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 two-state protein model is proposed to describe the salt effects on protein adsorption equilibrium on hydrophobic media. This model assumes that protein molecules exist in two equilibrium states in a salt solution, that is, hydrated and dehydrated states, and only the dehydrated-state protein can bind to hydrophobic ligands. In terms of the two-state protein hypothesis and the steric mass-action theory, protein adsorption equilibrium on hydrophobic media is formulated by a five-parameter equation. The model is demonstrated with the adsorption of bovine serum albumin to Phenyl Sepharose gels as a model system. The effects of salt type (sodium chloride, sodium sulfate and ammonium sulfate) on the model parameters are discussed. Then, the model formulism is simplified in terms of the small magnitude of the protein dehydration equilibrium constant in the model. This simplification has returned the model derived on the basis of the two-state protein hypothesis to its original mechanism of salt effects on the hydrophobic adsorption of protein. This simplified model also creates satisfactory prediction of protein adsorption isotherms.

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.

Novel dense composite adsorbents for expanded bed adsorption of protein have been fabricated by coating 4% agarose gel onto Nd-Fe-B alloy powder by a water-in-oil emulsification method. Two composite matrices, namely Nd-Fe-B alloy-densified agarose (NFBA) gels with different size distributions and densities, NFBA-S (50-165mm, 1.88g/ml) and NFBA-L (140-300mm, 2.04g/ml), were produced. Lysozyme was used as a model protein to test the adsorption capacity and kinetics for the NFBA gels modified by Cibacron blue 3GA (CB-NFBA gels). Liquid-phase dispersion behavior in the expanded beds was examined by measurements of residence time distributions, and compared with that of Streamline SP (Amersham-Pharmacia Biotech, Sweden). The dependence of axial mixing in the expanded beds on flow velocity, bed expansion degree, settled bed height, and viscosity of liquid phase was investigated. Breakthrough curves of lysozyme in the expanded beds of the CB-NFBA gels were also examined. The dynamic binding capacity at 5% breakthrough was 23.3mg/ml matrix for the CB-NFBA-S gels, and 16.7mg/ml matrix for the CB-NFBA-L, at a flow velocity of 220cm/h. The results indicate that the NFBA gels are promising for expanded bed adsorption of proteins.

The adsorption equilibria and kinetics of bovine serum albumin(BSA)and lysozyme to two Cibacron Blue 3GA(CB)modified agarose gels, that is, 6% agarose-coated steel (6AS)and Sepharose CL-6B, in 0.01 kmol•my- tris-HCl buffer(pH7.5)were studied. Effects of aqueous-phase ionic strength on both the adsorption equilibrium and uptake rate of the proteins were significant and distinctly different between BSA and lysozyme. The adsorption of lysozyme decreased monotonically with increasing ionic strength. Ionic strengths, however, maximized BSA adsorption capacities of the two CB-modified agarose gels in the tris-HCl buffer (about 0.2kmol•m-3 for CB-6AS and 0.05kmol•m-3 for CB-Sepharose), when the pore-size difference of the two matrixes and electrostatic interactions between the BSA and CB molecules of like charge were considered. The effective diffusivity of BSA, derived from a pore-diffusion model, to both the CB-modified agarose gels increased significantly with the increasing ionic strength at the ionic strength range of 0.01 to 0.3kmol•m-3, due to the electrostatic interactions between the BSA and CB molecules of like charge. In contrast, the effective diffusiities of lysozyme to CB-Sepharose in the buffer containing 0.1 and 0.3kmol/m-3 NaCl were nearly the same.

A poly(vinyl alcohol)-based magnetic gel entrapping Fe3O4 colloids has been prepared by an emulsification-crosslinking method. The gel was modified with Cibacron blue 3GA, and thus a magnetic affinity support was produced. The adsorption equilibrium studies showed that the adsorption isotherm of lysozyme was nearly rectangular, with a capacity of 254mg/ml, while the adsorption isotherm of bovine serum albumin obeyed the Henry's law. Uptake kinetics of the two proteins was investigated and analyzed with a pore diffusion model and a homogeneous diffusion model. Experimental results showed that the magnetic affinity gel had magnetic responsiveness and favorable properties in protein adsorption, and was mechanically and chemically stable.

The adsorption equilibria of bovine serum albumin (BSA), γ-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB cou piing density, and the net negative charges of proteins (or aqueous-phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4μmol/mL) at very low ionic strength (NaCI concentration below 0.05M in 10mM Tris-HCI buffer, pH 7.5). However, because CB 6AS and CB 4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2M. The electrostatic exclusion effect is even found for CB 6AS with a CB density as low as 2.38μmol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For γ-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.

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.