Separation of Ovotransferrin from Egg White by Immobilized Metal Affinity Chromatography

Abstract

Immobilized metal affinity chromatography (IMAC) was used to separate ovotransferrin (OVT) by a single chromatographic step. Ovotransferrin was bound strongly to immobilized copper ions, while other components of egg white passed through the column. Using acidic pH and /or a strong competing solute, imidazole, ovotransferrin was recovered in 94~98% pure form as indicated by SDS-polyacrylamide gel electrophoresis and Immunoelectrophoresis. The elution profiles from the IMAC column indicated the presence of two forms of ovotransferrin. The capacity of the IMAC column was 20 mg OVT/ml copper loaded gel. Saturation of the metal binding sites of ovotransferrin with Fe2+ and Cu2+ did not interfere with the binding of ovotransferrin to the IMAC column. However, modification of the histidine residues in ovotransferrin with diethyl pyrocarbonate almost completely destroyed the binding to the IMAC column.