Font Size: a A A

Theoretical Studies Of Protein Dye-Ligand Affinity Chromatography

Posted on:2004-07-07Degree:DoctorType:Dissertation
Country:ChinaCandidate:S P ZhangFull Text:PDF
GTID:1101360092980637Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
A reactive dye, Cibacron Blue 3GA (CB) was immobilized to three kinds of agarose gels: 6% agarose coated steel beads (6AS), home-made 4% agarose beads and Sepharose CL-6B to obtain dye-ligand adsorbents. Bovine serum albumin (BSA), hemoglobin (Hb) and lysozyme were chosen as model proteins. The effects of liquid-phase ionic strength, pH values and immobilized CB-ligand densities on the adsorption equilibrium and kinetics of proteins were studies in detail.It was found that as for BSA adsorption, on one hand, increase in ionic strength could weaken the electrostatic repulsion between like-charged BSA and CB modified gels, which favorable for BSA adsorption; on the other hand, high ionic strength results in decrease of ligand density accessible to protein adsorption induced by strong hydrophobic interactions between dye ligand and gel matrix, which is unfavorable for protein adsorption. As a result, there was an optimal ionic strength to maximize the adsorption of BSA to CB-ligand adsorbent. As for the CB-6AS and CB-home-made 4% Agarose beads with smaller pore sizes, the optimal salt concentration was 0.2 mol·L-1; while for CB-Sepharose CL-6B with larger pores, the optimal salt concentration was 0.05 mol·L-1. The adsorption kinetics was analyzed using a pore diffusion model and the effective pore diffusivities, De, of protein adsorption to CB-ligand agarose gels were obtained. It was shown that the De values of BSA increases with the increase of liquid-phase ionic strength and/or decrease of immobilized CB-ligand density. This was considered due to the decrease in electrostatic exclusion encountered by BSA during its diffusion to like-charged gel pores. While for the adsorption kinetics of Hb and lysozyme which with higher isoelectric points, little electrostatic hindrance effects were suffered. To quantitatively describe the effect of liquid-phase salt concentration on the adsorption equilibrium of protein to CB-modified agarose gel, a steric mass-action model (SMA) has been developed for the dye-ligand affinity system. This modelassumes that the decrease in adsorption of hydrophobic protein (i.e., BSA) at high salt concentration is mainly resulted from the lodge of the immobilized dye ligand to gel surface induced by the strong hydrophobic interaction between them. The effect of salt on the dye ligand lodging is presented by the equilibrium between salt and the dye ligand. Combining with the basic concept of steric-mass action theory, which considers both the multi-point nature and the macromolecule steric shielding of protein adsorption, an implicit SMA isotherm formulism for protein adsorption equilibrium on CB-Sepharose is formulated, involving salt concentration as a variable. It was showed that this model can be well used to predict the adsorption equilibrium of BSA to CB-Sepharose, as well as the adsorption of binary mixture system containing BSA and Hb at given salt concentration. In consideration of the difference in the mechanism of salt affecting the adsorption of acid protein (i.e., BSA) and basic protein (i.e., lysozyme), the SMA model developed above has been modified to account for the effect of salt on the dye-ligand adsorption of lysozyme. Incorporating with appropriate transport model equation, the SMA model was used to model the dye-ligand affinity chromatography process. It was proved that the model could describe the linear gradient elution of protein under non-linear adsorption condition fairly well.
Keywords/Search Tags:Dye-ligand adsorbents, Cibacron Blue 3GA, Protein adsorption equilibrium, Adsorption kinetics, Steric mass-action model, Dye-ligand affinity chromatography model
PDF Full Text Request
Related items