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.