Purification and characterization of catalase from chard (Beta vulgaris var. cicla).
Catalase is a major primary antioxidant defence component that primarily catalyses the decomposition of H(2) O(2) to H(2) O. Here we report the purification and characterization of catalase from chard (Beta vulgaris var. cicla). Following a procedure that involved chloroform treatment, ammonium sulfate precipitation and three chromatographic steps (CM-cellulose, Sephadex G-25, and Sephadex G-200), catalase was purified about 250-fold to a final specific activity of 56947 U/mg of protein. The molecular weight of the purified catalase and its subunit were determined to be 235 000 and 58 500 daltons, indicating 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 similar to that obtained for catalase from other plant sources. In the catalytic reaction, the apparent Km value for chard catalase was 50 mM. The purified protein has a broad pH optimum for catalase activity between 6.0 and 8.0. The enzyme had an optimum reaction temperature at 30 degrees C. Heme catalase inhibitors, such as azide and cyanide, inhibited the enzyme activity markedly and the enzyme was also inactivated by ?-mercaptoethanol, dithiothreitol and iodoacetamide.
Dinçer
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Celal Bayar University, Science and Arts Faculty, Chemistry Department, Muradiye-Manisa 45047 Türkiye. aadincer@superonline.com
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D000645:Ammonium Sulfate; D002374:Catalase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D019663:Chenopodiaceae / Q000201:enzymology*; D002712:Chlorides / Q000494:pharmacology; D002725:Chloroform; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019327:Copper Sulfate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005290:Ferric Compounds / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D017895:Manganese Compounds / Q000494:pharmacology; D008970:Molecular Weight; D011190:Potassium Cyanide / Q000494:pharmacology; D021122:Protein Subunits; D019810:Sodium Azide / Q000494:pharmacology; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013816:Thermodynamics
D002712:Chlorides; D004791:Enzyme Inhibitors; D005290:Ferric Compounds; D007202:Indicators and Reagents; D017895:Manganese Compounds; D021122:Protein Subunits; D013438:Sulfhydryl Compounds; C024823:ferric sulfate; D002725:Chloroform; D019810:Sodium Azide; D002374:Catalase; D019327:Copper Sulfate; D011190:Potassium Cyanide; C025340:manganese chloride; D000645:Ammonium Sulfate; D005976:Glutaral