title: The photolysis of disulfide bonds in IgG1 and IgG2 leads to selective intramolecular hydrogen transfer reactions of cysteine Thiyl radicals, probed by covalent H/D exchange and RPLC-MS/MS analysis.
abstract: OBJECTIVE
  The evaluation of photo-instability of biotherapeutic products is mandated by regulatory agencies. Photo-irradiation can induce oxidative modifications in proteins, which may lead to undesired biological and therapeutic consequences. Among the modifications, epimerization of amino acid residues can occur upon photo-irradiation of IgGs.
  
  
  METHODS
  We show here, that UV irradiation (λ = 253.7 nm) of IgG1 and IgG2 leads to the formation of intermediary carbon-centered radicals, validated by covalent incorporation of deuterium into the protein primary sequence.
  
  
  RESULTS
  By MS/MS analysis we identified the sites of deuterium incorporation, such as the sequence QD [303:304, HC], present in the peptide of VVSVLTVVHQDWLNGK [294:309, HC] in both IgG1 and IgG2, and V [111, LC] and K [116, LC], present in the peptide VTVLGQPK [109:116, LC] in IgG2. Both peptides are in the proximity of intrachain disulfide bonds.
  
  
  CONCLUSIONS
  The exposure of IgG1 and IgG2 to UV-light (λ = 253.7 nm) generates specific carbon-centered radicals. The latter were evidenced by a covalent H-D exchange reaction that likely occurred through a hydrogen atom transfer reaction between cysteine thiyl radical and C-H bond.
authors: Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047, USA.
authors: Shuxia
authors: S
authors: Zhou
authors: Olivier
authors: O
authors: Mozziconacci
authors: Bruce A
authors: BA
authors: Kerwin
authors: Christian
authors: C
authors: Schöneich
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D004220:Disulfides / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010782:Photolysis; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
chemical_list: D004220:Disulfides; D007074:Immunoglobulin G; D010455:Peptides; D006859:Hydrogen; D003545:Cysteine