Immobilization of human myeloperoxidase on silica gel for inhibitors assessment.

Myeloperoxidase (MPO), a hemo-protein present in neutrophils and monocytes, significantly contributes to immune responses against microbial pathogens. Increased MPO activity, however, promotes the production of highly reactive oxidized species in extracellular fluids that can damage host tissues causing inflammatory events. To explore the potential of MPO as a target for inhibitor screening, this study aimed to develop and evaluate a mini-reactor system based on immobilized MPO. Several immobilization strategies including physical adsorption and covalent binding, were tested using silica gel as the support material. Among them, immobilization via cyanogen bromide (CNBr) activation was selected as the optimal method based on long-term stability (> 90 % after 20 runs) and a relative activity of 60 % after immobilization. Kinetic parameters for HO were determined to characterize the enzymatic behavior post-immobilization, including K and V (46 ± 4 µM and 463 ± 38 µM/min, respectively). The system was then employed to evaluate the inhibitory potency (IC₅₀) of three model compounds-salicylhydroxamic acid (SHA), paroxetine (PARO), and 4-aminobenzohydrazide (4-ABAH)-on HOCl production in the presence of 10 µM H₂O₂. 4-ABAH emerged as the most potent irreversible inhibitor (IC₅₀ = 0.30 µM), while SHA was the least effective (IC₅₀ = 27.10 µM). Those results demonstrated that immobilized-MPO preserved its activity and exhibited good stability. This mini-reactor provides a reliable platform for inhibitor studies, offering potential applications in screening therapeutic compounds to mitigate MPO-related inflammatory conditions.

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