Disulfiram generates a stable N,N-diethylcarbamoyl adduct on Cys-125 of rat hemoglobin beta-chains in vivo

Disulfiram (DSF) is a drug used in aversion therapy to treat alcoholics and acts by inhibiting mitochondrial low-K-m aldehyde dehydrogenase. Investiga tions into the mechanisms for in vivo inactivation suggest that; the DSF me tabolite S-methyl-N,N-diethylthiocarbamate sulfoxide reacts irreversibly wi th an active site Cys. This work aimed to determine if DSF generates monoth iocarbamate adducts on cysteine residues in vivo by examining hemoglobin. S prague-Dawley rats were treated with DSF po for 2, 4, and 6 weeks. Rats hav e 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 fro m 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 experimen t, the globin mixture was digested for 2 h with Glu-C and reanalyzed by MAL DI-TOF MS. Results showed a peptide at m/z 2716.3 having a mass 99 Da highe r than a known Cys-containing peptide. Subsequently, the Glu-C digest was a nalyzed 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-se quence 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 the se beta-chains. To confirm the structure of the modification, globin was hy drolyzed 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 hyd rolysates of treated rats on the basis of comparison with the tandem MS spe ctrum of a standard. These results extend the findings of others obtained u sing glutathione conjugates and demonstrate the ability of DSF to covalentl y modify Cys residues of proteins in a manner consistent with the productio n of S-methyl-N,N-diethylthiocarbamate sulfoxide, or sulfone, intermediates .