Cu2+-immobilized magnetic poly(hydroxyethylmethacrylate-N-methacryloyl-(l)-histidinemethylester) (mPHEMAH) nanoparticles were prepared by surfactant-free emulsion polymerization for cytochrome C (cyt C) purification from rat liver. Elemental analysis, atomic force microscopy, zeta sizer, and vibrating sample magnetometer were used to characterize mPHEMAH nanoparticles. In addition to these characterization steps, surface area, average particle size, and size distribution of mPHEMAH nanoparticles were determined. Quantity of immobilized Cu2+ was measured using atomic absorption spectrophotometry. N-Methacryloyl-(l)-histidinemethylester and Cu2+ content of mPHEMAH nanoparticles were 0.18 mmol/g polymer and 0.11 mmol/g polymer, respectively. Specific surface area of Cu2+-immobilized mPHEMAH nanoparticles was 1180 m(2)/g. Effect of initial cyt C concentration, pH, temperature, and ionic strength on cyt C adsorption onto Cu2+-immobilized mPHEMAH nanoparticles was investigated. Maximum cyt C adsorption capacity of Cu2+-immobilized mPHEMAH nanoparticles was 311.9 mg/g polymer. Maximum adsorption was obtained at pH 8.0 and 4 degrees C. Cu2+-immobilized mPHEMAH nanoparticles were used ten times with 4.1% decrease in adsorption capacity. In the last stage, Cu2+-immobilized mPHEMAH nanoparticles were used to purify cyt C from rat liver tissue, and the purity of desorbed fractions was controlled by SDS-PAGE. (C) 2015 International Union of Biochemistry and Molecular Biology, Inc.