Demasking rate constants for tryptic hydrolysis of β-casein

Time-resolved studies were performed for tryptic proteolysis of β-casein. The accumulation and decay of the proteolysis products was shown to proceed in the form of nanoscale aggregates that exhibited considerable scattering cross sections for visible light. Monitoring the time course of degradation of these aggregates by static light scattering allowed the determination of the rate constants of aggregate degradation. Additionally, another set of rate constants was determined with fluorescence spectroscopy by monitoring the exposure of Trp residues to the aqueous polar medium. These constants were interpreted as the rate constants of peptide bond demasking, the process that implies the removal of steric obstacles shielding polypeptide sites against enzymatic attack. Both methods gave comparable values for the rate constants. It was concluded that for the trypsinolysis of β-casein, demasking proceeds in parallel with the degradation of aggregates that arise at the initial stage of proteolysis.