Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase.
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 hypochlorus acid (HOCl), 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.
Bozeman
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PM
Division of Cardiopulmonary Medicine, St. Jude Children's Research Hospital, Memphis, TN 38101.
Learn
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Thomas
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D000640:Amitrole / Q000494:pharmacology; D001560:Benzidines / Q000378:metabolism; D001965:Bromides / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D003622:Dapsone / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D047209:Eosinophil Peroxidase; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology; D007454:Iodides / Q000494:pharmacology; D007962:Leukocytes / Q000201:enzymology*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000097:blood*; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000097:blood*; D013861:Thiocyanates / Q000494:pharmacology
D001560:Benzidines; D001965:Bromides; D002712:Chlorides; D003902:Detergents; D007454:Iodides; D013861:Thiocyanates; C021758:3,3',5,5'-tetramethylbenzidine; D006997:Hypochlorous Acid; D003622:Dapsone; D047209:Eosinophil Peroxidase; D010544:Peroxidases; D009195:Peroxidase; D000640:Amitrole