H₂O₂ Consumption and Production Assay for the Characterization of U. maydis Oxidoreductases

This protocol describes a validated method to quantify hydrogen peroxide (H₂O₂) generated by oxidases and to measure H₂O₂ consumption by other enzymes or chemical scavengers. This method is a peroxidase-coupled assay using Amplex® Red, which is both fluorogenic and chromogenic and allows to monitor H₂O₂ formation in kinetic or end-point modes. The assay can be conducted in various volumes, and we describe here in detail a 96-well plate format. Typically, depending on the number of samples, the so-called Amplex Red assay can be carried out within 1-3 h. This document covers reagent preparation, calibration, kinetic measurements, data analysis, critical parameters, troubleshooting, and expected results.