title: Identification of the peroxidase-generated intermolecular dityrosine cross-link in bovine α-lactalbumin.
abstract: The peroxidase-mediated oxidation of calcium-depleted bovine α-lactalbumin generates a mixture of covalently bound protein oligomers with interesting foaming properties. Here, we isolated the initially formed covalent α-lactalbumin dimer and studied its mode of cross-linking. Liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) of proteolytic digests revealed the unambiguous identification of a peroxidase-catalyzed covalent link between Tyr18 and Tyr50. This shows that, although the radical reaction is often regarded as a random reaction, the initial product formation is specific. Protein structural modeling indicates that the conjugation reaction between these tyrosines is sterically favored and involves initial noncovalent protein complex formation through charge compensation, facilitating intermolecular cross-linking. The identification of the Tyr18-Tyr50 cross-link supports the view that the peroxidase-mediated oxidation of apo α-lactalbumin is a sequential process, involving the formation of linear trimers and higher order oligomers.
authors.affiliation: Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands.
authors.forename: Walter H
authors.initials: WH
authors.lastname: Heijnis
authors.forename: Henk L
authors.initials: HL
authors.lastname: Dekker
authors.forename: Leo J
authors.initials: LJ
authors.lastname: de Koning
authors.forename: Peter A
authors.initials: PA
authors.lastname: Wierenga
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.forename: Chris G
authors.initials: CG
authors.lastname: de Koster
authors.forename: Harry
authors.initials: H
authors.lastname: Gruppen
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
mesh_terms: D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry*; D011487:Protein Conformation; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D014443:Tyrosine; D007768:Lactalbumin; C007543:dityrosine; D009195:Peroxidase