An EPR study of the production of superoxide radicals by neutrophil NADPH oxidase.

Superoxide production by stimulated neutrophils and by preparations of NADPH oxidase has consistently been measured by an indirect method utilising the reduction of cytochrome c by superoxide radicals [4]. This reduction is inhibited by superoxide dismutase. EPR spectroscopy using the technique of spin trapping has been used to identify superoxide radicals formed by photo-irradiated spinach chloroplasts [5] and human neutrophils following stimulation [6]. This technique appears to be the least unambiguous method of detecting superoxide production by biological systems in their normal physiological state. Here, we provide the first direct demonstration of superoxide production by guinea-pig neutrophil NADPH oxidase. The results indicate that as seen by spin trapping, the superoxide radical is the only free radical produced by the enzyme in the presence of NADPH. 2. MATERIALS AND METHODS