Impacts of vesicular environment on Nox2 activity measurements in vitro

The production of superoxide anions (O2•-) by the phagocyte NADPH oxidase complex has a crucial role in destruction pathogens innate immunity. Majority vitro studies on functioning indirectly follows enzymatic reaction reduction cytochrome c (cyt c). Only few reports mention alternative approach consisting measuring consumption rate. When using membrane vesicles human neutrophils, enzyme specific activity is generally found twice higher monitoring oxidation than cyt reduction. Up to now, literature provides only little explanations about such discrepancy despite critical importance quantify exact activity. We deciphered reasons this disparity studying key parameters, including. and arachidonic acid concentrations, conjunction with an ionophore, detergent Clark electrode measure O2 rates. Our results show that O2•- low permeability vesicle as well secondary reactions (O2•- H2O2 disproportionations) are strong clues shed light inconsistency. These altogether indicate method underestimates accurate Nox2