A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin.
Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.
Colepicolo
P
P
Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138.
Camarero
V C
VC
Nicolas
M T
MT
Bassot
J M
JM
Karnovsky
M L
ML
Hastings
J W
JW
D000818:Animals; D000845:Annelida / Q000032:analysis; D002374:Catalase / Q000494:pharmacology; D003574:Cytochrome c Group; D005260:Female; D005609:Free Radicals; D006168:Guinea Pigs; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000302:isolation & purification; D008165:Luminol; D008264:Macrophages / Q000378:metabolism*; D008353:Mannitol / Q000494:pharmacology; D051379:Mice; D009504:Neutrophils / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000032:analysis*
D003574:Cytochrome c Group; D005609:Free Radicals; D008164:Luminescent Proteins; D013481:Superoxides; D008353:Mannitol; D008165:Luminol; D002374:Catalase; D013482:Superoxide Dismutase