Systematic Studies of the Interaction Behavior of Protein with

• Main Authors: Wu, Ching-Fa, 吳青發
• Other Authors: Chen Wen-Yih
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/72633455009932241423

博士 === 國立中央大學 === 化學工程學系 === 84 === ABSTRACT In this study, the binding mechanism of immobilized metal ion with amino acid, peptides, and proteins has been investigated systematically. A seriesof experiments divided into three subsystems were performed to fulfill the research. (a) In the homogenous phase, the binding constants of imidazole and histidine with Cu(II), that was immobilized on PEG-IDA, at various pH values,salt concentrations and temperatures of the solution were determined by using a differential UV spectrophotometer. A bimodel binding behavior of alkaline solutes was observed from the effect of salt concentration. However, the formation of the coordinated compound is dominated at pH values higher than the pKa of the deprotonation of imidazole nitrogen. Temperature had no obvious effects on the binding constant due to complexity of the binding mechanism. The binding behavior of several dipeptides and tripeptides with histidine at the C- or N-terminal was also investigated and the results were explained by the "metal ion transfer" (MIT) hypothesis. Furthermore, the binding constants of synthetic heptapeptides with two histidine residues separated by different amount of glycine residues were investigated to demonstrate the effect of histidine residues distance on the binding affinity. (b) In the heterogeneous phase, equilibrium binding analyses were performed toassess the influence of pH value, salt concentration and number of exposed histidine residues of protein on the binding affinity and capacity for imidazole and proteins with immobilized Cu(II) ion. The proteins under this investigation were lysozyme, ribonuclease A, and hemoglobin with the number of exposed histidine residues being 1, 2, and 27, respectively. Molecular modeling estimation was also performed to reveal the solvent-accessible surface area of each histidine residue. Also, the result obtained from the calculation on the number of the exposed histidines of the proteins were found to be compatible with that in the previous literature. Moreover, the impact of pH value on the binding affinity and capacity of imidazole and the proteins with immobilized metal ion was described by the deprotonation of N-imidazole and close proximity of adsorbed imidazole on the immobilized metal ion gel.However, the variety ofbinding capacities resulting from salt concentrationsindicated the existenceof a manifold binding mechanism involved with thehydrophobic and electrostatic interactions of solutes with immobilized Cu(II)ions and with the hydrophilic gel surface. Moreover, the different responses ofthe proteins toward the effects of pH values and salt concentrations were interpreted by the specific features of the protein surface structure and by the different expressions in the adsorption isotherm. (c) In this investigation, we measured the influence of pH value and salt econcentration on the heat of interaction of imidazole and lysozyme with CS-IDA-Cu(II) gel by a highly sensitive microcalorimeter. The direct enthalpy measurement of the interaction provides thermodynamic information regarding the binding behavior of solutes toward the immobilized metal ion. The binding enthalpy changed with the adsorbed amount of imidazole and lysozyme at various pH values and salt concentrations were measured and those results were discussedwith the reported binding isotherm The bindingand thermodynamic data obtained in this study can provide informationfor the binding mechanism and process of imidazole and protein with immobilizedmetal ions. In this study, there were several subprocedures to depictthe adsorption behavior of solutes to immobilized metal ion. Both enthalpydriven and entropy driven presented when solutes adsorbed onto immobilizedmetal ion. In addition, the effect of structure rearrangement would notbenegligible for immobilized metal ion adsorbing lysozyme since the enthalpyof adsorption on was positive at pH=7.0 and 8.5, 0.0M NaCl. In this study, the homogenous and heterogeneous results provided the details of adsorption behavior of amino acid, peptide and protein to immobilized metal ion. In addition, the binding mechanism of IMA was explored by the thermodynamic information (enthalpy of adsorption) measured by ITC. Therefore, the operating strategies of IMA for protein separation and purification were clear by this study.