Direct Observation of Trapping and Release of Nitric Oxide by Glutathione and Cysteine with Electron Paramagnetic Resonance Spectroscopy

AbstractWhile the biosynthesis of nitric oxide (NO) is well established, one of the key issues that remains to be solved is whether NO participates in the biological responses right after generation through biosynthesis or there is a “secret passage” via which NO itself is trapped, transported, and released to exert its functions. It has been shown that NO reacts with thiol-containing biomolecules (RSH), like cysteine (Cys), glutathione (GSH), etc., to form S-nitrosothiols (RSNOs), which then release nitrogen compounds, including NO. The direct observation of trapping of NO and its release by RSNO has not been well documented, as most of the detection techniques measure the content of NO as well as nitrite and nitrate. Here we use spin-trapping electron paramagnetic resonance (EPR) technique to measure NO content directly in the reaction time course of samples of GSH and Cys (∼ mM) mixed with NO (∼μM) in the presence of metal ion chelator, which pertains to physiological conditions. We demonstrate that NO is readily trapped by these thiols in less than 10min and ∼70–90% is released afterward. These data imply that ∼10–30% of the reaction product of NO does not exist in the free radical form. The NO release versus time curves are slightly pH dependent in the presence of metal ion chelator. Because GSH and Cys exist in high molar concentrations in blood and in mammalian cells, the trapping and release passage of NO by these thiols may provide a mechanism for temporal and spatial sequestration of NO to overcome its concentration gradient-dependent diffusion, so as to exert its multiple biological effects by reacting with various targets through regeneration