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 ccrsubunit contains one Cys residue and the ß-subunit contains two Cys residues, one of which (ß-Cys 93 ) 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 Snitrosocysteine 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 ß-Cys 93 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.

Hemoglobin βCys93 is essential for cardiovascular function and integrated response to hypoxia

“…The human β-subunit also contains a second Cys, βCys112, which can be S-nitrosylated in vitro (25). However, it has been established by previous mutagenic (26), mass spectrometric (27,28), and X-ray crystallographic analyses (29) that βCys93 is the predominant site of human β-subunit S-nitrosylation, consistent with the oxygenregulated disposition of NO within Hb in vivo (4,30,31). Additionally, the export of βCys93-derived NO bioactivity is based on an SNO cascade that involves the transfer of NO groups to RBC membrane proteins and to external sites that include smallmolecular-weight thiols (10,(14)(15)(16)(17).…”

The role of β93 Cys in the inhibition of Hb S fiber formation

“…To further examine the number of sites of NEM modification per Hb chain, ESI-MS was used, since it can sensitively differentiate mass changes as a result of NEM modification [14]. In both NEM-H Hb and NEM-L Hb samples, a peak was observed at 15,993 Da, corresponding to the mass of a β chain with one NEM molecule.…”

“…A variety of different methods, including FT-IR spectroscopy, ESI-MS and tandem MS, indicate NEM modification occurs at both β93 and α104 Cys residues and that the α104 residue is moderately reactive under relatively standard conditions [37]. Previously, modifications at Hb Cys residues other than β93 were only observed at high concentrations of modifying reagent [14,15,31]. Benesch and co-workers [31] observed that titration with an 100-fold molar excess of PMB results in the observation of 7.5 reactive -SH groups per mole of Hb, while lower PMB concentrations yield a value of 2.4 reactive -SH groups per mole of Hb.…”

“…Benesch and co-workers [31] observed that titration with an 100-fold molar excess of PMB results in the observation of 7.5 reactive -SH groups per mole of Hb, while lower PMB concentrations yield a value of 2.4 reactive -SH groups per mole of Hb. More recently, Ferranti and coworkers [14] demonstrated by ESI-MS that a 100-fold excess of NO relative to Hb leads to S-nitrosothiol formation at the α104 and β112 Cys residues. An important observation from the current work is that under conditions previously thought to only lead to β93 modification [37], modification of the α104 Cys residue also occurs (Fig.…”

“…Other investigators have determined that limited exposure of deoxy Hb to NO leads to nitrosylation of β-subunit hemes and subsequent oxygenation leads to the formation of SNO-Hb [12]. Formation of SNO-Hb primarily occurs when NO binds to the β93 Cys residues [13][14][15] and the S-nitroso form of hemoglobin has been observed to exhibit increased O 2 affinity [16]. A recent model for NO action, proposed by Stamler and co-workers [17,18] suggests that Hb quaternary state, and thus O 2 affinity, is mediated by the allosteric transfer of NO from the heme irons to the β93 Cys residues.…”