利用微卡計對於蛋白質與離子交換樹脂觸手間親疏水作用力的研究

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姓名

方瓊儀(Chiung-I Fang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

利用微卡計對於蛋白質與離子交換樹脂觸手間親疏水作用力的研究
(Microcalorimetric studies of the hydrophobic interaction between proteins and ligand of ion-exchanger resins)

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摘要(中)

中文摘要
由於蛋白質於高離子強度下與離子交換樹脂之吸附機制已被證實為疏水作用力，因此離子交換樹脂觸手親疏水不同對蛋白質吸附親和力之影響實為重要。本實驗主要探討於高離子強度下，離子交換觸手之親疏水性不同對蛋白質吸附親和力之影響。
實驗上我們選擇商業上之不同離子交換樹脂(CM Sepharose Fast Flow, SP Sepharose Fast Flow and SOURCE 30S)，將蛋白質Lysozyme和Myoglobin吸附至不同膠體上，建立批次操作之等溫吸附線，於等溫下，再藉由改變鹽濃度及鹽種類對吸附行為的影響探討其吸附行為影響之因子。另外，吸附焓值可直接被定量的量測出，從熱力學的觀點來探討蛋白質吸附行為受膠體觸手親疏水性不同以及鹽濃度的影響。
實驗結果顯示：蛋白質於高離子強度下其吸附機制為疏水作用力，於相同鹽濃度下，蛋白質和膠體上越疏水之觸手 (CM Sepharose) 其吸附親和力最大；於高鹽時，溶液之表面張力增加，使蛋白質越容易吸附到膠以表面上，因此膠體觸手之疏水性的影響更加明顯。於不同鹽之種類研究中於硫酸胺溶液中，則蛋白質的吸附親和力比在氯化鈉中大，而根據solphobic theory 因硫酸胺使溶液表面張力增加的程度比氯化鈉大，因此加強了蛋白質與吸附基材之疏水作用力。
而我們由熱力學的參數得知，於高離子強度下之吸附熱皆為吸熱，這是由於疏水作用力使去離子及去水合程序之所需能量貢獻最大，而使吸附行為中並不利於系統焓值降低，而以系統亂度增加以彌補焓值之需求，是為熵驅動之程序。

摘要(英)

Abstract
The interaction mechanism of proteins with various commercial available cation exchangers at different binding conditions were studied. The aim of this research is the study of the hydrophobic effect of the ion exchanger ligands on protein binding behavior at high ionic strength.
In this investigation, proteins, Lysozyme and Myoglobin, were bound to the absorbents which were selected to encompass the hydrophobic effects of three different ion exchanger ligand groups (CM Sepharose, SP Sepharose and SOURCE 30S). Under these experiment were modulated with different types of salt and at high ionic strength. The adsorption behavior were analyzed with batch isotherm and adsorption enthalpies, directly measured by isothermal titration calorimeter.
Experimental results indicated that the adsorption mechanism mainly by hydrophobic interaction occurs at high ionic strength and the binding enthalpies confirm this suggestion. In high ionic strength conditions, the enthalpy play a unfavior role in driving the process and the adsorption is driven by entropy gained from the dehydration and desolvation processes. In addition, binding affinities and enthalpies were found to be ligand-dependent, and the most hydrophobic lgands (CM Sepharose) has the highest binding affinitiy among the ligands studied.
The presented data shed light on the influence of the hydrophobic effect of the ion exchanger ligands on the protein binding at high ionic strength condition.

關鍵字(中)

★ 靜電作用力
★ 疏水作用力
★ 離子交換樹脂
★ 微卡計

關鍵字(英)

★ Microcalorimetric
★ ion-exchanger
★ hydrophobic interaction
★ electrostatic interaction

論文目次

目錄
中文摘要 I
英文摘要 Ⅲ
圖目錄 IV
表目錄 X
第一章緒論 1
第二章文獻回顧 3
2.1 層析之簡介 3
2.1.1 吸附層析法 4
2.1.2 膠體過濾層析 4
2.1.3 固定化金屬離子親和性系統 6
2.2 離子交換層析法 8
2.2.1 基本原理 8
2.2.2 離子交換樹脂之分類 8
2.2.3 離子交換樹脂的應用 13
2.2.4 離子交換樹脂 (stationary phases) 的型態 14
2.2.3 Retention of ion-exchange chromatography 19
2.3.1 Effect of salt 19
2.3.2 Effect of pH 23
2.4 疏水作用層析 25
2.5 Salt effect in HIC 29
2.5.1 Solvophobic theory 29
2.5.2 Preferential interaction analysis (P.I Model) 30
2.6 蛋白質吸附到膠體表面之吸附機制 32
第三章實驗動機與目的 37
第四章藥品與儀器設備 39
4.1 實驗藥品 39
4.2 儀器設備 41
4.3 恆溫滴定微卡計 46
5.2 吸附焓變化的量測 47
5.3 實驗數據分析 49
第六章實驗結果與討論 52
6.1 等溫吸附線之分析 52
6.1.1 Salt effect 62
6.1.2 Ligand effect 71
6.1.3 Species of protein effect 91
6.1.4 pH effect 93
6.2 利用微卡計探討熱力學數據 101
6.2.1 Salt effect 103
6.2.2 Ligand effect on gel dilution heat 105
6.2.3 Ligand effect on proteins binding 106
第七章結論與心得 114
參考文獻 116

參考文獻

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2002-7-3

推文