類澱粉胜肽於水溶液中聚集行為之動力學與熱力學探討

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蔡蕙婷(Hui-Ting Tsai) 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

類澱粉胜肽於水溶液中聚集行為之動力學與熱力學探討
(Kinetics and thermodynamics studies of β-amyloid peptide aggregation in aqueous solutionKinetics and thermodynamics studies of β-amyloid peptide aggregation in aqueous solution)

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摘要(中)

本研究主要是針對類澱粉胜肽（β-amyloid，Aβ）於不同的水溶液環境（培養溫度、緩衝液鹽濃度及pH值）中聚集行為的探討，藉以了解不同環境對於Aβ（1-40）聚集機制之影響。整個研究主題分為兩部分，第一部分是利用圓二色光譜儀及螢光光譜儀隨時間測量Aβ在不同培養環境中二級結構變化及纖維的生成，藉以探討其聚集的動力學機制，再輔以原子力顯微鏡得到其聚集後的形貌圖。第二部份是利用恆溫滴定微卡探討Aβ（1-40）於不同環境、不同聚集階段時稀釋熱的改變，以期得到Aβ（1-40）聚集時的熱力學機制。
在Aβ（1-40）聚集的過程中，其二級結構會由random coil直接轉變為β-sheet，可以two-state model來描述，且培養溫度及鹽濃度越高，Aβ越易摺疊成β-sheet，表示Aβ（1-40）是以疏水作用力來穩定β-sheet之二級結構。在Aβ（1-40）聚集動力學部分，其nucleation的速率常數遠小於elongation的速率常數，這說明在Aβ（1-40）聚集的過程中，成核反應為速率決定步驟。在熱力學部分，由熱量變化發現Aβ（1-40）成核反應是以疏水作用力為主導，而Aβ（1-40）形成纖維結構之後，纖維與纖維之間則帶有靜電排斥力。除此之外，環境鹽濃度也會影響到Aβ（1-40）的聚集機制，在低鹽的環境下單體的Aβ（1-40）與纖維兩側之間有靜電排斥力，因此只會與纖維的兩端結合，所產生之纖維較為細長。隨著環境鹽濃度提高，單體之Aβ（1-40）可能會與最初形成之纖維結構的兩側結合，除此之外，纖維外部所帶之靜電排斥力會被鹽類遮蔽，而導致纖維與纖維之間也會產生吸附聚集的情形，因此所產生之纖維較為粗短。

摘要(英)

In this study, we investigated the aggregation of Aβ（1-40） in various conditions including different incubated temperature、salt concentration and pH. The objectives of this investigation went in two fold and were achieved by the following studies : First, we used Circular Dichroism (CD) to observe the secondary structure transition during Aß aggregation, and we used Thioflavine T（ThT）fluorescence probe and AFM to monitor the fibrils’ formation and morphology. Second, we use Isothermal Titration Microcalorimetry (ITC) to obtain the dilution heat at different Aß aggregate states. The thermodynamic information during Aβ aggregation was then discussed with the CD and fluorescence data.
The variations of secondary structure of Aβ（1-40） obtained by CD exhibite a temperature and salt concentration dependent behavior. The transition from random coil to ß-sheet can be described by a two-state model. It indicates that the hydrophobic interactions is important for the intermolecular ß-sheet structure stability of Aβ（1-40） in solution. Besides, from the time dependent CD spectrum fitting, we found that the rate constant of nucleation is much smaller than that of elongation in the process of aggregation of Aβ（1-40）, indicating nucleation is the rate-determining step. From thermodynamic analysis, the dilution heat（ΔHdil） of Aß(1-40) monomer obtained by ITC is an endothermic reaction, and as temperature increases, the ΔHdil increases. The results demonstrate that hydrophobic interactions play a central role in the nucleation process of Aß(1-40). Moreover, we also observed that the mechanism of Aβ（1-40）aggregation in various salt concentration is different. In low salt concentration, monomer deposition is only on the ends of fibrils and resultes in long and thin fibrils. In contrast, monomer and fibril and fibril and fibril association occures in high salt concentration due to the reduced electrostatic repulsive forces, and the association involves lateral interactions resultes in short and thick fibrils. These results were demonstrated by AFM observation.

論文目次

中文摘要...................................................................I
Abstract .................................................................II
目錄 ......................................................................V
圖目錄 ..................................................................VII
表目錄 ................................................................ XIII
第一章序論............................................................... 1
第二章文獻回顧........................................................... 2
2.1 阿茲海默症與類澱粉胜肽................................................ 2
2.1.1 阿茲海默症（Alzheimer’s Disease，AD）.............................. 2
2.1.2 類澱粉胜肽（β-amyloid，Aβ） ........................................ 6
2.1.3 類澱粉胜肽的結構及特性.............................................. 9
2.1.4 影響類澱粉胜肽聚集之因素........................................... 16
2.1.4.1 Aβ 序列.......................................................... 16
2.1.4.2 Aβ 濃度.......................................................... 18
2.1.4.3 Aβ 培養溫度...................................................... 21
2.1.4.4 緩衝溶液pH 值.................................................... 23
2.1.4.5 緩衝溶液鹽濃度................................................... 25
2.1.4.6 Aβ 初始溶劑效應.................................................. 28
2.2 圓二色光譜儀（Circular Dichroism，CD） .............................. 30
2.2.1 圓二色光譜儀測量原理............................................... 30
2.2.2 蛋白質或多胜肽的圓二色光譜......................................... 32
2.3 恆溫滴定微卡計....................................................... 34
2.3 Thioflavine T（ThT）螢光............................................. 37
第三章實驗藥品與儀器設備................................................ 38
3.1 實驗藥品............................................................. 38
3.2 儀器設備............................................................. 39
3.3 實驗步驟............................................................. 40
3.3.1 溶液配置........................................................... 40
3.3.1.1 Aβ 溶液製備...................................................... 40
3.3.1.2 不同pH 值及鹽濃度之緩衝溶液...................................... 40
3.3.1.3 清洗溶液......................................................... 41
3.3.2 恆溫滴定微卡計實驗................................................. 41
3.3.3 圓二色光譜儀實驗................................................... 41
3.3.4 ThT 螢光光譜儀實驗................................................. 42
3.3.5 原子力顯微鏡實驗................................................... 42
第四章結果與討論........................................................ 43
4.1 Aβ（1-40）於水溶液中聚集機制之探討................................... 43
4.1.1 培養溫度對Aβ（1-40）聚集之影響..................................... 43
4.1.2 緩衝液鹽濃度對Aβ（1-40）聚集之影響................................. 56
4.1.3 緩衝液pH 值對Aß（1-40）聚集之影響.................................. 68
4.1.4 Aβ（1-40）二級結構變化數據之動力學分析............................. 74
4.2 Aβ（1-40）於水溶液中聚集熱力學之探討................................. 80
4.2.1 Aβ（1-40）不同培養溫度之熱力學探討................................. 80
4.2.2 Aβ（1-40）不同環境鹽濃度之熱力學探討............................... 83
4.3 綜合討論............................................................. 86
第五章結論.............................................................. 89
第六章參考文獻.......................................................... 92

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2006-7-11

推文