The fluorescein-derived dye aminophenyl fluorescein is a suitable tool to detect hypobromous acid (HOBr)-producing activity in eosinophils.
J Biol Chem
; 287(33): 27913-23, 2012 Aug 10.
Article
em En
| MEDLINE
| ID: mdl-22718769
ABSTRACT
The specific detection of peroxidase activity in human granulocytes is essential to elucidate their role in innate immune responses, immune regulation, and inflammatory diseases. The halogenating activity of myeloperoxidase in neutrophils can be determined by the novel fluorescent probe aminophenyl fluorescein (APF). Thereby non-fluorescent APF is oxidized by HOCl to form fluorescein. We successfully verified that APF equally detects the hypobromous acid (HOBr)-producing activity of eosinophil granulocytes. This was revealed by three different approaches. First, we investigated the conversion of non-fluorescent APF into fluorescein by HOCl and HOBr by means of fluorescence and mass spectrometry approaches. Thereby comparable chemical mechanisms were observed for both acids. Furthermore in vitro kinetic studies were used to detect the halogenating activity of myeloperoxidase and eosinophil peroxidase by using APF. Here the dye well reflected the different substrate specificities of myeloperoxidase and eosinophil peroxidase regarding chloride and bromide. Finally, peroxidase activities were successfully detected in phorbol ester-stimulated neutrophils and eosinophils using flow cytometry. Thereby inhibitory studies confirmed the peroxidase-dependent oxidation of APF. To sum up, APF is a promising tool for further evaluation of the halogenating activity of peroxidases in both neutrophils and eosinophils.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bromatos
/
Peroxidase
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Peroxidase de Eosinófilo
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Eosinófilos
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Fluoresceínas
/
Compostos de Anilina
Limite:
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Alemanha