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A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology
Mohamed Abderrahim1, Silvia M Arribas2, Luis Condezo-Hoyos3
1Universidad Carlos III de Madrid, Departamento de Ingeniería de Sistemas y Automática, Leganés, Madrid, Spain.
Talanta
|February 19, 2017
Summary
Pyrogallol red (PGR) is a novel, cost-effective optical probe for detecting hydrogen peroxide (H2O2). A new PGR-based assay offers a highly sensitive and stable method for measuring catalase (CAT) activity in biological samples.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Enzyme Assays
Background:
- Hydrogen peroxide (H2O2) is a reactive oxygen species implicated in various biological processes.
- Accurate measurement of catalase (CAT) activity is crucial for understanding oxidative stress and cellular defense mechanisms.
- Existing colorimetric assays for CAT activity can be limited by sensitivity, cost, or interference.
Purpose of the Study:
- To develop and optimize a novel, sensitive, and cost-effective optical probe for H2O2 detection.
- To establish a robust assay for quantifying catalase (CAT) activity using the developed probe.
- To compare the performance of the new assay against established methods.
Main Methods:
- Pyrogallol red (PGR) was employed as an optical probe for H2O2 detection via horseradish peroxidase (HRP)-catalyzed oxidation.
- Response surface methodology (RSM) was used to optimize PGR, HRP, and H2O2 concentrations for assay development.
- N-ethylmaleimide (NEM) was utilized to mitigate interference from thiol groups and preserve CAT activity.
- Assay parameters including incubation time and signal stability were evaluated.
- Assay validation involved linearity, sensitivity (LOD, LOQ), precision (RSD), and agreement with the Amplex Red assay using Bland-Altman and correlation analyses.
Main Results:
- The optimized PGR-based assay demonstrated high sensitivity with a linear range of 0-4 U/mL (R²=0.993).
- The assay achieved low limits of detection (LOD = 0.005 U/mL) and quantitation (LOQ = 0.01 U/mL).
- Adequate intra-day (0.6-9.5% RSD) and inter-day (2.4-8.9% RSD) precision were observed.
- The PGR-CAT assay showed good agreement with the Amplex Red assay and exhibited superior sensitivity.
- The cost of PGR is significantly lower (approx. 1/1000) compared to Amplex Red probes.
Conclusions:
- Pyrogallol red serves as a novel and effective optical probe for H2O2 detection.
- The developed PGR-based catalase (CAT) activity assay is highly sensitive, cost-effective, and reliable.
- This assay presents a valuable alternative to existing methods for studying CAT activity in biological samples.
- The assay's affordability and sensitivity suggest broad applicability in biochemical research.
