Background:

ADHP 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-1.The kobs also increases 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[1].

[1]. Jiansheng Huang, et al. Ordered Cleavage of Myeloperoxidase Ester Bonds Releases Active site Heme Leading to Inactivation of Myeloperoxidase by Benzoic Acid Hydrazide Analogs. Arch Biochem Biophys. 2014 Apr 15; 548: 74-85.