A comprehensive structural analysis of hemoglobin adducts formed after in vitro exposure of erythrocytes to butadiene monoxide

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 alpha- or beta -globin. The primary goals of the current research are to determine the degree of modification of alpha- and beta -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 less than or equal to 10 adducts per globin). The results indicate that BMO reacts faster and to a higher degree with alpha -globin than with beta -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 alpha- and beta -globin, and also provided information about their relative reactivity. Interestingly, the initial site of adduct formation on alpha -globin is located near the N-terminal peptide, whereas the initial site on beta -globin is located at the C-terminal region. Collectively, the results establish differences in the reactivities of alpha -and beta -globin toward BMO, demonstrate the formation of multiple adducts at several alpha- and beta -globin sites, and show that the N-terminal valine residues are not the first to be modified by BMO.