Purification and characterization of catalase from chard (Beta vulgaris var. cicla)

Catalase is a major primary antioxidant defence component that primarily ca talyses the decomposition of H2O2 to H2O Here we report the purification an d characterization of catalase from chard (Beta vulgaris var. cicla). Follo wing a procedure that involved chloroform treatment, ammonium sulfate preci pitation and three chromatographic steps (CM-cellulose, Sephadex G-25, and Sephadex G-200), catalase was purified about 250-fold to a final specific a ctivity of 56947 U/mg of protein. The molecular weight of the purified cata lase and its subunit were determined to be 235000 and 58500 daltons, indica ting that the chard catalase is a tetramer. The absorption spectra showed a soret peak at 406 nm, and there was slightly reduction by dithionite. The ratio of absorption at 406 and 275 nanometers was 1.5, the value being simi lar to that obtained for catalase from other plant sources. In the catalyti c reaction, the apparent Km value for chard catalase was 50 mM. The purifie d protein has a broad pH optimum for catalase activity between 6.0 and 8.0. The enzyme had an optimum reaction temperature at 30 degreesC. Heme catala se inhibitors, such as azide and cyanide, inhibited the enzyme activity mar kedly and the enzyme was also inactivated by beta -mercaptoethanol, dithiot hreitol and iodoacetamide.