Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol–aniline coupling hydrogen donor

The hydrogen donor couples pyrocatechol–aniline and phenol–aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV–Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol–aniline was converted to a pink-colored reagent with a λmax of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol–aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol–aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of Km (12.5 mM) and Vmax (12.2 mM min−1 mg−1) were calculated for pyrocatechol–aniline. Regarding the superiority of pyrocatechol–aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.