Purification and characterization of anionic peroxidases from cotton (Gossypium hirsutum L.)
نویسندگان
چکیده
Anionic isoperoxidases from cotton cotyledonary leaf tissue have been purified to electrophoretic homogeneity by chromatography on ion exchange, lectin affinity, and gel filtration matrices. The enzyme was stable and active at a wide range of temperatures with optimal activity at 55°C. Maximum activity was observed when the pH was adjusted from pH 5.5 to pH 7.5. Electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels indicated a relative molecular mass of 56 000 Da when the sample was treated with a disulfide reducing agent and heated. The electrophoretic mobility was altered when either the disulfide reducing agent or heat treatment was omitted. Isoelectric focusing under native conditions resolved the activity into three isozymes with isoelectric points of 4.2, 4.4 and 4.6. The K m of peroxidase for 4-aminoantipyrine and o-dianisidine was 350 and 36 ~M, respectively. Enzyme activity was inhibited by elevated concentrations of H202 and by disulfide reducing agents. Known inhibitors of peroxidase activity such as potassium azide, sodium cyanide and sodium sulfite inhibited cotton anionic peroxidases. The absorption spectrum showed maxima at 280 and 402 nm, indicating the presence of a heme group in the active enzyme. The extinction coefficient at 402 nm was 1.12 x 105 mol -I cm -I.
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