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

Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples.
Toxicol In Vitro. 2020 Sep 05;:104996
Authors: Grilo LF, Martins JD, Cavallaro CH, Nathanielsz PW, Oliveira PJ, Pereira SP
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 is 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 is 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 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, t...

Source: Toxicology in Vitro - September 4, 2020 Category: Toxicology Authors: Grilo LF, Martins JD, Cavallaro CH, Nathanielsz PW, Oliveira PJ, Pereira SP Tags: Toxicol In Vitro Source Type: research