Detection of the Brominating Activity of Myeloperoxidase Using Fluorescein

Abstract

A study was carried out on the spectralluminescent properties of fl uorescein after its reaction with various reactive oxygen and halogen species (\({O}_{2}^{\bullet-},\) H₂O₂, HOCl, HOBr, HOSCN, N-chloramine, taurine N-chloramine, and taurine N-bromamine) as well as in the myeloperoxidase (MPO)–H₂O₂–Cl^–/Br^–/SCN^– system. Reaction with only HOBr or with the MPO–H₂O₂–Br system turns fluorescein into a compound with an absorption maximum at 518 nm. The fluorescence maximum is recorded at 540 nm when excited at 520 nm, corresponding to eosin Y (brominated fluorescein). Conditions with phosphatebuffered saline (PBS) at pH 7.4 containing 137 mM NaCl, 5 mM fluorescein, 15–30 mM NaBr, and 25–50 mM H₂O₂ were found to be optimal for detecting HOBr in solution. A qualitative method for determining the brominating activity of MPO in vitro has been proposed. This method was used to study the effect of physiological and synthetic inhibitors as well as reactive oxygen and halogen species scavengers on the brominating activity of MPO. Our results indicate that fluorescein holds promise for use in a fluorescent method for detecting the brominating activity of mammalian hemecontaining peroxidases.