The effects of reactive oxygen species on cells have attracted much attention in relation to redox regulation and oxidative stress-related diseases. Superoxide (O2-) is the reactive oxygen species primarily formed in biological systems. However, no convenient O2 --generating device has been available for use in cell or tissue culture. The neutrophil NADPH oxidase, a professional enzyme for killing bacteria, has a high ability to produce O2-. However, the cell-free activation process requires several protein factors and an anionic amphiphile, and moreover, the activation is transient. To utilize the enzyme as an O2- generator, we improved the cell-free activation method by remodeling regulatory components, optimizing lipid composition, and modifying the mixing conditions. We established a new method to produce an active enzyme that is stable, efficient, and preservable. As an application, we examined the effect of the device on cultured HEK293 cells and observed that it caused cell death. This system has several advantages over the xanthine oxidase system often used. The new device will be useful for studies of oxidative stress and related diseases.
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