Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.
Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates.
Laboratory of Food Chemistry, Wageningen University, 6700 AA Wageningen, Wageningen, The Netherlands.
Surender Kumar
SK
Dhayal
Laboratory of Food Chemistry, Wageningen University, 6700 AA Wageningen, Wageningen, The Netherlands; Department of Food Science, University of Parma, Italy.
Stefano
S
Sforza
Laboratory of Food Chemistry, Wageningen University, 6700 AA Wageningen, Wageningen, The Netherlands.
Peter A
PA
Wierenga
Laboratory of Food Chemistry, Wageningen University, 6700 AA Wageningen, Wageningen, The Netherlands. Electronic address: harry.gruppen@wur.nl.
Harry
H
Gruppen
D001704:Biopolymers / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D007768:Lactalbumin / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D001704:Biopolymers; D003432:Cross-Linking Reagents; D014443:Tyrosine; D007768:Lactalbumin; D009195:Peroxidase
Dityrosine; Enzymatic oxidation; Protein oxidation; Protein polymers; Tetratyrosine; Trityrosine