Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples

Luís F. Grilo, João D. Martins, Chiara H. Cavallaro, Peter W. Nathanielsz, Paulo J. Oliveira, Susana P. Pereira

Research output: Contribution to journalArticle

Abstract

Oxidative stress biomarkers are powerful endpoints in toxicological research. Cellular reductive/oxidative balance affects numerous signaling pathways involving H2O2. Detoxification and control of H2O2 levels results mainly from catalase activity. The aim of this work was to develop a precise, simple, cost-effective microassay to measure catalase activity in small tissue samples and cell extracts. We developed a protocol that quantifies H2O2 decomposition by intrinsic catalase in biological samples. Catalase activity was calculated based on rate of decomposition of H2O2, following absorbance at 240 nm. We developed a multi-well spectroscopic approach, reducing sample quantity requirements and allowing simultaneous assessment of large number of samples. The protocol is sensitive across a wide range of catalase activity (11.5–7575 U). The assay presents a 95% confidence interval with an intra-assay coefficient of variation of 3.7%, an inter-assay coefficient of variation of 6.2% and good correlation with a commercial kit. The assay was established and validated for different biological samples, including sheep hepatic tissue and human tumor and non-tumor cell lines. This high-throughput method is robust, sensitive, time-saving and cost-effective, generating highly reproducible results with precision and good correlation with a commercial kit reinforcing the method's validity for research and toxicological applications.

Original languageEnglish (US)
Article number104996
JournalToxicology in Vitro
Volume69
DOIs
StatePublished - Dec 2020
Externally publishedYes

Keywords

  • Antioxidant defense
  • Catalase
  • Hydrogen peroxide
  • Oxidative stress
  • Toxicity screening

ASJC Scopus subject areas

  • Toxicology

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