Identification of Free Radicals on Hemoglobin from its Self-peroxidation Using Mass Spectrometry and Immuno-spin Trapping

Abstract

In an effort to understand the mechanism of radical formation on heme proteins, the formation of radicals on hemoglobin was initiated by reaction with hydrogen peroxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO nitrone adducts were analyzed by mass spectrometry (MS) and immuno-spin trapping. The spin-trapped protein adducts were then subjected to tryptic digestion and MS analyses. When hemoglobin was reacted with hydrogen peroxide (H 2 O 2 ) in the presence of DMPO, a DMPO nitrone adduct could be detected by immuno-spin trapping. To verify that DMPO adducts of the protein free radicals had been formed, the reaction mixtures were analyzed by flow injection electrospray ionization mass spectrometry (ESI/MS). The ESI mass spectrum of the hemoglobin/H 2 O 2 /DMPO sample shows one adduct each on both the ␣ chain and the ␤ chain of hemoglobin which corresponds in mass to the addition of one DMPO molecule. The nature of the radicals formed on hemoglobin was explored using proteolysis techniques followed by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) analyses. The following sites of DMPO addition were identified on hemoglobin: Cys-93 of the ␤ chain, and Tyr-42, Tyr-24, and His-20 of the ␣ chain. Because of the pi-pi interaction of Tyr-24 and His-20, the unpaired electron is apparently delocalized on both the tyrosine and histidine residue (pi-pi stacked pair radical).

Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry

“…Similar intermolecular electron transfer reactions between Tyr and Cys residues have recently been reported for the human myoglobin/H 2 O 2 [38] and human hemoglobin/peroxynitrite systems [39]. Regardless of the exact electron-transfer pathway, our observation that Cys-93 is the only residue on the β-chain of HuHb that forms DMPO trappable protein radicals confirms previous on-line LC MS/MS measurements performed in this laboratory [13] and is of significant interest. It reinforces the contention that cysteine residues in polypeptide chains can act as 'sinks' by providing reducing equivalents that limit heme-induced oxidative damage.…”

“…Methods using mass spectrometry in combination with spin-trapping methodology have been used by this laboratory [13][14][15]28,29] and by others [16][17][18][19][20] to detect and characterize peptideand protein-based radicals. A variant to this approach, presented here, combines chromatographic procedures and an immunology-based method to facilitate detection of peptide residues labeled by the nitrone spin-trap DMPO.…”

“…The nitrone adduct is the most stable end product and preserves the chemical bond formed between DMPO and the original free radical [26]. Previous investigations in this laboratory [13][14][15] have established that the protein-DMPO nitrone adducts are stable enough to permit peptide mass mapping and the identification of the spin-trapped species using standard on-line LC-MS/MS analysis. However, there is a lack of information regarding the long-term stability of those adducts.…”

“…This approach does not involve EPR, but uses the immunology-based methods of enzyme-linked immunosorbent assay (ELISA) and/or Western blotting to measure antigen-antibody binding. Immuno-spin trapping has greatly increased the sensitivity of radical detection and has been used by a number of groups to demonstrate protein radical formation in different proteins exposed to a variety of oxidants [7][8][9][10][11][12][13][14][15][16]. Nonetheless, the technology does suffer from some of the drawbacks/ limitations of spin trapping with EPR detection.…”

“…The use of tandem mass spectrometry allows the unequivocal identification of the actual amino acid(s) labeled by the spin trap and has the advantage that only small (picomole or less) amounts of material are required. A series of publications [13][14][15][16][17][18][19][20] has illustrated the utility and applicability of this technique in investigating radicals produced by the interaction of various oxidants with myoglobin, hemoglobin, NADH dehydrogenase and cytochrome c peroxidase. In the majority of cases reported [13][14][15][16][17], identification of the amino acid trapped by the spin-trapping agent was obtained on-line with tandem mass spectrometry (MS/MS).…”