Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase

doi:10.1016/0022-1759(90)90020-V

Journal of Immunological Methods

Volume 126, Issue 1, 24 January 1990, Pages 125-133

https://doi.org/10.1016/0022-1759(90)90020-V Get rights and content

Abstract

Conditions were optimized for measuring the activity of myeloperoxidase (MPO) and the eosinophil peroxidase (EPO) with tetramethylbenzidine (TMB) as the substrate. Detergents caused a small increase in the measured activity of the purified enzymes and were required when isolated neutrophils or eosinophils were assayed. Sharp concentration optima were observed with both ionic and non-ionic detergents. Activity was also influenced by halide ions. Bromide or iodide caused up to a 7-fold increase in EPO activity and a 1.5-fold increase in MPO activity. The effect of bromide is notable because the bromide-containing detergent CETAB is often used to extract the enzymes for assay and purification. Stimulation by bromide or iodide was consistent with peroxidase-catalyzed oxidation of the halides to hypohalous acids (HOBr and HOI), which oxidized TMB. MPO catalyzes the oxidation of chloride to hypochlorous acid (HOC1), which also oxidized TMB, but chloride up to 20 mM had little effect on the assay. Both MPO and EPO catalyze thiocyanate oxidation, but the product (HOSCN) was a poor oxidant for TMB, and thiocyanate inhibited the measured activities. Stimulation by bromide or iodide could be used to facilitate detection of EPO and to distinguish between MPO and EPO. Activities could also be distinguished based on the greater sensitivity of EPO to inhibition by thiocyanate, azide, aminotriazole, and dapsone. Methods reported here may prove useful for measuring leukocyte influx into inflamed tissues, detecting MPO or EPO deficiencies, and measuring enzyme synthesis and secretion.

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Citation Excerpt :
Fluorescence intensity was measured at Ex 485/Em 529 nm (Socci et al., 1999) and stated as fold changes in ROS levels. 3,3′,5,5′-Tetramethylbenzidine substrate (Bozeman et al., 1990; Menegazzi et al., 1992) was used for measuring Myeloperoxidase (MPO) and Eosinophil peroxidase (EPO) activity as described (Suzuki et al., 1983), and the activity was expressed as U/mg weight of lung tissue or U/ml BAL fluid. Envision microplate reader (PerkinElmer, USA) was used to measure the absorbance and fluorescence intensity of biochemical parameters.
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Here, we validated the anti-inflammatory and anti-asthmatic properties of traditional metallic medicine MS in asthmatic mice model and in LPS stimulated human monocytic THP-1 cells, by examining the relevant cellular, biochemical and molecular intermediates.
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^☆: This work was supported by an American Lung Association Training Fellowship, NIH Grants DE 04235, AI 16795, and CA 21765, and the American Lebanese Syrian Associated Charities.

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Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase☆

Abstract

Anal. Biochem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Invest. Dermatol.

J. Immunol. Methods

Biochim. Biophys. Acta

J. Biol. Chem.

Gastroenterology

Arch. Oral Biol.

Biochim. Biophys. Acta

FEBS Lett.

Blood

Anal. Biochem.

J. Biol. Chem.

Methods Enzymol.