Development of fluorescent probe for specific determination of halogenating activity of enzymes myeloperoxidase and eosinophil peroxidase

Author(s)

Luiza de Carvalho Bertozo

• Document type: Master's Dissertation
• Press: Bauru. 2017-03-16.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências. Bauru
• Defense date: 2017-02-23
• Advisor: Valdecir Farias Ximenes

Abstract

The enzyme myeloperoxidase (MPO) is able to catalyze the oxidation of chloride (Cl-) and bromide (Br-) ions from the reduction of hydrogen peroxide. The formed oxidizing agents, hypochlorous acid (HOCl) and hypobromous acid (HOBr) are highly reactive and involved in deleterious inflammatory processes that characterizes many diseases. The determination of the specific halogenating activity of MPO is not trivial, because due to high redox potential of the active form of compound I (MPO-I, 1.16 V), MPO is also capable to catalyze the oxidation of various organic compounds by the peroxidase mechanism. Thus, there are few methods used to date to measure only the halogenating activity. In this work, we developed a methodology for determining the halogenating activity of MPO using the fluorescent probe dansylglycine (DG), which proved insensitive to peroxidase action, but susceptible to electrophilic attack of HOCl and HOBr. The fast kinetics studies show that the reaction rate between DG and HOCl increases with the addition of Br-. It was possible to verify a linear decrease in fluorescence intensity of DG when increasing concentrations of HOCl were added to the reaction medium together with a fixed concentration of Br-. The same results were obtained using high performance lquid chromatography (HPLC) and, when coupled to mass spectrometry, it was possible to identify the product of the reaction, the compound bromo-dansylglycine. The abstraction of Cl- and Br- from the buffer blocked totally the fluorescence decay of dansylglycine. Eosinophil peroxidase is also capable of catalyzing the oxidation of halides, mainly producing HOBr. Thus, when applying the DG methodology in the determination of the halogenating activity of EPO, the production of HOCl / HOBr was also verified. By applying the DG methodology in studies with MPO and EPO inhibitors, it was possible to distinguish the mechanism of a reversible inhibitor from an irreversible one. In conclusion, this method based on the DG fluorescence bleaching has a great potential as a direct and selective analytical technique and for detection of MPO and EPO halogenating activity. (AU)

• FAPESP's process: 15/08267-1 - Fluorescent probe development for specific determination of halogenating activity of enzymes myeloperoxidase and eosinophil peroxidase
• Grantee: Luiza de Carvalho Bertozo
• Support Opportunities: Scholarships in Brazil - Master