Stationary Phase Molecular Arm Of Solute Retention And Protein Refolding And Ion Exchange Chromatography Refolding Of Lysozyme,

Stationary and mobile phases in liquid chromatography(LC) is the most important factors effecting the solute retention. So the studies on the effect of stationary phase on the retention of solute has a great significance. In was used stoichiometric displacement theory for retention ( SDT-R) to study on the effect of stationary phase on the solute retention, protein refolding and the refolding of the reduced/denatured lysozyme by both hydrophobic chromatography (HIC) and weak-cation exchange chromatography (WCX). The thesis includes five parts as the follows:1.Review: Solute retetion mechanism in LC attracts so many scientists attention that it is the foundation of separation and purification. It also provides a directions for protein separation and purification. In this chapter solute retention mechanism in LC, protein refolding mechanism and new developments in protein refolding kinetics were reviewed. It contains 61 references.2.Stoichiometric displacement theory for retention, parameters in SDT-R , the phyical meanings of every paramters and the methods of measuring four parameters in HIC were introduced.3.Different ligands of chromatogrphic stationary phase can effect solute retention. Four parameters logI Z j and logφ of proteins and aromatic alcohol homologues in SDT-R obtained from DIOL columns of HIC having same matrix and end group, but with and without molecular spacer were employed to find out the contribution of molecular spacer to protein and aromatic alcohol homologues retention. A conclusion was that the non-polar and polar amino acid residues of protein moleculars separately interact with non-polar and polar groups of the ligands including molecular spacer, resulting in increasing the interactions between proteins and the stationary phase.4.There is a great difference in mass and bioactivity recoveries when lysozyme refolding and recombinant human interferen-γ (rhINF-γ) using two different DIOLcolumns.The reason could be attributed to the exitence of hydrophilic molecular spacer in DIOL-1.5.We found the optimal chromatographic condition to refold the reduced/denatured lysozyme was that the urea concentration was 4molL*1, pH was 8.0 , the ratio of GSH/GSSG was 5:1 and refolding time was 4 hours. In this chromatographic conditions the mass and bioactivity recoveries approched to its maximun value. Compared to the effects of two preparations of reduced/denatured lysozyme sample —dialysed and without dialysed on refolding lysozyme each after, indicating the mass recoveries was no obvious difference between them but the bioactivity recovery by DIOL-2 column was far more great than the DIOL-1 column's.The reason should be: that the dialysed sample doesn't contain denaturant, part of denatured lysozyme refolding and becoming its intermediate without bioactivity. But the intermediate can be refolded by LC.