利用恆溫滴定微卡計探討聚乙二醇抗蛋白質吸附之作用機制

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許朝翔(Chao-Hsiang Hsu) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

利用恆溫滴定微卡計探討聚乙二醇抗蛋白質吸附之作用機制
(Isothermal Titration Calorimetric Studies of the Non-fouling Interaction Mechanism Between Proteins and Polyethyleneglycol)

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摘要(中)

由於聚乙二醇(polyethylene glycol or PEG)可以抵抗蛋白質的吸附，因此其擁有很好的生物相容性。目前已有相當多種聚乙二醇以及其相關的衍生物，被運用於各種不同的研究與應用的領域。然而聚乙二醇抗蛋白質吸附的作用機制尚未明確。我們認為由於聚乙二醇擁有良好的水合能力，具有從鄰近的水層優先排出其他生物分子的特性，因此可以有效的抵抗蛋白質的吸附。對於此機制在熱力學上的相關資訊並未被提出，因此在本次研究中，我們利用恆溫滴定微卡計在不同的鹽濃度、鹽離子、溫度以及不同PEG分子量等環境下，量測聚乙二醇的稀釋焓以及其與蛋白質之間的吸附焓。藉此來探討不同環境對於聚乙二醇水合能力的影響以及對聚乙二醇與蛋白質之間吸附行為的影響。
在稀釋焓結果方面，所有的稀釋焓都呈現放熱的結果，代表聚乙二醇分子在本研究當中所使用的不同溶液環境(包括不同鹽濃度、鹽種類、溫度及不同PEG分子量)下，均較傾向水合的狀態。其中在鹽濃度、溫度以及PEG分子量效應中，發現聚乙二醇水合能力會隨著溶液中的鹽濃度、溫度或者PEG分子量的提升而降低，從能量觀點上來說，在高鹽、高溫或高PEG分子量環境下，稀釋PEG為較不energy favorable的程序。而在鹽離子效應方面，發現影響PEG水合能力的離子序列符合了Hofmeister series。另外，我們發現Flory-Huggins parameter(χ12)在各種環境下的值均為負值，代表着聚乙二醇與水溶劑分子作用良好。
從恆溫吸附曲線的結果來看，當額外添加鹽類時，則降低了溶菌酶(lysozyme)吸附在Toyopearl Ether-650s上的吸附量以及吸附親合力。推測溶菌酶吸附於Ether-650過程中，除了疏水作用力外，靜電作用力的貢獻也必須考慮。此外，隨著溶液中鹽濃度或溫度的提升，溶菌酶與Ether-650間的吸附親合力有增加的趨勢。主要原因來自於溶菌酶與Ether-650間疏水作用力的提升。而在鹽離子效應方面，其影響溶菌酶吸附程度的序列也符合Hofmeister series。
在吸附焓的研究方面，在不同的鹽濃度、鹽種類及溫度環境下，溶菌酶與Ether-650間的吸附焓大多呈現吸熱，表示在吸附過程中大多為去水合的程序所主導。而在高鹽之1M KCl(25℃)下的溶液環境則呈現放熱，在此環境下溶菌酶的吸附則為焓驅動的非典型的疏水作用。在鹽離子的效應方面，發現添加銨鹽離子的溶液中，吸附焓的吸熱量較添加其他鹽類離子大。此外本研究也利用P.I. Model來計算在添加不同鹽類離子溶液下的吸附過程中，整體系統去水合的量。結果發現：(1)在添加銨鹽的溶液環境下，整體溶液系統去水合的量較鉀鹽的溶液環境下多。(2)對於溶菌酶與聚乙二醇吸附的系統而言，其吸附過程中去水合的量相較於文獻當中，溶菌酶與疏水觸手(如，C4,C8分子)吸附的系統還要來的少。從此點也證明聚乙二醇相較於其他非極性分子而言，其擁有較強大的水合能力，而此特性也是抵抗蛋白質吸附的重大因素之一。

摘要(英)

The characteristics of preventing nonspecific adsorption of protein has lead to extensive usage of PEG and its derivatives for biomedical applications. We consider that the interaction of water with the PEG is a major determinant of preventing protein adsorption. However, the thermodynamics aspect of the mechanism has not been well addressed. Therefore, in this study, we described the hydration behavior of PEG by measuring the dilution heat of PEG with various salt concentration, types of salt ions, temperature and molecular weight of PEG. In addition, we measured the isotherms and the interaction enthalpy between protein and Ether-650S with various salt concentrations, salt types and temperature by batch isotherms and ITC.
From the results of dilution heat, we observed that all the dilution heat are exothermic at all condition (i.e. salt conc. and types, temperature, PEG MW). It indicated that the PEG molecule is prefer to hydrate with water than aggregation in the conditions investigated. At high salt concentration, temperature and molecular weight of PEG, the dilution heat of PEG is less exothermic due to the poor hydration of PEG. In thermodynamics, the dilution of PEG is more energy unfavorable at high salt concentration, temperature and molecular weight of PEG. And the extent of salt ions which affect the hydration of PEG is consistent with the Hofmeister series. Besides, we also observed that all the values of Flory-Huggins parameter(χ) are negative at each condition. It also indicated that all the solvent which we used are good solvent for PEG.
From the results of isotherm, the amount of lysozyme adsorb on Ether-650 will decrease with increase the salt concentration. We considered that both of hydrophobic and electrostatic interaction affect the binding affinity of lysozyme.
The enthalpy of lysozyme adsorbed on Ether-650S are almost endothermic. It indicated that the hydrophobic force is the driving force during the adsorption process. However, the enthalpy of adsorption is exothermic at 1M KCl. This lead to the suggestion that the adsorption of lysozyme with the Ether-650 is of the “nonclassical” hydrophobic type interaction at 1M KCl. In this study, we also calculated the number of water molecules released during the adsorption by preferential interaction model. From the results of P.I Model, we can conclude : (1) when we adding
ammonium chloride to the solution, the system released more water molecules than add that of potassium chloride during the binding process.(2)compare with literature data, PEG ligand have stronger capability of hydration than other hydrophobic ligands.

關鍵字(中)

★ 聚乙二醇
★ 恆溫滴定微卡計
★ 稀釋焓
★ 恆溫吸附曲線

關鍵字(英)

★ isotherm
★ enthalpy of dilution
★ isothermal titration calorimetry
★ polyethylene glycol

論文目次

中文摘要 I
Abstract III
目錄 V
圖目錄 VIII
表目錄 XIV
第一章緒論 1
第二章文獻回顧 3
2.1 聚乙二醇的簡介與應用 3
2.2 蛋白質與聚乙二醇間存在的交互作用力 7
2.2.1 凡得瓦力 7
2.2.2 立體排斥力(Steric Repulsion Force) 10
2.2.3 疏水作用力 12
2.2.4 其他作用力 13
2.3 Kosmotrpoe and Chaotrope 14
2.4 Hofmeister series 17
2.5 蛋白質與聚乙二醇的吸附機制 18
2.6 其他影響蛋白質吸附機制的因素 21
2.6.1 聚乙二醇觸手鏈長及種類之影響 22
2.6.2 聚乙二醇於表面構形之影響 24
2.6.3 蛋白質種類之影響 28
2.6.4 鹽濃度及鹽種類之影響 30
2.7 恆溫滴定微卡計 33
2.7.1 VP-ITC的介紹 33
2.7.2 Flory-Huggins Theory於稀釋焓上的應用 36
2.7.3利用恆溫滴定微卡計研究吸附行為 37
第三章實驗藥品、儀器及方法 40
3.1 實驗藥品 40
3.2 儀器設備 42
3.3 實驗方法 43
3.3.1 等溫吸附線的量測 43
3.3.1.1 BCA Protein assay Kit操作步驟 44
3.3.2 vp-ITC操作步驟 46
3.3.3 稀釋焓量測 48
3.3.4 吸附焓的量測 48
第四章結果與討論 49
4.1 聚乙二醇稀釋焓的量測 49
4.1.1 鹽類效應 49
4.1.2 溫度效應 56
4.1.3 Flory-Huggins作用參數的分析 65
4.2 蛋白質與聚乙二醇間交互作用的研究 69
4.2.1恆溫吸附曲線的量測 69
4.2.1.1 鹽類效應 71
4.2.1.2 溫度效應 77
4.2.2 吸附驅動力的探討 81
4.2.3 吸附焓的量測 82
4.2.3.1 鹽類效應 82
4.2.3.2 溫度效應 92
第五章結論 94
第六章參考文獻 98

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指導教授

陳文逸(Wen-yih Chen)

審核日期

2007-7-24

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