Abstract
The detection of superoxide anion (O2●−) 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 O2●− 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 O2●−. In O2●− 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 O2●− generating NOXes versus NOX4, which produces H2O2. Moreover, there was no signal from cells transfected with NOS3 (NO●) and NOS2(ONOO−). To exclude the effects of altered tyrosine phosphorylation, O2●− 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 O2●− in biological systems.
Original language | English (US) |
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Article number | 1689 |
Journal | Antioxidants |
Volume | 12 |
Issue number | 9 |
DOIs | |
State | Published - Sep 2023 |
Keywords
- L-012
- NADPH oxidase
- ROS
- superoxide
ASJC Scopus subject areas
- Food Science
- Physiology
- Biochemistry
- Molecular Biology
- Clinical Biochemistry
- Cell Biology