Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbate-dependent reduction of nitroblue tetrazolium.
A method for the detection of ascorbate peroxidase activity in native electrophoretic gels is described. The assay is based on the ability of ascorbate peroxidase to prevent the ascorbate-dependent reduction of nitroblue tetrazolium in the presence of H2O2. The method was found to be both sensitive (detection of less than 0.01 units of ascorbate peroxidase activity) and specific for ascorbate peroxidase activity. The application of the method for the detection of ascorbate peroxidase activity in protein extracts from several plant sources was investigated by comparing staining for activities of ascorbate peroxidase, horseradish peroxidase, and ascorbate oxidase and by immunodetection of ascorbate peroxidase in these extracts.
Mittler
R
R
Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231.
Zilinskas
B A
BA
D000180:Acrylic Resins; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology; D004586:Electrophoresis; D004795:Enzyme Stability; D005782:Gels; D005990:Glycerol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D009580:Nitroblue Tetrazolium / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D010944:Plants / Q000201:enzymology
D000180:Acrylic Resins; D005782:Gels; D010940:Plant Proteins; C016680:polyacrylamide gels; D009580:Nitroblue Tetrazolium; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D005990:Glycerol; D001205:Ascorbic Acid