I. V. Gorudkoa,1, O. S. Tcherkalinab, A. V. Sokolovb, M. O. Pulinab, E. T. Zakharovab,
V. B. Vasilyevb, S. N. Cherenkevicha, and O. M. Panasenkoc
a Department of Biophysics, Belarusian State University, pr. Nezavisimosti 4, Minsk, 220030 Belarus
b Institute for Experimental Medicine, Russian Academy of Medical Sciences,
ul. Akademika Pavlova 12, St. Petersburg, 197376 Russia
c Research Institute of Physico-Chemical Medicine, ul. Malaya Pirogovskaya 1a, Moscow, 119992 Russia
Received March 2, 2009; in final form, March 20, 2009
Abstract—A novel method for spectrophotometrical measurement of myeloperoxidase (MPO) activity in
plasma with o-dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, includ-
ing the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in
plasma. Specific MPO inhibitors, salicylhydroxamic acid or 4-aminobenzoic acid hydrazide, are added to mea-
sure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract
their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a
new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised
in rats and rabbits. The sensitivity of this ELISA is high: 0.2–250 ng/ml. A direct and significant (P < 0.0001)
correlation was observed between the MPO activities measured spectrophotometrically and the MPO level
determined by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measure-
ment in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mech-
anisms by which MPO is regulated under physiological conditions and against the background of various
inflammatory diseases.
Key words: myeloperoxidase, plasma peroxidase activity, blood plasma, o-dianisidine, hemoglobin, enzyme-
linked immunosorbent assay.
DOI: 10.1134/S1068162009050057
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