| 摘要: | 本研究藉恆溫滴定微卡計(isothermal titration calorimetry)之量測，探 討蛋白質與吸附基材表面作用之機制。研究之系統包括疏水作用層析 (hydrophobic interaction chromatography, HIC)、固定化金屬親和性層析 (immobilized metal ion affinity chromatography, IMAC)、及離子交換層析 (ion-exchange chromatography, IEC)等。另外，也利用van’t Hoff plot 及相 關之熱力學關係式來分析以疏水作用力為主之程序(如疏水性氣體溶於水 和疏水性氨基酸滯留於疏水作用層析管柱之行為)。由此所獲致之結果與 數據將有助於更進一步瞭解生化反應程序中蛋白質於界面之行為，並可 應用於蛋白質之純化。 在HIC 系統的研究方面，ITC 的結果顯示鹽類對蛋白質作用機制主 要的影響在於降低去水合所需之熱量。固定不同疏水基(n-butyl 和n-phenyl) 之吸附基材之差別則在於後者與蛋白質間有p-p作用力，而導致兩吸附基 材間有明顯不同的焓和火商值。另外，蛋白質與固定n-butyl 之吸附基材之 吸附焓為負值，但與固定n-octyl 的吸附基材則為正值，顯示蛋白質與nbutyl 膠體之作用機制主要為吸附作用，而與n-octyl 膠體則為分布行為。 而且HIC 系統之焓值也隨吸附基材疏水基之密度或蛋白質之疏水表面積 之增加而增加。換言之，當HIC 系統之疏水作用增加，焓值也會隨著增 加，表示蛋白質之作用機制可能由吸附為主的作用轉為以分布為主的行 為。另外，本研究也利用ITC 的量測，探討蛋白質與蛋白質間之作用行 為。例如，蛋白質a-chymotrypsinogen A 和trypsinogen 與疏水性吸附基材 作用之焓值，皆隨蛋白質吸附量之增加而增加，而且trypsinogen 之焓值 隨吸附量增加的幅度較a-chymotrypsinogen A 的大，表示trypsinogen 分子 間之斥力可能較a-chymotrypsinogen A 間的大。 v 在IMAC 系統的研究方面，由ITC 所量得的數據得知，lysozyme 與 固定化Fe(III)作用之焓值隨pH 值或NaCl 濃度增加而降低，而且lysozyme 與固定化Cu(II)作用之焓值比Fe(III)小，表示lysozyme 與固定化Cu(II)間 產生之配位鍵所放出之熱量較Fe(III)多。另外，本研究也將一基因重組蛋 白穀胱甘月太轉移酵素(Schistosoma japonicum glutathion Stransferase, SjGST)融合一含六個histidine 的標籤，而成為SjGST/His 來探討IMAC 系統之作用機制。ITC 之結果顯示，SjGST/His 與IMA 膠 體(NTA-Ni)作用之吸附焓較SjGST 的小，這是因為SjGST/His 與NTA-Ni 間產生較多的配位鍵。另外，SjGST/His 在正常(nature)和失活(denaturing) 狀態下與IMA 膠體(TALON)作用之吸附焓相差不大，顯示蛋白質之作用 機制在此兩種狀態下差別不大。 在IEC 系統的研究方面，是利用ITC 的量測探討蛋白質b-lactoglobulin A & B 在蛋白質等電點(isoelectric point, pI)時與陰離子交換基材(QSepharose) 之作用機制。結果顯示在pH 值接近等電點時，蛋白質與QSepharose 間之作用主要受靜電力與疏水力之影響，而在高NaCl濃度(0.3 M) 下，則以疏水作用為主。另外，在不同溫度下所量得之吸附焓值顯示， 溫度對蛋白質與Q-Sepharose 作用機制之影響與鹽濃度有關。例如在高 NaCl 濃度(0.3 M)下，以疏水作用為主，因此對溫度的效應較明顯。 This study presents a thermodynamic framework employed by isothermal titration microcalorimetric (ITC) measurements to explore the interaction mechanisms between proteins and solid surfaces in various modes of liquid chromatography such as hydrophobic interaction chromatography (HIC), immobilized metal ion affinity chromatography (IMAC), and ion-exchange chromatography (IEC). The approach for evaluating thermodynamic parameters based on van’t Hoff analysis associated with hydrophobic interactions involving processes such as dissolution of non-polar gases in water and HIC retention of dansyl amino acids was also reported. The data obtained and conclusion reached in this manner provide valuable information about the behaviours of biomaterials at interfaces at the molecular level and facilitate the purification of proteins by liquid chromatography in the industrial application. Specifically, in the study of HIC, the adsorption enthalpies of the proteins with the hydrophobic sorbents were measured by ITC under various experimental conditions. The results show the dependence of the free energy change for protein adsorption to HIC sorbents on the salt composition can be mainly attributed to the enthalpy changes associated with the protein and sorbent dehydration and hydrophobic interactions. Differences in binding mechanisms between the n-butyland phenyl-HIC sorbents were evident. In the latter case, the participation of p-p hydrophobic interactions leads to significant differences in the associated enthalpy and entropy changes. Also, the adsorption of the proteins on butyl containing adsorbents was exothermic, while their adsorption on octyl ones was endothermic, indicating the binding of both the proteins with butyl ligand is basically an adsorption process, whilst the binding with octyl ligand is adsorption and partition processes. Moreover, the enthalpy and entropy changes became increasingly positive as the ligand density of the sorbents or exposed hydrophobic surface area of the proteins vii increased. As a consequence, an increased contribution from the entropy change to the respective change in free energy occurs when HIC sorbents or proteins of higher hydrophobicity are employed, with these larger entropy changes consistent with a change in the interaction mechanism from a binding event dominated by adsorption to a partitioning-like process. Moreover, as temperature is increased from 298 to 310 K, the enthalpy change of a-chymotrypsinogen A with butyl-Sepharose increases, while the value of trypsinogen is deduced. This is likely owing to a-chymotrypsinogen A has a higher area of exposed hydrophobic segments than trypsinogen does. This observation also implies that as temperature increases, the interaction mechanism of a- chymotrypsinogen A with butyl-Sepharose transfers from an adsorption dominated to a partitioning process. In addition, ITC measurements can be used to probe protein-protein interaction. For example, the enthalpy changes of the proteins increased as the amount of bound protein increased, and the enthalpy increase of trypsinogen appeared to be higher than that of a-chymotrypsinogen A. This observation indicates the protein-protein repulsion was stronger among trypsinogen molecules in this experiment. In IMAC, various effects on the interaction mechanisms between the proteins and the immobilized metal ions were investigated by ITC measurements. The results reveal that the enthalpy changes decreased with the pH values and NaCl concentrations. Furthermore, the enthalpy of lysozyme with Fe(III) is higher than that with Cu(II) implying the heat generated from the formation of the coordination bonds with Cu(II) is higher than with Fe(III). In addition, a recombinant protein, Schistosoma japonicum glutathione-S-transferase (SjGST) was fused with a Cterminal hexa-histidine tag to obtain SjGST/His. Both the proteins were also used to probe the interaction mechanisms with two commercial affinity resin, Ni-NTA and TALON (Co2+), under the conditions of with and without the presence of denaturant. The result demonstrates that SjGST/His had a lower enthalpy change with Ni-NTA than did SjGST, mainly attributed to the formation of more coordination bonds with viii or a stronger binding with Ni-NTA. Moreover, the difference between the enthalpy change of SjGST/His onto TALON under the nature and denaturing condition were insignificant, implying the binding topography of the hexa-histidine tail with immobilized Co2+ was not significantly changed with the presence of denaturant. As a consequence, the proposed binding models and the directly measured adsorption heat, can be combined to elucidate the difference in the interaction mechanisms of the proteins adsorption onto the sorbents containing transition metal ions in thermodynamic perspectives. In IEC study, the interaction mechanisms between b-lactoglobulin A and B (Lg A, Lg B) with an anion exchanger, Q-Sepharose at pH near isoelectric point (pI) were examined under various NaCl concentrations and temperature by the equilibrium binding analysis and the adsorption enthalpy directly measured by ITC. The results demonstrate that the involvement of electrostatic and hydrophobic forces collectively affect the interaction behaviors of Lg A or Lg B with Q-Sepharose at pH near pI, leading to the changes in the binding affinities, capacities and adsorption enthalpies. Specifically, at a higher NaCl concentration (0.3M), the hydrophobic interactions becoming a more important contributor to the adsorption, while at 0.03 M NaCl, the electrostatic attraction was dominated. Furthermore, the enthalpy change values measured at pH near pI under various NaCl concentrations and temperature have confirmed that the effects of temperatures on equilibrium binding behaviors of Lg A or Lg B with Q-Sepharose were salt concentration-dependent, due to their different interaction mechanisms at 0.03 M and 0.3 M NaCl. |
