The ion-exchange adsorption kinetics of bovine serum albumin (BSA) and g-globulin to an anion exchanger, DEAE Spherodex M, has been studied by batch adsorption experiments. Various diffusion models, that is, pore diffusion, surface diffusion, homogeneous diffusion and parallel diffusion models, are analyzed for their suitabilities to depict the adsorption kinetics. Protein diffusivities are estimated by matching the models with the experimental data. The dependence of the diffusivities on initial protein concentration is observed and discussed. The adsorption isotherm of BSA is nearly rectangular, so there is little surface diffusion. As a result, the surface and homogenous diffusion models do not fit to the kinetic data of BSA adsorption. The adsorption isotherm of g-globulin is less favorable, and the surface diffusion contributes greatly to the mass transport. Consequently, both the surface and homogenous diffusion models fit to the kinetic data of g-globulin well. The adsorption kinetics of BSA and g-globulin can be very well fitted by parallel diffusion model, because the model reflects correctly the intraparticle mass transfer mechanism. In addition, for both the favorably bound proteins, the pore diffusion model fits the adsorption kinetics reasonably well. The results here indicate that the pore diffusion model can be used as a good approximate to depict protein adsorption kinetics for protein adsorption systems from rectangular to linear isotherms.

A predictive model is developed to describe the salt effects on the adsorption equilibrium of protein to Cibacron Blue-modified agarose gel (Sepharose CL-6B). This model assumes that, with the addition of salt, a fraction of dye-ligand molecules will lodge to the surface of the agarose gel resulting from the induced strong hydrophobic interaction between the dye ligands and agarose surface. This leads to a decrease in the dye ligands accessible to protein adsorption and consequently decreases the adsorption capacity of protein. The effect of salt on the dye-ligand lodging is presented by the equilibrium between salt and the dye ligands. Combined with the basic concept of steric-mass action theory, which considers both the multipoint nature and the macromolecule steric shielding of protein adsorption, an implicit isotherm of the protein adsorption equilibrium on Cibacron Blue 3GA-modified Sepharose CL-6B is formulated, involving salt concentration as a variable. Good agreement between experimental data and the predicted adsorption isotherms for single-component protein systems has been demonstrated, and fairly good matching between the predicted and experimental data for the binary adsorption of bovine serum albumin and bovine hemoglobin is obtained under most conditions. This model is expected to be useful in the design and optimization of the salt-gradient elution process of dye-ligand affinity chromatography.

A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70mg/g MAS (wet weight) at a relatively low CB coupling density (3-5μmol/g). In addition, 1.0M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity.

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, there existed an optimum denaturant concentration of about 1M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3ml of the lysozyme (1mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54mg per l gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12mg per l gel perh), indicating the advantage of the present system for producing a high throughput in protein refolding operations.

Crude soybean lecithin was used as a novel surfactant to form reversed micelles in n-hexane. Cibacron Blue F-3GA (CB) was directly immobilized to the reversed micelles by a two-phase reaction. The reversed micellar system without CB showed low solubilizing capacity for low molecular weight proteins, lysozyme, and cytochrome c due to the weak electrostatic interactions. The introduction of CB significantly increased the solubilization of lysozyme because of its affinity binding to CB but showed no effect on the solubilization of cytochrome c since it did not bind to CB. Although bovine serum albumin had an affinity for CB, it was not extracted to the reversed micelles containing CB because its high molecular weight resulted in a significant steric hindrance effect. Thus the reversed micellar system had a high selectivity resulting from both biospecific and steric hindrance effects. The extraction yield of lysozyme decreased significantly with increasing ionic strength. Therefore, the back extraction of lysozyme was carried out using a stripping solution with an ionic strength of 0.865mol/L. The overall recovery yield of lysozyme after back extraction could be increased to 87% by stripping for 2 h. The recovered lysozyme exhibited an activity equivalent to native lysozyme, and its secondary structure was also unchanged.