title: A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology.
abstract: Pyrogallol red (PGR) was identified as a novel optical probe for the detection of hydrogen peroxide (H2O2) based on horseradish peroxidase (HRP)-catalyzed oxidation. Response surface methodology (RSM) was applied as a tool to optimize the concentrations of PGR (100µmolL-1), HRP (1UmL-1) and H2O2(250µmolL-1) and used to develop a sensitive PGR-based catalase (CAT) activity assay (PGR-CAT assay). N-ethylmaleimide -NEM- (102mmolL-1) was used to avoid interference produced by thiol groups while protecting CAT activity. Incubation time (30min) for samples or CAT used as standard and H2O2as well as signal stability (stable between 5 and 60min) were also evaluated. PGR-CAT assay was linear within the range of 0-4UmL-1(R2=0.993) and very sensitive with limits of detection (LOD) of 0.005UmL-1and quantitation (LOQ) of 0.01UmL-1. PGR-CAT assay showed an adequate intra-day RSD=0.6-9.5% and inter-day RSD=2.4-8.9%. Bland-Altman analysis and Passing-Bablok and Pearson correlation analysis showed good agreement between CAT activity as measured by the PRG-CAT assay and the Amplex Red assay. The PGR-CAT assay is more sensitive than all the other colorimetric assays reported, particularly the Amplex Red assay, and the cost of PGR is a small fraction (about 1/1000) of that of an Amplex Red probe, so it can be expected to find wide use among scientists studying CAT activity in biological samples.
authors.affiliation: Universidad Carlos III de Madrid, Departamento de Ingeniería de Sistemas y Automática, Leganés, Madrid, Spain.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Abderrahim
authors.affiliation: Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Fisiología, Madrid, Spain.
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Arribas
authors.affiliation: Universidad Carlos III de Madrid, Departamento de Ingeniería de Sistemas y Automática, Leganés, Madrid, Spain; Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Fisiología, Madrid, Spain. Electronic address: lcondezo@s-analyst-c.com.
authors.forename: Luis
authors.initials: L
authors.lastname: Condezo-Hoyos
mesh_terms: D000818:Animals; D002374:Catalase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D057230:Limit of Detection; D011748:Pyrogallol / Q000031:analogs & derivatives* / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D011748:Pyrogallol; C058731:pyrogallol sulfonphthalein; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D002374:Catalase
keywords: Catalase activity; Pyrogallol red; Response surface methodology