臺灣博碩士論文加值系統

蛋白質在溶液中受到高壓（>1 kbar）的變性變化在文獻中曾被報導，而中等壓力下（<350 bar）的影響卻往往被忽略，另外壓力對吸附在表面上之蛋白質的影響也因為技術問題而很少有相關報導。本實驗利用毛細管線上偵測直接得到溶質在當時（in-situ）滯留受壓力影響的情形，實驗的壓力範圍從20 bar到360 bar，且利用線上紫外光譜掃瞄來提供蛋白質三級結構變化的訊息。實驗中主要是探討壓力對蛋白質在等位沖提下滯留行為之影響，並分別研究在三種不同的靜相中：標準碳十八（standard C18）、碳四（C4）、低密度碳十八（low density C18），壓力對蛋白質滯留行為的影響，以及利用光譜方法（紫外光吸收比值 281nm/292nm）探討壓力對於蛋白質在管柱中的影響。
進行壓力探討之前，利用兩點線上偵測系統分別在標準碳十八、碳四及低密度碳十八管柱中，以等位沖提方式用不同比率的沖提溶劑（氰甲烷，甲醇）沖提lysozyme，層析圖譜顯示有兩根主要波峰。由容量因子（k'）對有機溶劑比率作圖顯示，lysozyme在三種靜相皆呈現脫附（desorbed）及吸附（adsorbed）的狀態，脫附狀態是指lysozyme和靜相的作用力很小（k'~0）時，而吸附狀態則是和靜相有明顯的作用，而壓力的實驗即是分別探討lysozyme在此兩種狀態下滯留行為的改變。
結果顯示，當lysozyme在脫附狀態時，壓力不會影響其在管柱中的滯留行為，但是當處於吸附狀態時，lysozyme滯留時間會隨著管柱壓力增大而增加，且波峰的寬度也會隨著壓力增加而變寬。經由熱力學原理以lnk'對壓力作圖所得之斜率，即反應出溶質分子在動相與靜相中莫耳體積的差（ΔV=Vsta－Vmob）。由一次方程式的實驗數據顯示，lysozyme在實驗的壓力範圍下，其莫耳體積差為一定值，在標準碳十八管柱中，lysozyme的莫耳體積變化在動相為氰甲烷及甲醇時分別為－110 mL/mol及－128 mL/mol，在碳四管柱中則分別為－116 mL/mol及－56 mL/mol，低密度碳十八管柱為－168 mL/mol及－163 mL/mol。而使用二次方程式所得到的結果顯示，lysozyme在高壓下的莫耳體積變化有減小的趨勢。另外，將在高壓(300 bar)作用後的lysozyme收集再重新注射進低壓狀態(20~30 bar)的管柱中時，由層析圖譜的結果顯示，壓力所造成的變化是可逆的。

Pressure-induced change of protein behavior has long been either ignored or observed to occur at high pressures (>1 kbar). In this study, the on-column detection system is used to directly investigated the effect of pressure on protein retention in situ along the column. The range of pressure in this study is from 20 bar to 360 bar. Meanwhile, in order to observe the tertiary structure change of lysozyme under pressure effect, the on-the-fly scan UV spectroscopy (A281nm/292nm) was acquired as well. In this work, three kinds of stationary phases, standard C18, C4 and low density C18 were investigated.
The isocratic elution behavior of lysozyme molecule was investigated by the dual on column detection system before pressure study. From the relationship between capacity factor (k') and the compositions of mobile phase (acetonitrile or methanol), the desorbed and adsorbed conditions were defined. In the desorbed condition, the lysozyme molecule is hardly retained on the stationary phase (k'~0), whereas, in the adsorbed condition the solute molecule is strongly retained (k'>0.5). In this study, the chromatographic retention of lysozyme molecule was measured directly on a transparent fused silica column as a function of the local pressure under both desorbed and adsorbed conditions.
Under the desorbed condition, both the capacity factor (k') and bandwidth of lysozyme are not changed significantly as the pressure increased. Under the adsorbed condition, the capacity factor (k') is substantially increased and the bandwidth is broadened upon pressurizing. From the thermodynamic theory, the molar volume change(ΔV=Vsta-Vmob) of the protein between stationary phase (Vsta) and mobile phase (Vmob) can be determined from the slope of the plot of lnk' versus pressure. The linear equation was attempted to fit the plots constructed under adsorbed conditions, resulting in R2 values greater than 0.95 for all fittings (except methanol as mobile phase for C4 column). For the standard C18 column, the molar volume changes were calculated to be around －110 mL/mol and －128 mL/mol for acetonitrile and methanol respectively. For the C4 column, the values were －116 mL/mol and －56 mL/mol. For the low density C18 column, the volume change was around －168 mL/mol and －163 mL/mol for acetonitrile and methanol, respectively. In addition, the quadratic equation was also used to fit the plot of lnk' versus pressure with R2 values greater than 0.99 for all fittings. Results indicate that the molar volume change of lysozyme was decreased as the pressure increased. Moreover, the fraction of the lysozyme eluted under high pressure (300 bar) was collected and reinjected into the column at low pressure (20~30 bar). Results show that the pressure-induced change of the chromatographic behavior of lysozyme is reversible.

目錄
中文摘要…………………………………………………………………Ⅰ
英文摘要…………………………………………………………………Ⅲ
表目錄…………………………………………………………………….IX
圖目錄…………………………………………………………………….X
第一章 緒 論
1-1 研究背景………………………………………………….1
1-2 本章參考文獻…………………………………………….5
第二章 毛細管液相層析線上螢光偵測系統之建立
2-1 研究背景………………………………………………….7
2-2 層析及偵測系統裝置…………………………………….7
2-2-1 毛細管柱的製備……………………………………9
2-2-2 毛細管柱效能測試……………………………..…10
2-2-3 層析及偵測系統…………………………………..12
2-3 毛細管液相層析之高壓系統的建立……………………..14
2-4 Moment Calculation……………………………………...16
2-5 本章參考文獻……………………………………………..18
第三章 壓力對蛋白質在標準碳十八管柱中滯留行為之探討
3-1 研究背景……………………………………..…………. .21
3-2 實驗部份……………..……………………………….…...21
3-2-1 試劑…………………………………………….…21
3-2-2 儀器裝置………………………………………….22
3-3 理論………………………………………………………23
3-4 管柱中的等位沖提行為探討……………………………25
3-4-1 前言……………………………………………….25
3-4-2 動相組成對lysozyme沖提的效應………………26
3-4-3 壓力實驗條件的選擇…………………………….27
3-5 壓力對容量因子之影響探討……………………………29
3-5-1 壓力對lysozyme在等位沖提下滯留行為
之影響………………………………………...…..29
3-5-2 壓力對lysozyme莫耳體積之影響……………...30
3-5-3 壓力對lysozyme在不同離子對試劑中之影響…32
3-6 利用光譜法探討壓力之影響……………………………34
3-6-1 前言……………………………………………….34
3-6-2 比較標準碳十八管柱及空管柱之紫外光吸收值比
率(281nm/292nm)…………………………………36
3-7 結論………………………………………………………39
3-8本章參考文獻……………………………………………40
第四章 壓力對蛋白質在碳四管柱中滯留行為之探討
4-1 研究背景………………………………………………….55
4-2 實驗部份………………………………………………….57
4-2-1 試劑…………………………………………….…..57
4-2-2 儀器裝置…………………………………………...57
4-3 管柱中的等位沖提行為探討…………………………….58
4-3-1 動相組成對lysozyme沖提的效應………………..58
4-3-2 壓力實驗條件的選擇………………………………59
4-4 壓力對容量因子之影響探討…..…………………………61
4-4-1 壓力對lysozyme在等位沖提下滯留行為之影響..61
4-4-2 壓力對lysozyme莫耳體積之影響………………..62
4-5 結論…………………………………………………………64
4-6 本章參考文獻………………………………………………65
第五章 壓力對蛋白質在低密度碳十八管柱中滯留行為之探討
5-1 研究背景……………………………………………………76
5-2 實驗部份……………………………………………………78
5-2-1 試劑…………………………………………………78
5-2-2 儀器裝置……………………………………………78
5-3 管柱中的等位沖提行為探討………………………………79
5-3-1 動相組成對lysozyme沖提的效應………………...79
5-3-2 壓力實驗條件的選擇………………………………80
5-4 壓力對容量因子之影響探討……………………………...81
5-4-1 壓力對lysozyme在等位沖提下滯留行為之影響..81
5-4-2 壓力對lysozyme莫耳體積之影響………………..82
5-5 結論………………………………………………………...83
5-6 本章參考文獻……………………………………………...84
第六章 總 結
6-1 比較三種管柱中的等位沖提行為探討……………….…..97.
6-2 比較壓力對lysozyme在三種管柱中的莫耳體積改變….98
附錄
一、 lysozyme的簡介……………………………………….……112
二、 利用光譜方法探討lysozyme在不同有機溶劑環境下
其構形的變化……………………………………………….113
三、 利用光譜方法探討lysozyme在層析環境中，以不同
動相沖提其構形的變化…………………………………….115
四、 本章參考文獻……………………………………………….118

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