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

• Published in: Analytical biochemistry 2007-03, Vol.362 (1), p.38-43
• Main Authors: Molaei Rad, A., Ghourchian, H., Moosavi-Movahedi, A.A., Hong, J., Nazari, K.
• Format: Article
• Language: English
• Subjects: Aniline; Aniline Compounds - chemistry; Aniline Compounds - metabolism; Catechols - chemistry; Catechols - metabolism; Enzyme assay; Horseradish peroxidase; Horseradish Peroxidase - chemistry; Horseradish Peroxidase - metabolism; Hydrogen donor; Kinetics; Protons; Pyrocatechol; Reproducibility of Results; Spectrophotometry - methods
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2006.11.035

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 λ max 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 K m (12.5 mM) and V max (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.