Hemoglobin (Hb) adducts have long been used as dosimeters of exposure to xenobiotics and endogenously formed reactive metabolites. In this study, hemoglobin chains were separated from each other and their prosthetic heme groups and reacted with 4-oxo-2-nonenal, a major breakdown product of lipid hydroperoxides. The adducts were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF/MS) analysis of the intact proteins and by a combination of liquid chromatography/electrospray ionization/tandem MS (MS/MS) and MALDI-TOF/MS/MS analysis of the tryptic peptides. Covalent modifications were found on both hemoglobin chains. The location was determined to be on H20 of the α-hemoglobin chain and on H63 of the β-hemoglobin chain. Molecular modeling revealed that these two residues were two most solvent accessible H residues present in intact Hb. The proposed reaction mechanism is based on that described for the reaction of 4-hydroxy-2-nonenal with proteins. Initial nucleophilic Michael addition is followed by hydration of the resulting aldehyde, cyclization, and two sequential dehydration reactions to give stable furan derivatives. This results in the addition of 136 Da from 4-oxo-2-nonenal to give adducts corresponding to 17VGAH* AGEYGAEALER31 from α-hemoglobin and 62AH* GK65 from β-hemoglobin. These hemoglobin modifications can potentially serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage and may reflect an indirect measurement of the potential for DNA damage in vivo.
|Number of pages||11|
|Journal||Journal of Mass Spectrometry|
|Publication status||Published - 2005 Jun|
- Electrospray ionization
- Lipid peroxidation 4-oxo-2-nonenal
- Protein adducts
ASJC Scopus subject areas