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-alk yl Edman method, which was developed to determine adducts formed at the ami ne group of the N-terminal valine of hemoglobin. Disadvantages of this proc edure include its limitation to detecting only one adduct per globin chain, despite the presence of numerous other, and potentially more reactive, nuc leophilic amino acids in hemoglobin. The method is also not suitable for de termining whether the reaction of BMO occurs at the N-terminal valine of al pha- or beta -globin. The primary goals of the current research are to dete rmine the degree of modification of alpha- and beta -globin chains by BMO a nd to localize the reactive residues to specific regions of the globin poly peptides. The reaction products after in vitro incubation of C57Bl/6 mouse erythrocytes with BMO were isolated by acid extraction of heme and micropre cipitation of globin, followed by the determination of the number and locat ion 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 eq ual to 10 adducts per globin). The results indicate that BMO reacts faster and to a higher degree with alpha -globin than with beta -globin. Structura l analysis was performed by peptide mapping of globin peptides after trypsi n digestion using liquid chromatography/mass spectrometry. These experiment s allowed the localization of BMO-hemoglobin adducts to specific regions wi thin alpha- and beta -globin, and also provided information about their rel ative reactivity. Interestingly, the initial site of adduct formation on al pha -globin is located near the N-terminal peptide, whereas the initial sit e on beta -globin is located at the C-terminal region. Collectively, the re sults 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 n ot the first to be modified by BMO.