title: Alkylation of Escherichia coli thioredoxin by S-(2-chloroethyl)glutathione and identification of the adduct on the active site cysteine-32 by mass spectrometry.
abstract: 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 adducts were separated by reverse phase chromatography, digested with trypsin, and peptide-mapped. The peptide containing the active site cysteines was collected and sequenced by tandem mass spectrometry. Results indicate that the site of alkylation was at Cys-32 exclusively with no alkylation at Cys-35. Raising the pH above the pKa of Cys-35 to ionize the thiol before reacting with the episulfonium ion of CEG did not lead to alkylation at Cys-35, suggesting that a steric factor prevents the alkylating moiety of CEG from accessing this cysteine. A tryptic digest of a minor bis-adduct yielded an alkylated peptide which contained tyrosine, an amino acid known to be alkylated at its hydroxyl group by CEG. Sequencing by tandem mass spectrometry, however, was unsuccessful due to fragmentation of the alkylating moiety from the peptide. Results of this study confirm that the episulfonium ion of CEG can adduct thioredoxin at the active site and may have important toxicologic significance regarding the mechanism of 1,2-dichloroethane toxicity.
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6502, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Erve
authors.forename: E
authors.initials: E
authors.lastname: Barofsky
authors.forename: D F
authors.initials: DF
authors.lastname: Barofsky
authors.forename: M L
authors.initials: ML
authors.lastname: Deinzer
authors.forename: D J
authors.initials: DJ
authors.lastname: Reed
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry*; D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry*; D005025:Ethylene Dichlorides / Q000633:toxicity; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013879:Thioredoxins / Q000737:chemistry*
chemical_list: D000477:Alkylating Agents; D018736:DNA Adducts; D005025:Ethylene Dichlorides; D011994:Recombinant Proteins; D013879:Thioredoxins; C024565:ethylene dichloride; D005978:Glutathione; D003545:Cysteine; C046570:S-(2-chloroethyl)glutathione