In-line quantification of peroxidase-catalyzed cross-linking of a-lactalbumin in a microreactor

Horseradish peroxidase can induce the oxidative cross-linking of proteins through the radicalization of tyrosine residues and subsequent formation of dityrosine bonds. The dityrosine bond absorbs light at 318 nm which can be used to monitor in-line the peroxidase-catalyzed cross-linking of proteins in a microfluidic system. In this study calcium-depleted a-lactalbumin is used as model protein. To quantify the progress of the reaction, the absorbance increase at 318 nm was monitored in-line and compared with the amount of reacted monomeric a-lactalbumin as determined with size-exclusion chromatography (SEC) at various residence times. The increase in absorbance at 318 nm shows a logarithmic relation with the extent of reacted monomer. The logarithmic relation can be explained using a reaction model describing minimum and maximum formation of dityrosine cross-links to reacted monomer. Since the size distribution of reaction products was found to be reproducible, the absorbance increase at 318 nm can be used as a fast in-line screening method for the peroxidase-mediated cross-linking of proteins