title: Characterisation of S-nitrosohaemoglobin by mass spectrometry.
abstract: Recent studies have demonstrated the biological importance of the interaction of S-nitrosothiols, which can be considered as nitric oxide (NO) protein donors, especially haemoglobin, at the level of Cys residues. It was recently proposed that S-nitrosohaemoglobin is formed within red blood cells and serves as a regulatory function. In human haemoglobin the alpha-subunit contains one Cys residue and the beta-subunit contains two Cys residues, one of which (beta-Cys93) is highly reactive and conserved among species, although its function has remained unknown. Electrospray ionization mass spectrometry was used to monitor the results of exposure of haemolysates to S-nitrosocysteine under different conditions and thus addressed some aspects of NO-haemoglobin interaction. When an equimolar ratio of S-nitrosothiol was added to haemoglobin, only a single NO molecule was added. Peptide mapping by liquid chromatography-mass spectrometry located the nitrosyl group at the level of beta-Cys93 demonstrating that this was the preferred site of formation of S-nitrosohaemoglobin. The present data also suggest that electrospray mass spectrometry can allow quantification and characterisation of S-nitrosoproteins in blood.
authors.affiliation: International Mass Spectrometry Facilities Centre of CNR, Napoli, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Ferranti
authors.forename: A
authors.initials: A
authors.lastname: Malorni
authors.forename: G
authors.initials: G
authors.lastname: Mamone
authors.forename: N
authors.initials: N
authors.lastname: Sannolo
authors.forename: G
authors.initials: G
authors.lastname: Marino
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D009569:Nitric Oxide / Q000737:chemistry
chemical_list: D006454:Hemoglobins; C103574:S-nitrosohemoglobin; D009569:Nitric Oxide; D003545:Cysteine