Quantifying Catalase Activity in Cell Extracts

Cells were grown to an OD_500nm of 0.6 and cytosolic cell extracts were prepared as described above. Right before starting the assay, a working solution of 25 µg/ml lactoperoxidase and 0.5 M dicarboxidine dihydrochloride (both Sigma-Aldrich) was prepared. To determine the degradation of H₂O₂ by cytosolic protein extracts, 5 µg of protein extract were filled up with catalase assay buffer (Catalase Assay Kit, BioVision) to 200 µl. H₂O₂ was added to a final concentration of 2 mM and the extracts were incubated at 30 °C. To measure the H₂O₂ concentration remaining in the culture at certain time points, 25 µl of the cell extract was mixed with 500 µl working solution and absorbance at 450 nm was measured as described [25 (link)]. The experiments were done in triplicates.