Quantitation of adsorption capacity of immunoglobulin G on histidine-aminohexyl sepharose and determination of affinity constant.
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 (KD) was determined by standard techniques such as frontal and zonal elution. The maximum capacity (QX) and KD were determined to be 11.6 mg IgG per ml gel and 2.4 x 10(-6) M, respectively, by frontal analysis. Using zonal elution with histidine as a competing soluble free ligand in the equilibrating buffer, the values KD between IgG and soluble free histidine and between IgG and immobilized histidine were determined to be 0.351 M and 2.4 x 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 KD and QX to be 4.6 x 10(-6) M and 9 mg/ml, respectively, which are very close to the frontal analysis results.
Mandjiny
S
S
Laboratoire de Technologie des Séparations, Université de Technologie de Compiègne, France.
Vijayalakshmi
M A
MA
D000327:Adsorption; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D006639:Histidine; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D008024:Ligands; D012685:Sepharose / Q000031:analogs & derivatives
D007074:Immunoglobulin G; D008024:Ligands; D006639:Histidine; C024903:aminohexyl-sepharose; D012685:Sepharose