Comparison of β-Casein and β-Lactoglobulin Proteolysis by Trypsin: Quantification of Demasking and Hydrolysis of Peptide Bonds

Prediction of polypeptide chain fragmentation of protein substrates during proteolysis by trypsin was performed for globular β-lactoglobulin (β-LG) and micellar β-casein (β-CN). Despite significant differences in the protein structures of these substrates, the concentra-tions of peptide fragments were calculated as functions of the time or degree of hydrolysis using the same equations derived from a general proteolysis model. This model consid-ered the opening of protein substrate, the so-called demasking, and the subsequent hy-drolysis of specific peptide bonds at different rates determined by the amino acid sequence of the hydrolyzed sites. The potential of this model for in silico proteolysis and the possi-bility of quantifying the rate constants for an arbitrary protein substrate are discussed. An algorithm for calculating demasking rate constants based on the experimental distribu-tion of peptide fragments is presented. The calculated concentration dependences on the degree of hydrolysis were compared with experimental data for the intermediate and final peptide fragments of β-LG and β-CN. The predicted and experimental concentration curves for the final products were compared based on their curvatures. For both sub-strates, the predicted distribution of peptide fragments was consistent with the experi-mental one.