NapH1 Monochlorodimedone (MCD) Assay.

The MCD assay was modified from the procedure described previously.¹⁶ Briefly, the reaction mixture contained 50 μM MCD and 1 μM enzyme in a reaction buffer containing 50 mM MES (pH 6.0), 10 μM vanadate, and 200 mM KCl or KBr. The reaction was initiated by supplying H₂O₂ to a final concentration of 1 mM. The decrease in absorbance at 290 nm was monitored with a Cary spectrophotometer (Varian/Agilent) while keeping the temperature at 30 °C. Data were analyzed with the Cary WinUV Kinetics program (Varian/Agilent).