Modified immunodiffusion and peroxidase staining methods to detect Escherichia coli nitrite reductase and other sirohaem-containing proteins.
A rapid, sensitive and stable staining procedure is described which has allowed the peroxidase activity associated with the sirohaem-containing NADH-dependent nitrite reductase from Escherichia coli to be detected. Sirohaem is rapidly lost from this enzyme, so conventional one- or two-dimensional immunoelectrophoresis techniques were modified to permit the detection of residual sirohaem associated with the protein. Reconstitution experiments established that a sirohaem-rich fraction from a nitrite reductase-deficient mutant could reactivate partially purified nitrite reductase eluted from a chromatography column. We suggest that the techniques described could readily be modified to detect immunoprecipitates containing other labile enzymes for which there is a specific activity stain, or for other proteins with an associated peroxidase activity.
D003378:Counterimmunoelectrophoresis; D004926:Escherichia coli / Q000032:analysis / Q000201:enzymology* / Q000235:genetics; D006418:Heme / Q000031:analogs & derivatives* / Q000032:analysis; D005779:Immunodiffusion / Q000379:methods; D007123:Immunoelectrophoresis, Two-Dimensional; D007527:Isoenzymes / Q000378:metabolism*; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism*; D009195:Peroxidase; D010544:Peroxidases / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
D007527:Isoenzymes; D006418:Heme; C009272:siroheme; D010544:Peroxidases; D009195:Peroxidase; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases