Registration of the Myeloperoxidase Brominative Activity Using Fluorescein

The change of spectral-luminescent properties of fluorescein after its interaction with various reactive oxygen and halogen species (О₂^•–, H₂O₂, HOCl, HOBr, HOSCN, taurine N-chloramine and taurine  N-bromamine), as well as in the myeloperoxidase (MPO)-H₂O₂-Cl^–/Br^–/SCN^– system has been studied. It has been found that after interaction only with HOBr as well as in the MPO-H₂O₂-Br^– system fluorescein turns into a compound having an absorption maximum at 518 nm. The fluorescence maximum of this compound is recorded at wavelength of 540 nm when excited at wavelength of 520 nm, that corresponds to the characteristics of brominated fluorescein — eosin Y. Optimal conditions (phosphate-buffered saline (pH 7.4) containing 137 mM NaCl, 5 μM fluorescein, 15—30 mM NaBr, and 25—50 μM H₂O₂) for registration HOBr in solution have been selected; a qualitative method for determining MPO brominative activity in vitro has been proposed. Using the developed method, the effect of physiological and synthetic inhibitors, as well as reactive oxygen and halogen species scavengers, on the MPO brominating activity has been studied. Based on the data obtained, it can be concluded that, fluorescein is a promising compound for the development on its basis of a fluorescent method for detecting mammalian heme-containing peroxidases brominating activity.

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