title: Site-specific detection of radicals on α-lactalbumin after a riboflavin-sensitized reaction, detected by immuno-spin trapping, ESR, and MS.
abstract: Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.
authors.affiliation: Department of Food Science, Faculty of Science and Technology, Aarhus University, P.O. Box 50, DK-8830 Tjele, Denmark. trine.dalsgaard@agrsci.dk
authors.forename: Trine K
authors.initials: TK
authors.lastname: Dalsgaard
authors.forename: Mathilde
authors.initials: M
authors.lastname: Triquigneaux
authors.forename: Leesa
authors.initials: L
authors.lastname: Deterding
authors.forename: Fiona
authors.initials: F
authors.lastname: Summers
authors.forename: Kalina
authors.initials: K
authors.lastname: Ranguelova
authors.forename: Grith
authors.initials: G
authors.lastname: Mortensen
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis / Q000737:chemistry; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000528:radiation effects; D007768:Lactalbumin / Q000737:chemistry* / Q000528:radiation effects; D008027:Light; D010084:Oxidation-Reduction; D012256:Riboflavin / Q000737:chemistry* / Q000528:radiation effects; D021241:Spectrometry, Mass, Electrospray Ionization; D019097:Spin Trapping; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000528:radiation effects
chemical_list: D005609:Free Radicals; D014443:Tyrosine; D006639:Histidine; D007768:Lactalbumin; D012256:Riboflavin