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 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 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.

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.

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.