Objective — the study of hypochlorous acid (HOCl) and its derivatives production, which catalyzed by human neutrophil myeloperoxidase, using “turn-on” fluorescent sensor — celestine blue B.
Materials and methods. Neutrophils were isolated from the venous blood of healthy donors. Phorbol 12-myristate 13-acetate, N-formyl-methionyl-leucyl-phenylalanine, plant lectins, HOCl-modified proteins were used as agonists. N-acetylcysteine, 4-aminobenzoic acid hydrazide, isoniazid and ceruloplasmin were used as regulators of neutrophil myeloperoxidase activity and/or HOCl scavengers.
Results. Using a wide range of agonists and inhibitors, it has been shown that celestine blue B is oxidized in vitro by HOCl and its derivatives as a result of neutrophil myeloperoxidase activity. The oxidation of celestine blue B by HOCl-modified human serum albumin (HSA-Cl) and inhibition of this process by monoclonal antibody against HSA-Cl (IgM class) was also found.
Conclusion. Based on the developed method using celestine blue B, it is possible to conduct a sensitive analysis for the presence of HOCl-modified proteins (chloramines, etc.), to investigate the effect of various agonists and drugs on myeloperoxidase activity and exocytosis from the neutrophil granules.
The development of adequate methods for monitoring the functional state of neutrophils is an urgent task due to their important role in the development of the inflammatory response. In this study, the functional activity of neutrophils has been assessed using a complex approach, including the registration of generation of ROS by neutrophils as a result of the functioning of the NADPH oxidase complex; degranulation of azurophilic granules; as well as production of AFG, formed in reactions catalyzed by the enzyme azurophilic granules of neutrophils – MPO in the presence of substrates (Н2О2 and halide ions).
Reactive oxygen species (ROS) have a crucial role in human physiological and pathophysiological processes. Prolonged exposure to high ROS concentrations may lead to cardiovascular, neurodegenerative, etc. diseases. In this study, gallocyanine has been proposed to register the ROS production. The gallocyanine spectral properties changes under ROS (•О2ˉ, H2O2) and reactive halogen (HOCl) species are analyzed. It is shown that the dye is oxidized in solution, both under the action of ROS and reactive halogen species. Based on the data obtained, it is possible to suggest that superoxide anion radicals make a major contribution to chemical conversion of the dye in suspensions of activated neutrophils. It is that gallocyanine can be used to assess the functional activity of neutrophils, namely, the NADPH-oxidase, as well as to design and test novel therapeutic agents for diseases associated with developing oxidative stress.
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