A Comprehensive Structural Analysis of Hemoglobin Adducts Formed after in Vitro Exposure of Erythrocytes to Butadiene Monoxide

• Published in: Chemical research in toxicology 2000-11, Vol.13 (11), p.1103-1113
• Main Authors: Moll, Thomas S, Harms, Amy C, Elfarra, Adnan A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; butadiene monoxide; Carcinogens - chemistry; Carcinogens - metabolism; Carcinogens - toxicity; Chromatography, Liquid; Dose-Response Relationship, Drug; Epoxy Compounds - blood; Epoxy Compounds - chemistry; Epoxy Compounds - toxicity; Erythrocytes - drug effects; Erythrocytes - metabolism; Globins - chemistry; Globins - metabolism; Hemoglobins - chemistry; Hemoglobins - metabolism; Hydrogen-Ion Concentration; Kinetics; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Molecular Sequence Data; Mutagens - chemistry; Mutagens - metabolism; Mutagens - toxicity; Peptide Mapping; protein adducts; Spectrometry, Mass, Electrospray Ionization
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx000151f

A widely used method for assessing occupational and environmental exposure to 1,3-butadiene involves the detection of hemoglobin adducts formed by the reactive metabolite butadiene monoxide (BMO). This assay employs the N-alkyl Edman method, which was developed to determine adducts formed at the amine group of the N-terminal valine of hemoglobin. Disadvantages of this procedure include its limitation to detecting only one adduct per globin chain, despite the presence of numerous other, and potentially more reactive, nucleophilic amino acids in hemoglobin. The method is also not suitable for determining whether the reaction of BMO occurs at the N-terminal valine of α- or β-globin. The primary goals of the current research are to determine the degree of modification of α- and β-globin chains by BMO and to localize the reactive residues to specific regions of the globin polypeptides. The reaction products after in vitro incubation of C57Bl/6 mouse erythrocytes with BMO were isolated by acid extraction of heme and microprecipitation of globin, followed by the determination of the number and location of adducts by mass spectrometry. The modification degree was monitored by electrospray mass spectrometry, which was used to measure the time- and concentration-dependent formation of BMO−hemoglobin adducts (≤10 adducts per globin). The results indicate that BMO reacts faster and to a higher degree with α-globin than with β-globin. Structural analysis was performed by peptide mapping of globin peptides after trypsin digestion using liquid chromatography/mass spectrometry. These experiments allowed the localization of BMO−hemoglobin adducts to specific regions within α- and β-globin, and also provided information about their relative reactivity. Interestingly, the initial site of adduct formation on α-globin is located near the N-terminal peptide, whereas the initial site on β-globin is located at the C-terminal region. Collectively, the results establish differences in the reactivities of α- and β-globin toward BMO, demonstrate the formation of multiple adducts at several α- and β-globin sites, and show that the N-terminal valine residues are not the first to be modified by BMO.