During enzymatic hydrolysis of proteins, some cleavage sites will be accessible in the intact protein. Others may only become accessible after demasking of the cleavage site, i.e. after cleavage of other bonds in the protein. Studies on demasking so far have used data of the release of few peptides in a hydrolysate. To obtain a more complete understanding of the hydrolysis kinetics and to validate the demasking model, this study used the absolute quantification of all peptides formed during hydrolysis of whey protein isolate by Bacillus licheniformis protease. This allowed the determination of the rate constants of hydrolysis of each individual cleavage site in the substrate protein. For five cleavage sites, no cleavages were found. For nine cleavage sites, the hydrolysis was best described with first order kinetics addition. The other 13 cleavage sites were better described with the two-step demasking model, which consists of two consecutive first order reaction equations. In this way, the demasking concept was validated, showing that some peptide bonds are only cleaved when present in intermediate peptides.
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