ADHP

Kinase experiment:

ADHP, 4-ABAH, 2-ABAH, 4-BAH, 4-FBAH, 4-NBAH, 4-TFMBAH, 3-DMABAH, NaN3 and isoniazid are dissolved in DMSO and subsequently diluted into assay buffer. The final concentration of DMSO in the reaction is less than 0.5 % (v/v), which does not affect fluorescence of the oxidized ADHP product 7-hydroxyl-3H-phenoxazin-3-one (resorufin). Reactions of ADHP (20 μM) are incubated with MPO (2.8 nm) in assay buffer and initiated by the addition of 1/10th volume H2O2 from a serial dilution basin. To determine the effect that the simplest benzoic acid hydrazide inhibitor or its analog 4-TFMBAH has on the heme catalytic ability of MPO, MPO (1.2 μM) is incubated for 10 min with different concentrations of BAH inhibitor (0, 0.025, 0.25, 2.5, 12.5 and 25 mM) with ADHP (40 μM) and timing of the reaction is measured following addition of H2O2 (20 μM) ADHP. All reactions are measured in assay buffer at room temperature. Samples of 20 μL are added to non-reducing sample loading buffers, and then loaded without prior heating and resolved by 4-15% gradient SDS-polyacrylamide gel electrophoresis[1].

References:

[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.