title: Disulfiram generates a stable N,N-diethylcarbamoyl adduct on Cys-125 of rat hemoglobin beta-chains in vivo.
abstract: Disulfiram (DSF) is a drug used in aversion therapy to treat alcoholics and acts by inhibiting mitochondrial low-K(m) aldehyde dehydrogenase. Investigations into the mechanisms for in vivo inactivation suggest that the DSF metabolite S-methyl-N, N-diethylthiocarbamate sulfoxide reacts irreversibly with an active site Cys. This work aimed to determine if DSF generates monothiocarbamate adducts on cysteine residues in vivo by examining hemoglobin. Sprague-Dawley rats were treated with DSF po for 2, 4, and 6 weeks. Rats have four different globin beta-chains, of which three (beta-1-3) contain two cysteine residues each. MALDI-TOF MS analysis of two new globin species from DSF-treated rats collected by HPLC revealed increments of 99 Da above the mass of the unmodified chains (beta-2 and beta-3). In a separate experiment, the globin mixture was digested for 2 h with Glu-C and reanalyzed by MALDI-TOF MS. Results showed a peptide at m/z 2716.3 having a mass 99 Da higher than a known Cys-containing peptide. Subsequently, the Glu-C digest was analyzed using Q-TOF tandem MS, enabling observation of the +4 charge state of the peptide with m/z 2716.3. This peptide was fragmented to produce y-sequence ions that located the modification to Cys-125 (present on both beta-2 and beta-3). Cys-125 is the most reactive of two cysteine residues on these beta-chains. To confirm the structure of the modification, globin was hydrolyzed with 6 N HCl at 110 degrees C for 18 h. The adduct survived these conditions so that S-(N,N-diethylcarbamoyl)cysteine was detected in the hydrolysates of treated rats on the basis of comparison with the tandem MS spectrum of a standard. These results extend the findings of others obtained using glutathione conjugates and demonstrate the ability of DSF to covalently modify Cys residues of proteins in a manner consistent with the production of S-methyl-N, N-diethylthiocarbamate sulfoxide, or sulfone, intermediates.
authors.affiliation: Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Erve
authors.forename: O N
authors.initials: ON
authors.lastname: Jensen
authors.forename: H S
authors.initials: HS
authors.lastname: Valentine
authors.forename: V
authors.initials: V
authors.lastname: Amarnath
authors.forename: W M
authors.initials: WM
authors.lastname: Valentine
mesh_terms: D000427:Alcohol Deterrents / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004221:Disulfiram / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000427:Alcohol Deterrents; D006454:Hemoglobins; D004221:Disulfiram