| Summary: | 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.
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