Determination of the myeloperoxidase-hydrogen peroxide-halide system activity following the respiratory burst of human neutrophils.
A sensitive assay was developed for the determination of the activity of cell derived myeloperoxidase-hydrogen peroxide-halide system associated with the respiratory burst of human neutrophils. The methodological approach relies on the oxidation of 2-nitro-5-thiobenzoic acid (TNB) which could be continuously monitored by means of a chart recorder attached to a photometer. The oxidation of TNB by neutrophils stimulated with either phorbol myristate acetate or N-formyl-Met-Leu-Phe showed the following features: a) Superoxide dismutase failed to modify the rate of TNB oxidation; b) it was strongly inhibited by catalase but not by boiled catalase; c) azide and cyanide inhibited the reaction; d) it required a halide cofactor, namely chloride, and e) the onset of TNB oxidation was strongly potentiated by the calcium ionophore A23187. These results support the involvement of the myeloperoxidase system in the oxidation of TNB. Therefore, the procedure described here seems to be a suitable method for the above indicated purpose.
Lucas
M
M
Dpto. de Bioquímica, Facultad de Medicina, Hospital Universitario, Sevilla, Spain.
Solano
F
F
D001386:Azides / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D006219:Halogens / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009579:Nitrobenzoates / Q000378:metabolism*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D013481:Superoxides / Q000378:metabolism*
D001386:Azides; D003486:Cyanides; D003574:Cytochrome c Group; D006219:Halogens; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D013481:Superoxides; C011136:thionitrobenzoic acid; D003571:Cytochalasin B; D006861:Hydrogen Peroxide; D009195:Peroxidase