劉懷勝臺灣大學：化學工程學研究所張棨翔Chang, Chi-HsiungChi-HsiungChang2007-11-262018-06-282007-11-262018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/52268本研究所探討之重點在於觀察變性(denaturation)程序中，母雞蛋白溶菌酶(Hen Egg White Lysozyme，HEWL)結構的變化，並針對變性劑中重要的還原劑成份，二硫代蘇糖醇(DTT)之氧化情形做定量分析，進而探討其對變性程序之影響。實驗方法主要是利用大小排阻層析法(Size Exclusion Chromatography，SEC)來分析溶菌酶在不同變性時間下結構大小的變化，同時收集波峰分液進行生物活性測定以及半胱胺酸(cysteine)濃度測定，藉以了解溶菌酶活性區域以及雙硫鍵之破壞程度，並針對在不同變性條件下之變性溶菌酶，進行直接稀釋法復性以觀察其結構差異與DTT殘留對於復性產率之影響。 實驗結果顯示，DTT在空氣中之氧化反應為一級反應(25℃；pH＝8.2)，其反應速率常數為0.0994 day-1。此外，DTT在空氣中之氧化反應受到氧氣擴散作用所控制，因此改變盛裝容器之規格以及氧氣濃度均會影響其氧化速率。 而在溶菌酶之變性程序中，發現所添加之DTT不足量時會因尿素之持續作用而造成變性溶菌酶波峰向後偏移。根據實驗結果與文獻之輔助，我們推測在溶菌酶變性過程中，最先斷裂之雙硫鍵應為Cys76-Cys94，而最後斷裂者則為Cys30-Cys115。根據不同變性條件下所得變性溶菌酶之復性實驗結果顯示，當雙硫鍵斷裂數越少，則復性效果越佳；而當DTT在復性緩衝液中之殘餘濃度高於0.8mM時，則幾乎沒有任何復性效果。除此之外，當殘留量較低時，D/L比值則為決定復性產率之重要指標，當D/L比值越低，則復性產率越佳。其中D代表復性緩衝液中DTT之殘餘濃度；L則代表復性緩衝液中溶菌酶稀釋後之濃度。Observation of the conformational change of hen egg-white lysozyme during the denaturation procedure and quantitative analysis of oxidative kinetics of dithiothreitol (DTT) and refold lysozyme under different denaturation conditions via direct dilution method were investigated in this research. By size-exclusion chromatography (SEC), we were able to compare the apparent sizes of denatured lysozyme at different denaturation times with that of the native conformer. Furthermore, the measurements of activity and cysteine concentration also helped us to monitor the disruptions of active region and disulfide linkages of lysozyme during the course of denaturation and renaturation processes. As shown in our results, the reaction of DTT oxidation at 25℃ and pH 8.2 followed the first order kinetics with a rate constant of 0.0994 day-1. In addition, we found that the rate of DTT oxidation was controlled by the diffusion rate of oxygen. That is, the magnitude of the rate constant of DTT oxidation was determined by the diffusion condition which is affected by the geometry of reaction vessel in use (e.g.: the height and cross-sectional area of tube) and the concentration of oxygen. In the denaturation procedure, we found that, with insufficient DTT, the presence of urea would shift the peak of denatured lysozyme to the right (larger retention time), which means a more compact lysozyme conformer was formed resulted from the presence of urea after the complete depletion of DTT. According to our experimental results along with others found in the literature, we suggested that the first and the last reduced disulfide bond of lysozyme during the denaturation procedure were Cys76-Cys94 and Cys30-Cys115, respectively. Moreover, it appeared that the less the numbers of disulfide bond being reduced, the better the activity recovered in the renaturation procedure. Our results showed that the activity recovery of lysozyme would be obviously suppressed when the concentration of DTT carried over from the denaturing buffer to renaturation solution was higher than 0.8mM.中文摘要 I Abstract I 圖目錄 VII 表目錄 XII 第一章 緒論 1 1-1 研究背景與方向 1 第二章 文獻回顧 2 2-1 結構生物學 2 2-1-1 定義及應用 2 2-1-2 蛋白質組成及結構 2 2-1-3 穩定蛋白質結構之作用力 10 2-1-4 蛋白質結構測定方法 12 2-2 蛋白質工程 13 2-2-1 定義及應用 13 2-2-2 蛋白質之摺疊與聚集 13 2-2-3 蛋白質之變性程序 16 2-2-4 蛋白質之復性程序 19 2-3 溶菌酶(LYSOZYME) 24 2-3-1 溶菌酶之發現與簡介 24 2-3-2 溶菌酶之結構 25 2-3-3 溶菌酶之活性中心 27 2-3-4 溶菌酶之雙硫鍵 31 2-4 二硫代蘇糖醇(DL-DITHIOTHREITOL，DTT) 33 2-4-1 性質與簡介 33 2-4-2 穩定性 35 2-5 雙硫鍵偵測方法簡介(ELLMAN’S METHOD) 36 2-5-1 DTNB(Ellman’s Reagent)之性質與簡介 36 2-5-2 Ellman’s Method之偵測原理與限制 37 第三章 實驗裝置、藥品及步驟 39 3-1 實驗裝置 39 3-2 實驗藥品 39 3-3 實驗步驟 41 3-3-1 溶菌酶之變性 41 3-3-2 變性試劑中DTT之氧化速率分析 41 3-3-3 溶菌酶之活性測定 42 3-3-4 半胱胺酸濃度測定 43 3-3-5 溶菌酶之直接稀釋法復性 43 第四章 變性緩衝液中DTT之氧化動力學分析 45 4-1 實驗動機 45 4-2 實驗方法 47 4-3 實驗結果與討論 47 4-3-1 氧化態DTT之濃度校正曲線 47 4-3-2變性緩衝液中DTT之氧化動力學分析 49 4-4 結論 61 第五章 大小排阻層析法分析DTT與溶菌酶作用之氧化速率 62 5-1 實驗動機 62 5-2 實驗方法 62 5-3 實驗結果與討論 63 5-3-1 氧化態DTT之濃度校正曲線 63 5-3-2 DTT與溶菌酶作用之氧化速率分析 65 5-4 結論 69 第六章 大小排阻層析法分析溶菌酶在變性過程中結構之變化 71 6-1 實驗動機 71 6-2 實驗方法 72 6-3 實驗結果與討論 72 6-3-1 變性蛋白質液中不足量DTT對溶菌酶波峰滯留時間之影響 72 6-3-2 變性蛋白質液中高濃度過量DTT對溶菌酶變性過程之影響 82 6-3-3 不同DTT濃度及8M尿素作用下對溶菌酶結構之影響 84 6-4 結論 94 第七章 直接稀釋法分析溶菌酶在不同變性條件下之復性效果 96 7-1 實驗動機 96 7-2 實驗方法 96 7-3 實驗結果與討論 96 7-3-1 變性溶菌酶結構差異對復性產率之影響 97 7-3-2 復性緩衝液中DTT殘留量對直接稀釋法復性之影響 99 7-4 結論 102 參考文獻 1037682304 bytesapplication/pdfen-US溶菌酶二硫代蘇糖醇雙硫鍵變性復性大小排阻層析法lysozymedithiothreitol(DTT)disulfide bondsdenaturationrenaturationsize-exclusion chromatography(SEC)thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52268/1/ntu-96-R94524057-1.pdf