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研究生： 郭俊毅
Kuo, Chun-Yi
論文名稱： 以時間相關單光子計數系統與圓二色光譜法研究聚脯胺酸鏈長對於色胺酸對的螢光及光學活性之影響
Roles of polyprolines in the fluorescence kinetics and optical activity of tryptophan pair monitored with circular dichroism spectroscopy and time-correlated single photon counting system
指導教授： 朱立岡
Chu, Li-Kang
口試委員: 洪嘉呈
Horng, Jia-Cherng
周佳駿
Chou, Chia-Chun
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2017
畢業學年度： 106
語文別： 中文
論文頁數： 82
中文關鍵詞： 色胺酸 、聚脯胺酸 、螢光生命期 、光學活性
外文關鍵詞： tryptophan, polyproline, fluorescence lifetime, optical activity
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    • 摘要
    改變色胺酸所處的化學環境時，其螢光強度、最強螢光波長及螢光生命期也隨之變化，故此特性常應用於鑑定蛋白質局部結構。此外色胺酸也用於研究發光團間的交互作用，包括激子轉移(exciton transfer)以及激子耦合(exciton coupling)。吾人於本研究中使用不同長度的聚脯胺酸作為兩色胺酸之間隔分子(WPnW，n=7-10、13、16)，以時間相關單光子計數系統觀察WPnW分別在純水、甘油-水混合溶液、正丙醇及異丙醇中是否具有福斯特共振能量轉移現象，並且討論溶劑對於色胺酸螢光性質之影響。此外，吾人亦利用近紫外圓二色光譜觀察WPnW於水中是否具有色胺酸間的激子-激子耦合現象。
    除了研究聚脯胺酸長度的影響，吾人亦合成WP、WPW、PW三種不同序列的胜肽，以觀察色胺酸改變氮端或碳端基團後其螢光生命期與光學活性的變化。其中當色胺酸接上脯胺酸時螢光生命期由一個變為兩個，且隨著胜肽序列不同，其螢光生命期也不相同。比較WP及PW的近紫外圓二色光譜顯示到當色胺酸的碳端接上脯胺酸時會增強色胺酸的光學活性，但在色胺酸的氮端接上脯胺酸時會降低色胺酸的光學活性。若比較WPW與WPnW之近紫外圓二色光譜，吾人發現聚脯胺酸的二級結構會改變色胺酸之光學活性，與Nagai等人推論色胺酸之光學活性來自於胜肽鍵磁偶極躍遷之微擾的論點相符。

    Abstract
    The fluorescence properties of tryptophan, in terms of intensity, maximum wavelength and lifetime, are related to the chemical surroundings and used to identify the local environments of the proteins. Furthermore, tryptophan is commonly used to observe the interactions of two chromophores, in terms of exciton transfer and exciton coupling. In this work, polyproline (PP) of different lengths serve as the spacers between two tryptophans to investigate Förster resonance energy transfer and exciton-exciton coupling.
    Three peptides, WP, WPW and PW, manifested significant changes in their fluorescence lifetimes and optical activities upon changing amino group or carbonyl groups of tryptophan. When tryptophans are attached to proline, there are two time constants, whereas only one time constant was observed in the absence of proline. The near-UV circular dichroism spectra of WP and PW showed that optical activity of L-Trp decreases when modified at the amino group and increases when modified at the carbonyl group. In addition, the influence of secondary structures of polyproline on the optical activity of constituent tryptophan has been discussed. By comparing the near-UV circular dichroism spectra of WPW and WPnW, we found that polyproline would influence the optical activity of tryptophan. It is consistent with the result of Nagai et al. that the magnetic transition dipole of peptide bond will perturb the indole’s electronic transition via the chiral carbon of tryptophan.

    目錄 第一章 緒論 1 1.1 前言 1 1.2 文獻簡介與實驗動機 1 參考文獻 3 第二章 色胺酸及聚脯胺酸的性質 4 2.1色胺酸之性質 4 2.1.1 色胺酸之螢光性質 4 2.1.2. 色胺酸於胜肽或蛋白質中螢光性質的變化 5 2.1.3 吲哚螢光之溶劑效應 5 2.2色胺酸之近紫外光圓二色光譜 6 2.3 聚脯胺酸的性質 7 2.4福斯特共振能量轉移(Förster Resonance Energy Transfer，FRET) 7 2.5 固相胜肽合成法(Solid Phase Peptide Synthesis，SPPS) 8 2.5.1 樹脂與連接體 9 2.5.2 去保護 9 2.5.3 活化 9 2.5.4 耦合 10 2.5.5 切除 10 參考文獻 26 第三章 儀器原理與樣品製備 28 3.1 儀器介紹 28 3.1.1 穩態紫外/可見吸收光譜(Steady-state ultra-violet/visible absorption spectroscopy, UV-Vis) 28 3.1.2 圓二色光譜(Circular dichroism spectroscopy，CD spectroscopy) 28 3.1.3 螢光光譜(Fluorescence spectroscopy) 31 3.1.4 時間相關單光子計數系統 (Time-correlated single photon counting system) 32 3.2 樣品合成與配製 33 3.2.1 WPnW合成廠商 34 3.2.2 WP、WPW、PW胜肽合成 34 3.2.3 樹脂切除與胜肽純化 34 3.2.4 不同比例之甘油水溶液配製方法 35 3.3 胜肽特徵光譜鑑定 35 3.4 儀器型號與參數設定 36 3.4.1 微波胜肽合成儀 36 3.4.2 高效能液相層析儀 36 3.4.3 基質輔助雷射脫附游離飛行時間質譜儀 36 3.4.4 冷凍乾燥機 36 3.4.5 穩態紫外/可見光吸收光譜 36 3.4.6 圓二色光譜 36 3.4.7 螢光分光光譜 37 3.4.8 時間相關單光子計數系統 37 參考文獻 50 第四章 實驗數據與討論 51 4.1 以遠紫外圓二色光譜鑑定胜肽的二級結構 51 4.2 穩態光譜鑑定 51 4.2.1 穩態紫外/可見光吸收光譜 51 4.2.2 穩態螢光光譜 52 4.3時間相關單光子計數系統(Time-Correlated Single Photon Counting，TCSPC) 52 4.3.1 在甘油-水混合溶液及異丙醇中色胺酸與WPnW螢光衰減數據 52 4.3.2不同溶劑對於色胺酸以及WPnW之螢光生命期機制探討 54 4.3.3 短胜肽的螢光衰退機制 55 4.4 近紫外光圓二色光譜(near-UV Circular Dichroism Spectrum) 55 4.4.1 近紫外光圓二色光譜數據 55 4.4.2 含色胺酸之近紫外圓二色光譜討論 56 參考文獻 80 第五章 結論 81

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