Nitric Oxide Interaction with Lactoferrin and its Production by Macrophage Cells Studied by EPR and Spin Trapping

Abstract

The production of nitrate (NO₃^-) and nitrite (NO₂^-) from macrophage-derived NO• was studied using EPR and spin trapping. The formation of NO₃^- was determined via EPR in reactions involving the iron-binding protein, lactoferrin. The formation of NO₂^- was determined via EPR/spin trapping in the reaction between NO₂^- and H₂O₂. Dissolved nitric oxide (NO•) was reacted with lactoferrin yielding an EPR spectrum (77°K) different from the normal EPR spectrum obtained for lactoferrin, suggesting that NO• interacts with the ferric ions bound to lactoferrin forming a ferric-nitrosyl type complex. The EPR spectrum (77°K) of this ferric-nitrosyl type complex was also observed in the supernatant fluid of macrophage cell suspensions following their stimulation with lipopolysaccharide (LPS). During LPS stimulation of macrophages, these cells generate NO• which in turn produces NO₃^- and NO₂^-. The ferric-nitrosyl type complex is formed in a reaction mixture containing apolactoferrin and bicarbonate following the reaction of Fe⁺² with NO₃^-, generated from macrophage-derived NO•, to produce Fe⁺³ and NO•. Furthermore, in an acidic medium, NO₂^- reacts with H₂O₂ forming peroxynitrous acid (HOONO) which rapidly decomposes into hydroxyl radicals (•OH) and the nitrogen dioxide (NO₂•) radical. In the supernatant fluid of LPS-stimulated macrophage suspensions, the production of •OH was verified by spin trapping using 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) as the spin trap and ethanol as the •OH scavenger. The EPR spectra corresponding to the DMPO-OH and the DMPO-hydroxyethyl adducts were identified. These results suggest that the peroxynitrous acid decomposes via the formation of •OH and NO₂• and that NO₂^- was formed from macrophage-derived NO•

Key Words:

• nitric oxide
• lactoferrin
• macrophage
• EPR
• spin trapping