A Reappraisal of the Utility of L-012 to Measure Superoxide from Biologically Relevant Sources

The detection of superoxide anion (O-2(center dot-)) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O-2(center dot-) since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O-2(center dot-). In O-2(center dot-) producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O-2(center dot-) generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO center dot) and NOS2(ONOO-). To exclude the effects of altered tyrosine phosphorylation, O-2(center dot-) was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O-2(center dot-) in biological systems.