Multiple adduction reactions of nitroso sulfamethoxazole with cysteinyl residues of peptides and proteins: implications for hapten formation

Chem Res Toxicol. 2009 May;22(5):937-48. doi: 10.1021/tx900034r.


Sulfamethoxazole (SMX) induces immunoallergic reactions that are thought to be a result of intracellular protein haptenation by its nitroso metabolite (SMX-NO mass, 267 amu). SMX-NO reacts with protein thiols in vitro, but the conjugates have not been defined chemically. The reactions of SMX-NO with glutathione (GSH), a synthetic peptide (DS3), and two model proteins, human GSH S-transferase pi (GSTP) and serum albumin (HSA), were investigated by mass spectrometry. SMX-NO formed a semimercaptal (N-hydroxysulfenamide) conjugate with GSH that rearranged rapidly (1-5 min) to a sulfinamide. Reaction of SMX-NO with DS3 also yielded a sulfinamide adduct (mass increment, 267 amu) on the cysteine residue. GSTP was exclusively modified at the reactive Cys47 by SMX-NO and exhibited mass increments of 267, 283, and 299 amu, indicative of sulfinamide, N-hydroxysulfinamide, and N-hydroxysulfonamide adducts, respectively. HSA was modified at Cys34, forming only the N-hydroxysulfinamide adduct. HSA modification by SMX-NO under these conditions was confirmed with ELISA and immunoblotting with an antisulfonamide antibody. It is proposed that cysteine-linked N-hydroxysulfinamide and N-hydroxysulfonamide adducts of SMX are formed via the reaction of SMX-NO with cysteinyl sulfoxy acids. Evidence for a multistep assembly of model sulfonamide epitopes on GSH and polypeptides via hydrolyzable intermediates is also presented. In summary, novel, complex, and metastable haptenic structures have been identified on proteins exposed in vitro to the nitroso metabolite of SMX.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid
  • Cysteine / chemistry*
  • Enzyme-Linked Immunosorbent Assay
  • Glutathione / chemistry
  • Glutathione Transferase / chemistry
  • Haptens / chemistry*
  • Haptens / metabolism
  • Humans
  • Mass Spectrometry
  • Peptides / chemistry*
  • Serum Albumin / chemistry
  • Sulfamethoxazole / chemistry*
  • Sulfamethoxazole / toxicity


  • Haptens
  • Peptides
  • Serum Albumin
  • Glutathione Transferase
  • Glutathione
  • Sulfamethoxazole
  • Cysteine