| Summary: | 碩士 === 國立中央大學 === 化學工程研究所 === 89 === 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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