title: Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates.
abstract: Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λmax = 390 nm, ɛ = 32 880 M(-1) cm(-1) at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05 nmol mL(-1) was achieved for peroxidase.
authors.affiliation: a Department of Chemistry , Islamic Azad University of Mashhad , Mashhad , The Islamic Republic of Iran .
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Mirazizi
authors.affiliation: b National Institute for Genetic Engineering and Biotechnology , Tehran , The Islamic Republic of Iran .
authors.forename: Azita
authors.initials: A
authors.lastname: Bahrami
authors.affiliation: c Biochemistry and Biophysics, National Institute for Genetic Engineering and Biotechnology , Tehran , The Islamic Republic of Iran .
authors.forename: Kamahldin
authors.initials: K
authors.lastname: Haghbeen
authors.affiliation: d National Institute for Genetic Engineering and Biotechnology , Tehran , The Islamic Republic of Iran .
authors.forename: Hossein
authors.initials: H
authors.lastname: Shahbani Zahiri
authors.affiliation: e Department of Chemistry , Ferdowsi University of Mashhad , Mashhad , The Islamic Republic of Iran , and.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Bakavoli
authors.affiliation: f Department of Chemical Engineering , University of Waterloo , Waterloo , Ontario , Canada.
authors.forename: Raymond L
authors.initials: RL
authors.lastname: Legge
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D001391:Azo Compounds / Q000378:metabolism*; D007700:Kinetics; D009195:Peroxidase / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods; D013379:Substrate Specificity
chemical_list: D000814:Aniline Compounds; D001391:Azo Compounds; D009195:Peroxidase
keywords: Diazo of aniline; direct assay; peroxidase; spectrophotometry