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Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye–ligand adsorbents
Author(s) -
Zhang Songping,
Sun Yan
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10067
Subject(s) - ionic strength , chemistry , adsorption , agarose , bovine serum albumin , aqueous solution , sepharose , chromatography , covalent bond , protein adsorption , ionic bonding , hydrophobic effect , phase (matter) , ion , organic chemistry , enzyme
Abstract 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 coupling 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 (15.4 μmol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 m M Tris‐HCl 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 0.2 M . The electrostatic exclusion effect is even found for CB–6AS with a CB density as low as 2.38 μmol/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. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 710–717, 2001.