蛋白質與疏水性基材間交互作用的溫度效應及其吸附機制探討; The Effect of Temperature on Hydrophobic Interaction between Proteins and Hydrophobic Adsorbents

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题名:	蛋白質與疏水性基材間交互作用的溫度效應及其吸附機制探討;The Effect of Temperature on Hydrophobic Interaction between Proteins and Hydrophobic Adsorbents
作者:	林健成;Chien-Cheng Lin
贡献者:	化學工程與材料工程研究所
关键词:	熱力學參數;van't Hoff關係式;疏水交互作用;澱粉水解酵素;溶菌酵素;肌紅蛋白;焓熵補償;恆溫滴定微卡計;hydrophobic inter;amylase;lysozyme;myoglobin
日期:	2001-07-10
上传时间:	2009-09-21 12:18:49 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	本研究主要是針對蛋白質及疏水性吸附基材在不同溫度時的吸附機制進行探討，實驗上主要是利用等溫吸附線的建立及配合van't Hoff方程式，期望從熱力學的角度來研究分析，以瞭解溫度、觸手鏈長、鹽濃度等對蛋白質與疏水基材之作用機制及其影響因素，並應用恆溫滴定微卡計(Isothermal Titration Calorimetry，ITC)量測吸附熱，以獲得ITC吸附焓值與van't Hoff焓值的差異比較。在等溫吸附的研究方面，是利用批次(batch)操作來量測溶菌酵素(lysozyme)，肌紅蛋白(myoglobin)和澱粉水解酵素(amylase)吸附於Butyl and Octyl Sepharose 4 Fast Flow gel的等溫吸附行為。在固定鹽濃度下，藉著改變溫度以探討疏水性吸附基材與蛋白質之間的吸附行為在不同觸手鏈長的改變，以及在固定溫度下，改變鹽濃度以探討鹽離子對吸附行為的影響。實驗結果顯示 : 當結構穩定度較柔軟時，也就是較容易進行構形改變或重排的蛋白質(即肌紅蛋白)，當其處在漸漸增高的溫度或鹽濃度時，由於處在高溫或高鹽的情況下，更容易展露出埋藏的疏水性殘基，亦即在高溫或高鹽時，疏水交互作用的增強對蛋白質的吸附行為有較明顯的影響，當這三種蛋白質處在低鹽濃度(0及0.1M)狀態時，則溫度的影響對肌紅蛋白尤其較其它兩種結構穩定性較剛硬的蛋白質而言，其吸附親和力和鍵結量都較來的大而且明顯。另外，實驗結果也顯示肌紅蛋白和溶菌酵素處在高溫且高鹽(60C及1M)的狀態時，其吸附行為不論是在butyl ligand或是octyl ligand，都明顯的與澱粉水解酵素不同，其吸附曲線非常陡峭，推測可能是二三級結構的喪失，使得蛋白質展開(unfolding)成鬆散的構形，顯示蛋白質的構形穩定性亦是扮演足以影響吸附行為的一個重要因子。在熱力學數據的量測方面，我們將整個吸附作用分成四個子程序，藉由先前所量測的等溫吸附線，配合van't Hoff及Kirchoff關係式，以獲得各種吸附焓值、熵值及熱容量的變化，並輔以Preferential Interaction方程式以推測驅動吸附作用的能量來源及分析每一個子程序受溫度、鹽濃度、觸手鏈長及蛋白質種類影響所產生的能量變化情形。實驗結果顯示 : 三種蛋白質在butyl和octyl的條件下，其吸附主要驅動力分別各為吸附焓值和吸附熵值，亦即其吸附行為模式各為butyl為adsorption binding(點對點鍵結) 和octyl為partition binding(溶入分散鍵結)，因此，在octyl上的吸附機制主要為去水合。在溫度效應及鹽濃度效應的實驗結果方面，在低溫(20~30C)或低鹽(0~0.1M)所得到的熱力學數據可知，其吸附行為發生的驅動力為吸附焓值;而當在高溫(40~60C)或高鹽(0.5~1M)時，亂度對於吸附行為發生的貢獻提高，因此可推論在此條件下，吸附機制主要是受去水合和構形改變所驅動。另外，由於蛋白質的去水合程度與蛋白質表面的疏水區大小成正比關係，但若為熱穩定性高的澱粉水解酵素，由於去水合需構形改變才能達成，故其在高溫高鹽時，其吸附焓值和由P.I.方程式所獲得的去水合量皆比另外兩種蛋白質來的小，其原因可能就在此。 The aim of this research is related to the study of the effects of temperature, ligand chain length and salt concentration on adsorption mechanism of protein interacting with a Sepharose based hydrophobic interaction support by adsorption isotherm analysis and by adsorption enthalphy measurement. In this investigations, the influence of a range of experimental parameters on the isothermal characteristics of hen egg white lysozyme, horse heart myoglobin and Bacillus licheniformis amylase adsorbed to several different adsorbents has been examined. The adsorbents were selected to encompass the same basic types of agarose support matrices, but with the ligand chain length adjusted so that the dominant mode of interaction between the protein and the ligand involved hydrophobic binding. The effect of temperature and the ionic strength of the adsorption buffer on the isothermal adsorption behaviour under batch equilibrium binding conditions of the three proteins were determined.The affinities and the capacities of the agarose-based butyl ligand adsorbents, as well as the octyl ligand adsorbents, for the test proteins were increased as the salt concentration was increased under batch equilibrium binding conditions. Moreover, with both butyl and octyl adsorbents, as the ionic strength was increased under constant temperature conditions, the isothermal adsorption dependencies progressively approximated more closely a Langumuir model of independent binding site interactions, typical of a mono-layer binding process. In parallel experiments, the effect of changes in temperature under iso-ionic strength conditions was examined. With increasing temperature , different patterns of isothermal adsorption behaviour for the test proteins were observed, with the magnitude of these trends depending on the the type of interaction involved between the hydrophobic ligand and the protein. Utilizing second order van’t Hoff relationships to analyse the experimental data for these protein-ligand interactions, the apparent changes in enthalpy, entropy and heat capacity for these interactions have been derived from the dependency of the change in the apparent Gibbs free energy on 1 / T. Additionally, we also applied the isothermal titration microcalorimetry (ITC) to analyse the discrepancies between van’t Hoff and calorimetric enthalpies by measuring adsorption enthalphy of protein interacting with hydrophobic adsorbents directly. The thermodynamic parameters presented herein have important implication, both for providing further insight into the binding mechanism of protein adsorption and for improving theoretical approaches to HIC.
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