Kinetic method for assaying the halogenating activity of myeloperoxidase based on reaction of celestine blue B with taurine halogenamines

Abstract

Myeloperoxidase (MPO) is a challenging molecular target which, if put under control, may allow regulating the development of inflammatory reactions associated with oxidative/halogenative stress. In this paper, a new kinetic method for assaying the halogenating activity of MPO is described. The method is based on measuring the rate of iodide-catalyzed oxidation of celestine blue B (CB) by oxygen and taurine N-chloramine (bromamine). The latter is produced in a reaction of taurine with HOCl (HOBr). CB is not a substrate for the peroxidase activity of MPO and does not react with hydrogen peroxide and superoxide anion radical. Taurine N-chloramine (bromamine) reacts with CB in molar ratio of 1:2. Using the new method, we studied the dependence of MPO activity on concentration of substrates and inhibitors. The specificity of MPO inhibition by non-proteolyzed ceruloplasmin is characterized. The inhibition of taurine N-chloramine production by neutrophils and HL-60 cells in the presence of MPO-affecting substances is demonstrated. The new method allows determining the kinetic parameters of MPO halogenating activity and studying its inhibition by various substances, as well as screening for potential inhibitors of the enzyme.

Keywords::

• myeloperoxidase
• halogenating activity
• celestine blue B
• taurine
• HOCl
• ceruloplasmin

Acknowledgments

The authors are grateful to Professor V. N. Kokryakov (Institute of Experimental Medicine, St. Petersburg) for granted materials, valuable advice, and constructive discussions; to Professor A.A. Karyakin and Ph.D. A.V. Borisova (“RUSENS”) for granted H₂O₂-sensors. Estimation of rate constants was done with kind assistance of Dr. A.V. Bulatov (Department of Analytical Chemistry, Faculty of Chemistry, Saint-Petersburg State University). This study was supported by RFBR grants No. 13-04-01186, 14-04-00807, 14-04-90007, and 15-04-03620, and by the RAMS Program “Human Proteome”.

Declaration of interest

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