An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase.
When plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD-dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)-mediated reduction of NBT. This reduction was enhanced by H2O2 and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS-mediated reduction of NBT and for the peroxidase-catalyzed NADH-dependent formation of H2O2. This peroxidase-catalyzed reaction has been proposed for the plant peroxidases involved in lignification.
Fieldes
M A
MA
Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
D000180:Acrylic Resins; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D006735:Horseradish Peroxidase; D006861:Hydrogen Peroxide; D007527:Isoenzymes / Q000302:isolation & purification*; D008291:Malate Dehydrogenase / Q000032:analysis*; D008773:Methylphenazonium Methosulfate / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D009580:Nitroblue Tetrazolium; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000302:isolation & purification*; D013194:Staining and Labeling
D000180:Acrylic Resins; D005782:Gels; D007527:Isoenzymes; C016680:polyacrylamide gels; D009243:NAD; D009580:Nitroblue Tetrazolium; D008773:Methylphenazonium Methosulfate; D006861:Hydrogen Peroxide; D008291:Malate Dehydrogenase; D006735:Horseradish Peroxidase; D010544:Peroxidases