| 摘要: | 中文摘要 在生物辨識的相關程序當中，疏水作用扮演關鍵的角色，而疏水作用易受環境因子影響而改變，因此可能使得生物分子鍵結行為有所不同。為了探討不同條件下，疏水作用對生物辨識系統的影響，因此本研究從簡單的model systems著手，討論疏水作用對於蛋白質鍵結程序的貢獻，並且期望藉由鍵結作用機制的分析，提供研究生物辨識上基礎熱力學的資訊，以用來解釋溶液中蛋白質分子的行為。本研究針對幾個較少被探討的因子，例如：溫度、疏水鏈長以及與溶液分子作用的貢獻來進行討論，我們利用恆溫微卡計直接量測蛋白質和疏水基材的反應焓，量測蛋白質以及月生 月太 的稀釋熱，配合等溫吸附線的分析推求出相關的熱力學參數，以了解疏水作用對蛋白質和基材的交互作用貢獻，以及對蛋白質溶液行為的影響，在研究中的三大主題將可分為五個部分來進行討論： (1) 對於蛋白質和基材間疏水交互作用的研究當中，我們選擇兩種分子量相似、等電點相近的蛋白質(α-Chymotrypsinogen A和Trypsinogen)進行分析。結果顯示提高溫度會影響較疏水的α-Chymotrypsinogen A和Butyl-Sepharose之間吸附機構，使吸附程序由adsorption-dominated binding process轉變為partition-dominated binding process，但對於Trypsinogen來說，溫度的貢獻僅造成蛋白質吸附力的增加。蛋白質稀釋熱研究中指出α-Chymotrypsinogen A是溫度敏感的蛋白質。 (2) 為更了解溫度對蛋白質鍵結程序的影響，我們選擇三種蛋白質(Lysozyme、α-amylase、Myoglobin)和兩種疏水基材(Butyl-Sepharose、Octyl-Sepharose)進行蛋白質鍵結機構的研究。我們一併討論鹽效應、觸手疏水鏈長效應、蛋白質特性和溫度效應的影響，我們發現鹽類、觸手疏水鏈長和溫度的增加，都會造成蛋白質和基材間疏水作用增強，這些都是由於分子間疏水交互作用面積增加所致。溫度效應方面，因溫度升高而降低水溶液的極性，並造成蛋白質結構調整而使得表面水合狀態改變，則是造成蛋白質吸附機構不同的主因。 (3) 由於疏水作用在離子交換層析系統中也具有貢獻，因此我們選擇兩種蛋白質(Lysozyme、Myoglobin)和三種商用陽離子交換樹脂(CM-Sepharose、SP-Sepharose、Source-30S)，對蛋白質鍵結機構進行研究，我們討論pH影響、鹽效應、觸手和基質效應和蛋白質特性的影響，並以scatchard plot和Freundlich-Langmuir model來分析蛋白質的吸附模式。我們發現高pH值時，蛋白質和膠體之間的疏水作用影響吸附程序，此時鹽效應的貢獻將促進蛋白質吸附；而低pH值則由於靜電作用的影響較大，因此鹽效應減少蛋白質的吸附親和力。此外，研究發現Source-30S是疏水的膠體，且其較親水的觸手和強疏水性的基質的貢獻分別造成不同吸附程序下蛋白質和基材間都具有最強的鍵結作用。 (4) 為了解月生月太 鏈中芳香族疏水胺基酸對疏水交互作用的貢獻，我們設計不同Tryptophan鏈長的月生月太 (Gly-Trp-Gly：GWG、Gly-Trp-Trp-Gly：GWWG、Gly-Trp-Trp-Trp-Gly：GWWG)進行初步的研究。我們發現GWG和疏水基材有最強的吸附且其稀釋熱為吸熱，而GWWG或GWWWG和疏水基材的吸附則相對較低，且其稀釋為放熱行為，這是由於月生月太 上tryptophan的增加使月生月太 和溶液分子的cation-π interaction增強所致。另外，在月生月太 和帶電基材的吸附實驗當中，我們發現當tryptophan增加到三個時(GWWWG)，由於月生月太 和基材間的疏水作用貢獻超越靜電作用的貢獻，而使得吸附機構轉變為疏水作用主導。 (5) 為了解蛋白質溶液行為，我們探討不同蛋白質濃度、鹽濃度、pH值下溶菌酉每 的稀釋熱，我們發現在溶菌酉每 濃度25g/L時，蛋白質之間有強作用力而使得稀釋為energy unfavorable。當增加鹽濃度或是調整溶液pH值使其接近等電點時，由於蛋白質間靜電斥力減少使得稀釋的吸熱量增加。對於較低濃度5g/L而言，蛋白質之間存在的靜電斥力則是造成蛋白質稀釋為放熱的主因，而鹽類的貢獻在於遮蔽分子間的靜電斥力。對於更低濃度0.5g/L而言，稀釋焓仍然是負值，但沒有發現明顯的鹽效應，這表示在此稀釋過程中，蛋白質和溶液的交互作用較蛋白質分子間的作用來的重要。 藉由相關的熱力學分析，我們可以清楚了解不同環境因子對於蛋白質鍵結行為和機構的影響，這些討論對於研究蛋白質吸附行為有相當的幫助，研究結果可以提供生物辨識作用方面的基礎資訊。 Abstract To understand the roles of hydrophobic interaction in the biorecognition system, we studied the simple model systems of hydrophobic interaction between protein and interaction surface in this investigation. We discussed the effects of environment factors such as temperature, hydrophobic chain length and the contribution of solution molecules in the hydrophobic interaction between biomolecules. By the thermodynamic analysis, we estabished an interaction mechanism to explain the protein-resin and protein-protein interaction behavior in solution. As summary, we divide into five related topics as following: (1) In the study of hydrophobic interaction between protein and matrix, we studied two kinds of proteins with similar molecule weight and at nearly isoelectrical point (α-Chymotrypsinogen A and Trypsinogen) of solution. Results showed that the increasing temperature significantly influence the binding mechanism of α-Chymotrypsinogen A with Butyl-Sepharose from an adsorption-dominated binding process to a partition-dominated binding process because α-Chymotrypsinogen A is a temperature sensitive protein. Evidences showed in the dilution enthalpy measurements confirmed this suggestion. (2) To understand the contribution of temperature effect on protein binding process, we chose proteins (Lysozyme、α-amylase、Myoglobin) and hydrophobic resins (Butyl-Sepharose、Octyl-Sepharose) to study the protein binding mechanism. We discussed the effects of salt, ligand chain length, protein characteristics, temperature and so on. Results showed that the increased salt concentration, hydrophobic ligand chain length and temperature promote the hydrophobic interaction between proteins and resins because the increased hydrophobic interaction regions between these suspended reactants. (3) We selected proteins (Lysozyme、Myoglobin) and cation resins (CM-Sepharose、SP-Sepharose、Source-30S) to study the contributons of hydrophobic interaction between ligand and protein in IEC system. We studied the effect of pH, salt, ligand and matrix, protein characteristics by ITC and isotherm experiments. We also utilized scatchard plot and Freundlich-Langmuir model to analyze the protein-binding model. In higher pH solution condition, results showed that the hydrophobic interaction between proteins and resins could significantly influence the protein binding process and the protein binding affinity was promoted by salt effect. In contrast, the electrostatic interaction dominated the protein binding process in lower pH solution condition and the increased salt concentration decreased the protein binding affinity. Moreover, we revealed that Source-30S, which has the most hydrophilic ligand and the most hydrophobic aromatic matrix, could make stronger hydrophobic interaction with protein in different solution conditions. (4) To study the effect of aromatic ligand chain length in the peptide-resin binding process, we designed three kinds of peptides with different tryptophan chain length (Gly-Trp-Gly：GWG、Gly-Trp-Trp-Gly：GWWG、Gly-Trp-Trp-Trp-Gly：GWWG). Results showed that GWG has stronger binding affinity with hydrophobic resins, and the dilution of GWG is an endothermic reaction. In contrast, the dilutions of GWWG or GWWWG are exothermic reactions. This is because the increased tryptophan on the peptides increase the cation-π between peptide and solution molecules. Furthermore, we demonstrated that the hydrophobic interaction between GWWWG and CM-Sepharose dominated the peptide binding process whereas GWG and GWWG interact with CM-Sepharose is a electrostatic dominated binding process. (5) We also discussed the protein solution dilution behaviors in varied lysozyme concentration. At higher protein concentration (25g/L), we found that the stronger protein-protein interactions make an energy unfavorite protein dilution reaction. In contrast, the dilution of lysozyme at lower protein concentration (5g/L) is an exothermic reaction because the existence of electrostatic repulsion between protein molecules. Furthermore, we observed the decreased electrostatic repulsion between protein molecules at higher salt concentration could result in the increased protein dilution enthalpy. In this investigation, we studied the protein solution behavior and protein binding mechanism in varied solution conditions by thermodynamic analysis. The results and discussions provide useful knowledge to understand the protein binding behaviors in solution and fundamental thermodynamic information in biorecognition system. |
