3,3’,5,5’-Tetramethylbenzidine (TMB) oxidation assay

hPx as indicated was added into 100 μL of TMB solution (TMB Liquid Substrate System, Sigma-Aldrich), which contains H₂O₂. Reaction mixture was incubated at RT for 30 min. TMB oxidation was recorded at absorbance 650 nm. In some experiments, LPS (Sigma-Aldrich Cat #L2880, from E. coli 055:B5) or GN bacteria were mixed with 400 nM/heme of recombinant PXDN at RT for 30 min. 20 μL of mixture was added into 100 μL TMB solution. After 30 min, absorbance at 650 nm was recorded.