Intracellular spin-trapping of oxygen-centered radicals generated by human neutrophils.
Phagocytosing neutrophils secrete superoxide into a vacuole generally inaccessible for direct study. However, the spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enters the cytoplasm of several cell types where it can report free radical species including superoxide and hydroxyl radical. In the present study we employed a variety of experimental conditions to eliminate extracellular ESR signals and/or free radicals generated by stimulated neutrophils so that DMPO adducts reported events inside the cell. We identified a concentration of poly(ethylene glycol)-modified superoxide dismutase that permitted measurement of intracellular superoxide as determined by several criteria. It seems likely that poly(ethylene glycol)-modified superoxide dismutase is too large to enter the neutrophil phagosome. Under these conditions no hydroxyl radical was detected, as would be predicted from earlier studies with spin-trapping. Use of poly(ethylene glycol)-modified superoxide dismutase should allow on-line measurement of phagosomal events, thereby improving our understanding of microbicidal and inflammatory processes.
Pou
S
S
Department of Medicine, University of North Carolina, Chapel Hill 27599-7030.
Rosen
G M
GM
Britigan
B E
BE
Cohen
M S
MS
D003497:Cyclic N-Oxides; D003574:Cytochrome c Group / Q000097:blood; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000378:metabolism*; D010070:Oxalates; D019815:Oxalic Acid; D010100:Oxygen / Q000097:blood*; D011092:Polyethylene Glycols / Q000097:blood; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000097:blood
D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D005609:Free Radicals; D010070:Oxalates; D013113:Spin Labels; D011092:Polyethylene Glycols; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D019815:Oxalic Acid; D013482:Superoxide Dismutase; D010100:Oxygen