Aromatic azo compounds as spectrophotometric kinetic assay substrate for HRP

Aromatic azo compounds were efficiently cleaved by the horseradish peroxidase (HRP) into aromicdiazonium ions, during which an intense special absorption of the substrate disappeared completely. This furnished a sensitive spectrophotometric detection of 0.025–6.0 nm peroxidase. Kinetic characteristics of enzyme reaction were investigated with 14 different chemical structures of aromatic azo compounds as the substrates for HRP. Then the structure requirements for substrates were elucidated. PH dependence of enzymatic catalysis was studied with pKa′of 6.54 and 8.40 for Eriochrome black T, and 3.22 and 3.83 for Nitrosulfophenol S as the substrate of HRP, respectively.