Nanostructure development during peroxidase catalysed cross-linking of a-lactalbumin

Whereas extensive work has been done on the food functional and chemical aspects of enzymatic protein cross-linking, relatively little is known about the nanostructure and physical-chemical properties of enzymatically cross-linked protein. We investigate how nanostructure develops during enzymatic cross-linking of the 4 tyrosine residues of the globular protein apo a-lactalbumin. Protein cross-linking is catalysed by Horseradish Peroxidase, under the periodic addition of peroxide. We use on-line static and dynamic light scattering, combined with on-line UV-spectroscopy to simultaneously probe the development of nanostructure, the extent of dityrosine formation, and the catalytic state of the enzyme, as a function of the number of peroxide additions. It is found that initially, the rate of dityrosine formation is high, whereas the increase in the solution size of the cross-linked protein is limited. At later stages, the increase in solution size is significant whereas dityrosine formation slows down. Finally, the reaction stops due to enzyme inactivation. Off-line size exclusion chromatography shows that the initial phase corresponds to a fast cross-linking of monomers into small oligomers, followed by a slower joining of oligomers into large protein polymers. Consistent with this, Atomic Force Microscopy shows very heterogeneous polymers, apparently consisting of subunits that we identify with the oligomers formed in the first phase of the reaction. The dependence of the solution size on the molar mass of the cross-linked protein is determined using static and dynamic light scattering on fractionated reaction products. For sizes ranging from 30 nm to 80 nm, the protein polymers consist of 100–1000 a-lactalbumin subunits, and have molar masses of 106–107 g/mol. Apparent internal protein densities of the protein polymers calculated from these numbers are only a few weight percent, indicating a very dilute, open architecture of the cross-linked protein