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 alpha chain and the beta 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 beta chain, and Tyr-42, Tyr-24, and His-20 of the alpha 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).
Highlights
From the ‡Laboratory of Structural Biology, ¶Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
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-dimethyl1-pyrroline N-oxide (DMPO)
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)
Summary
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-dimethyl1-pyrroline N-oxide (DMPO). When hemoglobin was reacted with hydrogen peroxide (H2O2) in the presence of DMPO, a DMPO nitrone adduct could be detected by immuno-spin trapping. In a previous report using the immuno-spin trapping approach [16], we demonstrated that hemoglobin reacted with H2O2 in the presence of DMPO and suggested that the tyrosine. To identify the precise amino acid residues trapped by the DMPO, we have utilized mass spectrometry-based sequencing This approach includes proteolysis of the hemoglobin-derived DMPO adducts by trypsin, followed by MS peptide mapping, and MS/MS analyses of the peptides. We have identified the specific location of the DMPO adducts on hemoglobin and report our results here
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