Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-lin ked by horseradish peroxidase in the presence of hydrogen peroxide. As prod ucts, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. T o a GYG-containing incubation mixture was added ferulic acid in small aliqu ots, therewith keeping the molar ratio of the substrates favorable for hete ro-cross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, fu rthermore, two ferulic acids linked to peptide oligomers, ranging from dime rs to pentamers. Also, mono- and dimers of the peptide were linked to one m olecule of ferulic acid. A mechanism explaining the formation of all these products is proposed.