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.