title: Glutathione-induced radical formation on lactoperoxidase does not correlate with the enzyme's peroxidase activity.
abstract: Lactoperoxidase (LPO) is believed to serve as a mediator of host defense against invading pathogens. The protein is more abundant in body fluids such as milk, saliva, and tears. Lactoperoxidase is known to mediate the oxidation of halides and (pseudo)halides in the presence of hydrogen peroxide to reactive intermediates presumably involved in pathogen killing. More recently, LPO has been shown to oxidize a wide diversity of thiol compounds to thiyl free radicals, which ultimately lead to the formation of a protein radical characterized by DMPO-immunospin trapping. In the same study by our group the authors claimed that a consequence of this protein radical formation was the inactivation of LPO (Guo et al., J. Biol. Chem.279:13272-13283; 2004). Here we demonstrate that although thiyl radical formation does lead to LPO radical production, the formation of this radical is unrelated to the enzyme's activity. We suggest the source of this misleading interpretation to be the binding of GSH to ELISA plates, which interferes with ABTS and guaiacol oxidation. In addition, DMPO-GSH-nitrone adducts bind to ELISA plates, leading to ambiguities of interpretation since we have demonstrated that DMPO-GSH nitrone does not bind to LPO, and only LPO-protein-DMPO-nitrone adducts can be detected by Western blot.
authors.affiliation: Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, MD, USA. bonini@niehs.nih.gov
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Bonini
authors.forename: Arno G
authors.initials: AG
authors.lastname: Siraki
authors.forename: Suchandra
authors.initials: S
authors.lastname: Bhattacharjee
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D004566:Electrodes; D004578:Electron Spin Resonance Spectroscopy; D004797:Enzyme-Linked Immunosorbent Assay; D005609:Free Radicals; D005978:Glutathione / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007784:Lactoperoxidase / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D006861:Hydrogen Peroxide; D007784:Lactoperoxidase; D005978:Glutathione