Microplate assay for the quantification of catalase activity in biological samples

Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (H₂O₂). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by H₂O₂, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10-0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.