ALKYLATION OF ESCHERICHIA-COLI THIOREDOXIN BY S-(2-CHLOROETHYL) GLUTATHIONE AND IDENTIFICATION OF THE ADDUCT ON THE ACTIVE-SITE CYSTEINE-32BY MASS-SPECTROMETRY

Alkylation of reduced Escherichia coli thioredoxin by the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG) at physiologic pH resulted in at least three different alkylation products. These adduc ts were separated by reverse phase chromatography, digested with tryps in, and peptide-mapped. The peptide containing the active site cystein es was collected and sequenced by tandem mass spectrometry. Results in dicate that the site of alkylation was at Cys-32 exclusively with no a lkylation at Cys-35. Raising the pH above the pK(a) of Cys-35 to ioniz e the thiol before reacting with the episulfonium ion of CEG did not l ead to alkylation at Cys-35, suggesting that a steric factor prevents the alkylating moiety of CEG from accessing this cysteine. A tryptic d igest of a minor bis-adduct yielded an alkylated peptide which contain ed tyrosine, an amino acid known to be alkylated at its hydroxyl group by CEG. Sequencing by tandem mass spectrometry, however, was unsucces sful due to fragmentation of the alkylating moiety from the peptide. R esults of this study confirm that the episulfonium ion of CEG can addu ct thioredoxin at the active site and may have important toxicologic s ignificance regarding the mechanism of 1,2-dichloroethane toxicity.