Mixing artifacts from the bolus addition of nitric oxide to oxymyoglobin: implications for S-nitrosothiol formation

The addition of nitric oxide (NO) solution to oxygenated heme proteins has been used to measure NO concentration and as an experimental model to investigate the biochemical mechanism of NO metabolism. In this paper we demonstrate that bolus addition of NO to oxymyoglobin (oxyMb) results in the artifactual formation of nitrosating intermediates. When NO is added as a bolus, using fully aerated oxyMb solutions, the measured NO concentration is half as much as that when the oxyMb solution is partially degassed (0.86 ± 0.01 mM vs. 1.61 ± 0.02 mM, mean ± SD). Similar results are found when calibrating NO concentration using a nitronyl nitroxide-type NO scavenger. The apparent stoichiometry of NO to oxyMb increases when the solution oxygen concentration increases. A fraction of the added NO generates nitrite or, in the presence of glutathione (GSH), S-nitrosoglutathione (GSNO). When using an NO donor, which slowly releases NO, oxyMb oxidation shows no dependence on the presence of oxygen in solution, and no nitrosating intermediate is formed. Bolus NO addition causes a local high concentration of NO. Kinetic calculations under this condition using known rate constants indicate that both the NO/oxyMb reaction and the NO/O2 reaction can occur before it is possible fully to mix the solution. Our results suggest that the presence of the NO/O2 reaction is an artifact of bolus NO addition, and leads to the formation of nitrite, or GSNO in the presence of GSH.