Quantitation of adsorption capacity of immunoglobulin G on histidine—aminohexyl Sepharose and determination of affinity constant

Abstract

Histidine, a pseudobiospecific ligand, had been utilized to purify several proteins such as chymosin, acidic protease, carboxypeptidase Y and immunoglobulin G (IgG). A detailed study was undertaken to purify IgG on histidine coupled to aminohexyl Sepharose [A. El-Kak and M. A. Vijayalakshmi, J. Chromatogr., 510 (1991) 29]. To better understand the force of interaction between IgG and histidine coupled to aminohexyl Sepharose, the equilibrium dissociation constant ( K D) was determined by standard techniques such as frontal and zonal elution. The maximum capacity ( Q x) and K D were determined to be 11.6 mg IgG per ml gel and 2.4 · 10 −6 M, respectively, by frontal analysis. Using zonal elution with histidine as a competing soluble free ligand in the equilibrating buffer, the values K D between IgG and soluble free histidine and between IgG and immobilized histidine were determined to be 0.351 M and 2.4 · 10 −5 M, respectively. The zonal elution value is approximately ten times higher than that estimated by frontal analysis. It was verified again by equilibrium binding analysis. Using this technique we determined K D and Q x to be 4.6 · 10 −6 M and 9 mg/ml, respectively, which are very close to the frontal analysis results.