4 aminobenzoic acid hydrazide

PIC1 (IALILEPICCQERAA-dPEG24) was manufactured by PolyPeptide Group (San Diego, CA) to ≥ 95% purity verified by HPLC and mass spectrometry analysis. Lyophilized PIC1 was solubilized in normal saline with 0.01 M Na₂HPO₄ buffer to 15 mM. Pure MPO (MPO) was purchased from Lee Biosolutions (Maryland Heights, MO). TMB as a solution containing H₂O₂ was purchased from Thermo Fisher Scientific (Waltham, MA). 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) solution was purchased from Thermo Fisher Scientific. O-dianisidine and 4-aminobenzoic acid hydrazide (ABAH) were purchased from Sigma-Aldrich (St. Louis, MO).

WT mice were intraperitoneally injected with the irreversible MPO inhibitor 4-Aminobenzoic acid hydrazide (4-ABAH, Sigma-Aldrich, St. Louis, MO, USA; 20 mg/kg bodyweight) dissolved in 10% DMSO (14 mg/ml) or vehicle 2 days before DOX/NaCl injection and every other day thereafter until organ dissection at day 14 post treatment. Such administration scheme has been previously reported to sufficiently inhibit MPO [54 (link)].

MPO from human neutrophils was obtained from Athens Research and Technology (Athens, GA, According to the Certificate of Origin, the material used has been obtained from an FDA licensed collection center. Further the donors has given by signature full informed consent for the material to be used in commercial scientific research). Catalase form Corynebacterium, hydrogen peroxide, sodium hypochlorite, 4-aminobenzoic acid hydrazide (ABAH), NaN₃, umbelliferone (COH), fluorescein, N,N,N’,N’-tetramethylbenzidine, NaCl, taurine, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich and were of the purest grade available. Fluorescent probes were synthetized according to the procedures described in the literature. The synthesis of fluorescein pinacol boron ester (Fl-B) was performed based on the procedures published by Dickinson et al. and Rios et al.^{58 (link),59 (link)}. The obtained boronate was then hydrolyzed to fluorescein boronic acid (FLBA). The synthesis of APF was performed based on the procedure published by Setsukinai and cooworkers^{39 (link)}. The APC probe was synthesized according to the procedure published by Kavani and cooworkers^{60 (link)}. The detailed description of synthetic procedures can be found in the Supplementary Information. The CBA synthesis is described elsewhere^{37 (link),61 (link)}.