ADHP (10-Acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic peroxidase substrate (λex=530 nm, λem=590 nm).

To obtain the parameters Km and kcat for Compound I, two independent methods are used. Initially, the oxidation of ADHP using the injector functionality built-in to the fluorescence plate reader is studied. The auto-injector dispenses the H2O2 to initiate the reaction, as a means of generating a set of progress curves. Analysis for MPO-mediated oxidation of ADHP gives a Km of 31±4 μM and the kcat of 186± 6/s. The kobs also increase over the experimental range of ADHP concentrations from 1 to 80 μM and for the converse experiment holding substrate constant over 3 to 45 nM MPO. The apparent second-order rate constant obtain from the slope of kobs against ADHP concentration Kappon is 2.1±0.2 mM/s.