Biological fate of sulphur mustard: in vitro alkylation of human haemoglobin by sulphur mustard.
1. Human blood was incubated in vitro with a 1:1 mixture of [35S,12C4]- and [13C4]-sulphur mustard. Alkylated globin, containing the 2-hydroxyethylthioethyl (HETE) moiety, was isolated from the blood incubate following lysis of the erythrocytes and acidification with HCl in isopropanol. 2. The alkylated globin was hydrolysed with Pronase E to give a digest containing alkylated amino acids and alkylated dipeptides. A number of these were partially purified by hplc and identified by gc-ms and lc-ms. 3. The alkylated globin was hydrolysed with trypsin to give a digest containing alkylated peptides. Ten of these were partially purified by hplc, tentatively identified by lc-electrospray mass spectrometry, and the sequences and sites of alkylation determined using lc-electrospray tandem mass spectrometry. 4. (2-Hydroxyethylthioethyl)glutathione was also shown to be present in the pronase and trypsin digests of alkylated globin. 5. N-terminal valine, on both the alpha and beta chains, and histidine residues were identified as the key sites of interaction for targeting as biological markers of sulphur mustard poisoning.
Black
R M
RM
Chemical and Biological Defence Sector, Defence Evaluation and Research Agency, Porton Down, Salisbury, UK.
Harrison
J M
JM
Read
R W
RW
D000478:Alkylation; D000595:Amino Acid Sequence; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005914:Globins / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009151:Mustard Gas / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D010949:Plasma / Q000378:metabolism; D011402:Pronase; D014357:Trypsin
D006454:Hemoglobins; D005914:Globins; D014357:Trypsin; D011402:Pronase; D009151:Mustard Gas