Immobilized metal affinity monolithic cryogels for cytochrome c purification.

A monolithic cryogel column was prepared to obtain an efficient, cost effective and rapid purification of cytochrome c from rat liver homogenate. N-Methacryloyl-(l)-histidine methyl ester (MAH) was used as the metal-chelating ligand. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] monolithic cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. PHEMAH cryogel was characterized by surface area measurements, swelling studies, scanning electron microscopy and elemental analysis. The PHEMAH cryogel had a specific surface area of 38.6 m(2)/g. The PHEMAH cryogel containing 113.7 μmol MAH/g was complexed with the Cu(2+) ions directly via MAH for the adsorption of cytochrome c from aqueous solutions and rat liver homogenate. The cytochrome c adsorption on the PHEMAH cryogel was 3.1 mg/g. The maximum cytochrome c adsorption capacity of the Cu(2+)-chelated PHEMAH cryogel (carrying 58.7 μmol Cu(2+) per gram of polymer) was found to be 20.8 mg/g at pH 8.0 in phosphate buffer. The cytochrome c adsorption amount from rat liver homogenate was 37.7 mg/g with a purity of 93.8%. It was observed that cytochrome c could be repeatedly adsorbed and eluted with the PHEMAH cryogel without significant loss in the adsorption capacity.