Dansylglycine, a fluorescent probe for specific determination of halogenating activity of myeloperoxidase and eosinophil peroxidase

Author(s)

Bertozo, Luiza de Carvalho ; Zeraik, Maria Luiza ; Ximenes, Valdecir Farias

• Document type: Journal article
• Source: Analytical Biochemistry; v. 532, p. 29-37, SEP 1 2017.
• Web of Science Citations: 2

Abstract

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are enzymes present in neutrophil and eosinophil leukocytes, respectively. Here, we present the development of a sensitive and specific assay for determination of the halogenating enzymatic activity of MPO and EPO based on the electrophilic attack of HOCI and HOBr on aromatic ring of dansylglycine (DG). We found that the intrinsic fluorescence of DG was promptly depleted by the action of these acids. In the presence of the enzymes, the fluorescence bleaching was dependent of chloride (Cl-) and bromide (Br-), which makes the assay able to distinguish the halogenating from the peroxidase activity. A linear correlation was obtained between the hydrogen peroxide (H2O2) concentration and the fluorescent decay. Similarly, the enzyme activity was measured by keeping constant H2O2. The method was applied for studding MPO/EPO specific inhibitors as 5-fluortryptamine (reversible inhibitor) and 4-hydroxybenzhydrazide (irreversible inhibitor). Differently of the taurine chloramine/3,3',5,5'-tetramethylbenzidine assay, which is among the most used technique, the dansylglycine assay was able to differentiate these inhibitors based on their kinetic behavior. In conclusion, this assay can differentiate the peroxidase and halogenating activity of MPO and EPO. Moreover, the method is adequate for real-time measurement of the production of HOCI and HOBr. (C) 2017 Elsevier Inc. All rights reserved. (AU)

• FAPESP's process: 16/20549-5 - Development and application of fluorescent probes and probes based on circular dichroism for the interaction studies of ligands with protein, characterization of amyloid proteins and determination of enzymatic activity
• Grantee: Valdecir Farias Ximenes
• Support Opportunities: Regular Research Grants
• FAPESP's process: 15/21693-0 - Gallic acid and its esters as anti-Helicobacter pylori agents and scavenger of oxidants produced by neutrophils
• Grantee: Vanessa Gonçalves Wolf
• Support Opportunities: Scholarships in Brazil - Master