Detection and identification of oxidants formed during ^•NO/O₂^•– reaction: A multi-well plate CW-EPR spectroscopy combined with HPLC analyses

Abstract

New techniques and probes are routinely emerging for detecting short-lived free radicals such as superoxide radical anion (O₂^•–), nitric oxide (^•NO), and transient oxidants derived from peroxynitrite (ONOO^–/ONOOH). Recently, we reported the profiles of oxidation products (2-hydroxyethidium, ethidium, and various dimeric products) of the fluorogenic probe hydroethidine (HE) in the ^•NO/O₂^•– system (Zielonka et al. 2012). In this study, we used HPLC analyses of HE oxidation products in combination with continuous wave electron paramagnetic resonance (CW-EPR) spin trapping with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) to define the identity of the oxidizing species formed in the ^•NO/O₂^•– system. EPR spin-trapping technique is still considered as the gold standard for characterization of free radicals and their intermediates. We monitored formation of BMPO-superoxide (BMPO-^•OOH) and BMPO-hydroxyl (BMPO-^•OH) radical adducts. Simultaneous analyses of results from EPR spin-trapping and HPLC measurements are helpful in the interpretation of the mechanism of formation of products of HE oxidation.

Keywords::

• BMPO
• EPR/ESR
• HPLC
• diethidium
• ethidium
• hydroethidine
• 2-hydroxyethidium
• nitric oxide
• peroxynitrite
• spin-trapping
• superoxide radical anion

Acknowledgment

Authors are grateful to Dr. Neil Hogg in the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, for his help in discussing ESR simulation analysis with use of SVD treatment.

Declaration of interest

The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.

This research was supported by NIH grant R01 HL063119 (to B.K.).