Detection of the halogenating activity of heme peroxidases in leukocytes by aminophenyl fluorescein

Abstract

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.

Keywords::

• myeloperoxidase
• hypochlorous acid
• neutrophils
• aminophenyl fluorescein
• eosinophil peroxidase
• hypobromous acid
• eosinophils

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

The authors gratefully acknowledge the financial support provided by the Sächsische Aufbaubank from a funding of the European Regional Development Fund (ERDF) (SAB Project Nr: 100116526) and the German Federal Ministry of Education and Research (BMBF 1315883).