The peroxidase-mediated oxidation of calcium-depleted bovine a-lactalbumin generates a mixture of covalently bound protein oligomers with interesting foaming properties. Here, we isolated the initially formed covalent a-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 a-lactalbumin is a sequential process, involving the formation of linear trimers and higher order oligomers.
- microbial transglutaminase