Visualization of peroxidase isozymes with eugenol, a noncarcinogenic substrate

Plant peroxidase isozymes utilize hydrogen peroxide to oxidize redox dyes. Benzidine, the substrate most commonly used for identification of peroxidase isozymes, is a potent carcinogen and has been banned from laboratory use. O-Dianisidine, the other common substrate for peroxidase isozymes, is structurally related to benzidine and is a possible carcinogen. A peroxidase zymogram stain has been developed which uses eugenol, a substrate which is safe to use in the laboratory. Peroxidase isozymes are recognized as bright blue fluorescent bands under ultraviolet light and develop within 1 min after staining. Eugenol may also be used in a quantitative fluorimetric assay of peroxidase activity.