Catalase activity assay

10 µg of mice liver was homogenized in 1.15% KCl using a Dounce homogenizer. The samples were centrifuged at 12,000 rpm for 15 minutes. The supernatant was extracted and diluted with 50 mM phosphate buffer pH 7.0. The colorimetric assay to measure catalase activity in liver samples was done by using Sigma-Aldrich Catalase Assay Kit (Cat. CAT100). This assay in the presence of hydrogen peroxide (H₂O₂) and horseradish peroxidase (HRP) uses a substituted phenol (3,5-dichloro-2-hydroxybenzenesulfonic acid) that combines with 4-aminoantipyrine to form a red quinoneimine dye. All solutions were prepared as instructed by the manufacturer. Briefly, this assay measured the H₂O₂ substrate that remains after the catalase action. In first step, catalase via its catalytic pathway converted H₂O₂ to water and oxygen, and the reaction was stopped using sodium azide. Next, a small volume of reaction mix is then analyzed for the amount of H₂O₂ remaining by a colorimetric method. The absorbance was measured using a spectrophotometer at 520 nm, and the actual concentration was measured using Beer’s Law: [H₂O₂] (mM) = A240/0.0436.