研究生: |
簡詩展 |
---|---|
論文名稱: |
蛋白質摺疊的研究 Protein folding |
指導教授: |
張一知
Chang, I-Jy |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2004 |
畢業學年度: | 92 |
語文別: | 中文 |
中文關鍵詞: | 蛋白質摺疊 、黃嘌呤氧化酶 、細胞色素 c |
英文關鍵詞: | protein folding, xanthine oxidase, cytochrome c |
論文種類: | 學術論文 |
相關次數: | 點閱:197 下載:0 |
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蛋白質具有獨特的空間結構,造成不同蛋白質具有不同的特定功用。錯誤摺疊的蛋白質常會造成身體的病害,如狂牛症和庫賈氏病都是一種蛋白質錯誤摺疊造成的神經退化症。所以設計有效的方法來追蹤蛋白質的摺疊是一門很重要的課題。
本實驗研究的黃嘌呤氧化酶 (XOD) 在生物體嘌呤和嘧啶的代謝作用中扮演很重要的角色。使用圓二色分光光譜儀偵測 XOD 在不同濃度的變性劑中摺疊的程度。由熱力學公式推算出摺疊和變性兩狀態間自由能的差。利用黃嘌呤當作基質來偵測 XOD 在不同摺疊程度下反應活性的變化。並推測探討反應活性的變化和蛋白質構形的關係。
酵母菌細胞色素 c 的羧端具有一個半胱胺酸。本實驗室合成出一個發藍光的發光團,其螢光光譜和細胞色素 c 的吸收光譜有很好的重疊。將發光團修飾在細胞色素 c 上並純化後可以得到在半胱胺酸位置接上一個發光團的細胞色素 c。當加入不同的變性劑會使蛋白質的摺疊改變,並有不同程度的展開。利用螢光放射光譜可以分析細胞色素 c 中血基質和發光團的距離。
Protein has a unique three-dimensional structure that reflects its function. A misfolded protein appears to be the cause of a number of rare degenerative brain diseases in mammals such as the infamous mad cow diseases and Creutzfeldt-Jakob disease (CJD). It is important to understand the protein folding processes.
Xanthine Oxidase (XOD) plays a critical role in metabolism. It converts hypoxanthine to xanthine, and then to uric acid. Circular Dichroism has been used to detect the α-helix folding of XOD in various concentrations of guanidine hydrochloride. Thermodynamic equations were employed to calculate the folding free energy. Xanthine was taken as the substrate to determine the enzyme activity of XOD at a variety of folding conditions. The change on the α-helix percentage and the enzyme activity were compared.
Yeast iso-1 cytochrome c has a cysteine in C terminal. A blue fluorophore whose emission spectrum has excellent overlap with heme protein absorption was synthesized. It was covalently attached to cytochrome c and the modified protein was purified by FPLC. Emission intensity was measured in various concentration of guanidine hydrochloride. Förster fluorescence energy transfer theory was used to calculate the mean distance between the fluorophore and the heme.
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