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 H₂O₂ as 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^-1 and 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.

Keywords: Catalase activity; Pyrogallol red; Response surface methodology.