Impacts of vesicular environment on Nox2 activity measurements in vitro

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 1; p. 129767
• Main Authors: Serfaty, Xavier, Lefrançois, Pauline, Houée-Levin, Chantal, Arbault, Stéphane, Baciou, Laura, Bizouarn, Tania
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2021; Elsevier
• Subjects: arachidonic acid; Arachidonic Acid - metabolism; Biochemistry; Biochemistry, Molecular Biology; Cell-free assay; Cells, Cultured; Cytochrome c reduction; Cytochromes c - metabolism; detergents; electrodes; enzymatic reactions; Humans; Hydrogen Peroxide - metabolism; innate immunity; Life Sciences; NAD(P)H oxidase (H2O2-forming); NADP - metabolism; NADPH oxidase (NOX); NADPH Oxidase 2 - metabolism; neutrophils; Neutrophils - metabolism; oxidation; Oxidation-Reduction; Oximetry; Oxygen Consumption; permeability; Superoxide anion; AA; NADPH; PBS; Superoxide anion; DTT; Oximetry; Cytochrome c reduction; SOD; cyt c; cyt b558; dPBS; PMSF; Nox; Cell-free assay; DOC; MF; NADPH oxidase (NOX); DPI
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129767

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