利用恆溫滴定微卡計量測蛋白質分子於溶液中之第二維里係數與自我聚集之行為

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姓名

黃俞強(Yu-chiang Huang) 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

利用恆溫滴定微卡計量測蛋白質分子於溶液中之第二維里係數與自我聚集之行為
(Second Virial Coefficient Measurements by Isothermal)

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摘要(中)

第二維里係數（second virial coefficient，B22）為一熱力學參數，可以利用它來描述一稀薄蛋白質溶液中之兩分子間交互作用行為。藉由深入暸解蛋白質於不同溶液中之交互作用行為，將可對於蛋白質藥物於生產程序及蛋白藥物於配方( formulation )、藥導( delivery )上，提供關鍵之基礎與應用知識。
因此本研究我們利用恆溫滴定微卡計（Isothermal Titration Calorimetry， ITC）之理論與實驗設計，發展出量測第二維里係數之熱力學方法。並以肌紅蛋白( Myoglobin )與核醣核酸酶（Ribonuclease A）為研究之模型蛋白，來探討於不同溶液條件(pH值、鹽種類與鹽濃度)下之蛋白質與蛋白質交互作用行為。期望以 ITC獲得之研究結果與現今常被使用於量測第二維里係數之自我反應管柱層析法( Self Interaction Chromotagraphy，SIC）去進行比較。
由實驗結果可得知，其蛋白質分子間的作用力為多種作用力所貢獻，會對於不同溶液條件下之蛋白質作用行為有不同的影響。當溶液條件接近蛋白質pI值及低鹽濃度時，如果此蛋白質表面電荷分布均勻時，主要會因靜電排斥而造成分子間作用力為排斥力；相反地，當一蛋白質表面電荷分佈不均勻時，因ITC (蛋白質分子間交互作用之環境為溶液系統) 與SIC( 固定相上之蛋白質分子與移動相之蛋白質分子交互作用 )之量測第二維里係數方法的差異性，而造成所產生的偶極-偶極作用力會對第二維里係數有顯著的影響。另外，蛋白質分子在不同種鹽類溶液中的行為亦會受到鹽類之鹽析或鹽溶效應影響，且分子間行為符合Hofmeister Series之論述。本研究更進一步地，藉由利用其它關於蛋白質聚集(aggregation)及結晶(crystallization)實驗，如SEC等，以對ITC與SIC之研究結果進行驗證與討論。

摘要(英)

Understanding the proteins self-interactions in solution system has great potential value due to its relevance in several research and applications fields, such as bio-separation、protein conformational disease and biopharmaceutical development. Particularly in several stages of biopharmaceutical process, protein aggregation has been observed frequently. Therefore, a priori prediction of protein pair potentials will provide crucial information for analyzing or screening suitable solution conditions. The second virial coefficient (B22), a thermodynamic parameter that characterizes the non-ideal solution behavior arising from two body solute-solute interactions, is a potential predictive tool. Determinations of B22 proteins have been extensively and mostly done by self-interaction chromatography (SIC). In this study, a novel platform, isothermal titration calorimetry (ITC), is established to describe the B22 of proteins with polarized surface charges distribution in aqueous solutions by means of measure the dilution enthalpy of protein solution. With the aim, B22 of Myoglobin and Ribonuclease A are measured by ITC in different solution conditions(pH、salt concentration、salt type), and compared to results obtained from self-interaction chromatography (SIC). Furthermore, size-exclusion chromatography and crystallization results offer the efficient way to let us verify whether or not proteins are stable in solution. Detailed discussion as following: in the research of Myoglobin, the protein-protein interactions are repulsive with increasing salt concentration in ITC and SIC system. Moreover, different kinds of salt in solution affect the proteins’ behavior, which are consistent with Hofmeister Series. On the other hand, the research of Ribonuclease A shows that protein-protein interactions change from repulsive to attractive with increasing NaCl concentration in ITC system. Furthermore, comparing the experimental results from ITC and SIC, we discover the adverse results as the pH value of solution is close to the pI value of protein and at lower salt concentration. Under this circumstance, the charge distribution on protein surface is anisotropic. Meanwhile, B22 measurement in SIC system is not convincing because the immobilized proteins can’t move freely. On the contrary, we can describe protein behaviors in solution precisely by ITC measurement, because of the degrees of freedom of protein are not restricted. Additionally, at the aforementioned conditions, the less aggregation of Ribonuclease A is detected from the size-exclusion chromatography measurements. Consequently, we successfully establish a platform to measure B22 of proteins with isotropic or anisotropic surface charges distribution in ITC system.

關鍵字(中)

★ 第二維里係數
★ 恆溫滴定微卡計
★ 自我反應管柱層析

關鍵字(英)

論文目次

中文摘要 I
AbstractIII
致謝 V
目錄 VI
圖目錄 IX
表目錄 XI
第一章緒論 1
第二章文獻回顧 4
2.1 第二維里係數 (Second virial coefficient) 4
2.1.1 維里方程式及第二維里係數之簡介 4
2.1.2 第二維里係數之應用 6
2.2 蛋白質與蛋白質間的交互作用力及其應用相關性 8
2.3 鹽離子水合效應及Hofmeister 序列 14
2.4 第二維里係數之量測方法 16
2.4.1 自我反應管柱層析 ( Self-interaction Chromatography，SIC ) 16
2.4.1.1 實驗方法之簡介 16
2.4.1.2 第二維里係數之理論計算 18
2.4.1.3 蛋白質於SIC之固定化 23
2.4.1.4 第二維里係數於SIC量測之缺點 25
2.4.2 恆溫滴定微卡計 ( Isothermal Titration Calorimetry，ITC ) 27
2.4.2.1 實驗方法之簡介 27
2.4.2.2 第二維里係數之理論計算 28
2.4.3 其它量測方法 33
2.5 SIC於蛋白質分子間作用力之量測 38
2.6 恆溫滴定微卡計之介紹與應用 44
第三章實驗藥品、儀器設備與方法 48
3.1 實驗藥品 48
3.2 儀器設備 50
3.3 實驗步驟 51
3.3.1 溶液配製 51
3.3.2 恆溫滴定微卡計實驗 52
3.3.2.1 操作步驟 52
3.3.2.2 稀釋熱之量測 54
3.3.3 螢光光譜儀實驗 55
3.3.4 圓二色光譜儀實驗 55
3.3.5 尺寸大小排除層析實驗 55
3.3.6 批式(Batch)結晶實驗 56
第四章結果與討論 57
4.1 恆溫滴定微卡計量測蛋白質分子間之作用力 57
4.1.1 肌紅蛋白 (Myoglobin)之研究 57
4.1.2 核醣核酸酶 (Ribonuclease A)之研究 64
4.2 藉由尺寸大小排除層析法(Size-exclusion chromatography)於蛋白質聚集之分析 71
4.2.1 肌紅蛋白之研究 71
4.2.2 核醣核酸酶之研究 74
4.3 批式結晶實驗(Batch crystallization experiment) 81
4.3.1 肌紅蛋白之研究 81
4.3.2 核醣核酸酶之研究 83
第五章結論 85
第六章參考文獻 87

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指導教授

陳文逸(Wen-yih Chen)

審核日期

2014-7-22

推文