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Title: Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples
Authors: Grilo, Luís F
Martins, João D 
Cavallaro, Chiara H
Nathanielsz, Peter W 
Oliveira, Paulo J
Pereira, Susana P
Keywords: Antioxidant defense; Catalase; Hydrogen peroxide; Oxidative stress; Toxicity screening
Issue Date: Dec-2020
Volume: 69
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.
ISSN: 08872333
DOI: 10.1016/j.tiv.2020.104996
Rights: openAccess
Appears in Collections:I&D CNC - Artigos em Revistas Internacionais

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