title: Electrochemistry/liquid chromatography/mass spectrometry to demonstrate irreversible binding of the skin allergen p-phenylenediamine to proteins.
abstract: BACKGROUND
  para-Phenylenediamine (PPD) is a potent and well-known allergen, which is commonly used in hair or fur dyes and can cause severe allergic contact dermatitis. In this work, the skin-sensitizing potential of PPD with respect to the conjugation of proteins was evaluated using an approach without animal testing.
  
  
  METHODS
  Electrochemistry (EC) coupled offline to liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS) was employed to convert the pre-hapten PPD into its reactive hapten analogs. A previous study had already shown that this purely instrumental method is suitable for accelerating and simulating the various oxidation processes, which PPD may undergo, and that the emerging products are prone to react with soft thiol groups of small nucleophiles like glutathione and cysteine.
  
  
  RESULTS
  This investigation was extended by successfully demonstrating adduct formation between EC-generated PPD oxidation products and the three proteins β-lactoglobulin A (β-LGA), human serum albumin and human hemoglobin. A tryptic digest of modified β-LGA provided evidence for irreversible protein binding of monomeric PPD, a PPD dimer and the PPD trimer known as Bandrowski's Base. It was shown that the main oxidation product p-phenylene quinone diimine, and the reactive oligomerized species, primarily attack the free thiol function of proteins rather than other nucleophilic amino acid residues.
  
  
  CONCLUSIONS
  The pre-hapten PPD was efficiently activated upon EC oxidation and the resulting species were further reacted with different proteins leading to diverse hapten-protein complexes. Thereby, problems related to the complex matrix present in conventional in vitro or in vivo methods could effectively be avoided.
authors.affiliation: University of Münster, Institute of Inorganic and Analytical Chemistry and NRW Graduate School of Chemistry, Corrensstr. 30, 48149 Münster, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Jahn
authors.forename: Helene
authors.initials: H
authors.lastname: Faber
authors.forename: Raniero
authors.initials: R
authors.lastname: Zazzeroni
authors.forename: Uwe
authors.initials: U
authors.lastname: Karst
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods*; D006241:Haptens; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007477:Ions / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D007782:Lactoglobulins / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010655:Phenylenediamines / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D006241:Haptens; D006454:Hemoglobins; D007477:Ions; D007782:Lactoglobulins; D010446:Peptide Fragments; D010655:Phenylenediamines; D012709:Serum Albumin; D014357:Trypsin; C029728:4-phenylenediamine