A kinetic assay for eosinophil peroxidase activity in eosinophils and eosinophil conditioned media.
The activity of eosinophil peroxidase (EPO) is commonly employed as a measure of eosinophil activation in biologic fluids. Determination of product formation by this enzyme by end-point measurement may be affected profoundly by substrate concentrations, reaction time and degradation of end-product and enzyme. To determine more accurately EPO concentrations in media conditioned by isolated, purified eosinophils, we have developed a kinetic, colorimetric assay to measure EPO concentration as a function of maximum velocity of reaction (Vmax). An automated method for determining Vmax in a 96-well microplate colorimetric assay was utilized over a wide range of substrate concentrations. Concentrations greater than or equal to 3 x 10(-8) g/ml could be determined reliably with this assay. Peroxidase activity was inhibited in a concentration-dependent manner by the addition of 3-amino-1,2,4-triazole (AMT). The EPO concentration in eosinophils determined by this kinetic method was approximately 1.1 x 10(-5) g/10(6) eosinophils. Eosinophil activation with 10(-6) M f-Met-Leu-Phe (fMLP) caused substantial EPO secretion (9.0 +/- 1.7% vs. 2.9 +/- 0.6% total EPO content for control, P = 0.05) and decrease in eosinophil EPO concentration (92.3 +/- 4.2% of control, P = 0.038). Secretion was enhanced by the addition of 5 micrograms/ml cytochalasin B to 10(-6) M fMLP (25.9 +/- 12.7% total EPO content, P = 0.043 vs. control); similar decreases were noted in eosinophil EPO concentration (71.7 +/- 16.1% of control, P = 0.043). These data demonstrate that determination of EPO secretion by measurement of Vmax is a reliable, accurate method for precise quantification of this enzyme in media containing purified eosinophils or eosinophil products in the absence of other forms of peroxidase activity.
White
S R
SR
Department of Medicine, University of Chicago, IL 60637.
Kulp
G V
GV
Spaethe
S M
SM
Van Alstyne
E
E
Leff
A R
AR
D000640:Amitrole / Q000494:pharmacology; D003470:Culture Media; D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009195:Peroxidase / Q000032:analysis; D010544:Peroxidases / Q000032:analysis* / Q000037:antagonists & inhibitors
D003470:Culture Media; D009240:N-Formylmethionine Leucyl-Phenylalanine; D047209:Eosinophil Peroxidase; D010544:Peroxidases; D009195:Peroxidase; D000640:Amitrole