Researchers in this study developed a rapid high throughput-compatible assay for determining generation of O2⨪ and H2O2 using two probes: coumarin boronic acid and hydropropidine probes. During the study, researchers found they were able to identify Nox enzyme inhibitors with reliability and accuracy using a medium-throughput plate reader-based oximetry and electron paramagnetic resonance (EPR) spin trapping.
A Bruker X-band EPR spectrometer was used in this experiment to help identify short-lived free radicals via spin trapping. This spectrometer enabled a better monitoring of reaction oxygen species (ROS) activity in the studied samples. Bruker EPR spectrometers include:
- EMXnano, a benchtop instrument with best in class sensitivity and stability, microwave and digital technologies and an integrated reference standard, nitrogen variable temperature unit.
- EMXmicro, a compact spectrometer with multi-frequency capabilities and a helium variable temperature unit
- EMXplus which provides multi-frequency and multi-resonance options/capabilities and helium variable temperature unit
Additionally, this method, among others used in this study, helped in understanding the mechanisms of action of the discovered selective inhibitors of Nox isoenzymes. Researchers concluded that the rigorous and swift identification of O2⨪ and H2O2 will enable a deeper comprehension of the biology behind H2O2 and O2⨪-producing enzymes, potentially assisting in the discovery of selective Nox isoform inhibitors and the advancement of care for patients with inflammatory- and fibrosis-related diseases.