Manipulation of the affinity between protein and metal ions by imidazole and pH for metal affinity purification of protein C from Cohn fraction IV-1.

Protein C (PC) is an important anticoagulant in blood plasma. Cohn Fraction IV-1 (CFIV-1) is an inexpensive PC source but contains a large amount of factor II (FII). Immobilized metal affinity chromatography (IMAC) utilizes metal ions to adsorb proteins primarily via their surface histidine. Two major operation parameters for IMAC are imidazole concentration and pH: imidazole is a histidine analog and pH controls the protein surface protonation level. The effects of these two parameters on the adsorption and elution of PC and FII were studied for each protein individually and also together as a mixture. For the individual proteins, low FII (16%) and high PC (98%) adsorption were achieved at 8 mM imidazole, pH 8.0. At 11 mM imidazole, 92% of the adsorbed FII was eluted, with only a 3% PC loss. At 40 mM, 97% of the adsorbed PC was recovered. For the protein mixture, very similar adsorption and elution results were obtained, but slightly greater PC loss (16%) during elution at 11 mM imidazole. This result shows that there is a high potential for the PC purification from CFIV-1 by appropriately adjusting the imidazole concentration and pH in the IMAC process.