多胜肽間的作用力由實驗結果推測主要可能為氫建和疏水作用力。由研究結果，有助於了解類澱粉纖維生成的過程中，蛋白質分子結構變化及詳細機制，並可更進一步對治療類澱粉症提供有效的策略。

關鍵字

溶菌酶胰島素 ； 木瓜酵素 ； 類澱粉纖維 ； 共聚多胜肽抑制 ； 囊泡

並列摘要

Proteins are essential elements for living organisms and play a crucial role in various physiological activities. An enzyme is a protein molecule that serves as a biological catalyst. Proteins/enzymes with correct conformations are able to serve appropriate biological functions. Proteins that misfold may not only lose their normal biological function but also form aggregates which lead to a variety of diseases.
In this thesis, we attempt to explore the interactions between the polypeptides and protein molecules. The preservation of protein conformation was carried out by copolypeptides with different compositions through various processes such as entrapment, or adsorption. Moreover, we examined the effects of polypeptides on folding, structural changes, aggregation, and amyloid fibrillation of proteins.
In the first part of the thesis, we report the immobilization of a model enzyme, papain, within silica matrices by combining vesiclization of poly-L-lysine-b-polyglycine block copolypeptides with following silica mineralization. The polypeptide mediated silica-immobilized enzyme exhibits enhanced pH and thermal stability and reusability, comparing with the free enzyme and the vesicle encapsulated enzyme (e.g. after 48 hr incubation at 25°C, the percentage residual activities for the immobilized and the untreated papain samples were found to be ~68.5% and ~29.6% of that of the free papain at 0 hr, respectively). The enhanced enzymatic activity in the immobilized enzyme is due to the confinement of the enzyme in the polypeptide mediated silica matrices. Kinetic analysis shows that the enzyme functionality is determined by the structure and property of silica/polypeptide matrices.
In the second part of the thesis, with hen egg-white lysozyme and bovine insulin as the model systems, we show the results regarding the influences of two random copolypeptide D,L-lysine-co-glycine and D,L-lysine-co-L-phenylalanine on the in vitro protein fibrillation. Our TEM and ThT fluorescence results show that the observed inhibitory effects on amyloid fibrillation are significantly dependent on the amount and the composition ratio of polypeptide chains. For instance, the percentage reduction in hen lysozyme fibrillation was found to be approximately 35 % or 65% for the case of 1 mM or 2 mM random copolypeptide, respectively. The addition of 0.5 mM or 1 mM random copolypeptide results in approximately 25 % or 80% reduction, respectively, in fibrillogenesis derived from bovine insulin. The copolypeptides with a higher fraction of glycine or L-phenylalanine residue exhibit higher inhibitory potency against fibril formation. Moreover, we examine the structural changes in both proteins and inhibition mechanisms through CD spectroscopy, ANS fluorescence, intrinsic fluorescence spectroscopy, and SDS-PAGE. The major driving forces for the association of HEWL and copolypeptides are likely hydrogen bonding and hydrophobic interactions. We believe that the outcome of this work may contribute to the understanding of molecular mechanism(s) of the fibril formation and provide potential treatment strategies against the amyloid formation associated with amyloid disease.

並列關鍵字

lysozyme ； amyloid fibril ； insulin ； papain ； copolypeptide ； inhibition ； vesicle

參考文獻

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