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Time-resolved studies were performed for tryptic proteolysis of β-casein in media containing 10–40 % (v/v) ethanol at 37 °C and pH 7.9. The peptide bond demasking, the process which implies the removal of steric obstacles shielding polypeptide sites against enzymatic attack, was quantitatively evaluated with fluorescence spectroscopy by monitoring the exposure of Trp residues to the aqueous polar medium. This process obeys a first-order kinetic law that allowed us the determination of the rate constants of demasking k d . The fraction of initially masked bonds m, being able to convert during proteolysis to the demasked ones, and the fraction of unhydrolysable bonds n were calculated within the framework of a two-step model with consecutive demasking and hydrolysis steps. Parameters m and n were shown to decrease and increase, respectively, with the addition of ethanol.

Demasking of peptide bonds during proteolysis of β-casein and β-lactoglobulin by trypsin was monitored by the measurement of the overall spectral shift of intrinsic protein fluorescence. A clear shift of the apparent emission maxima from approximately 340–345 nm to 355–360 nm during proteolysis was observed, with a time course, which follows protein degradation and structural opening. In contrast to procedures using extrinsic fluorescence labels, this label-free procedure does not bear the risk of structural alterations. It is easy to perform, fast, and has a relatively high accuracy of determination. Proteolysis was modelled as simple two-step process with consecutive demasking and hydrolysis stages. It was shown that the fluorescence shift can be attributed to the demasking stage. Formally, kinetics of the peptide bond demasking obeys a first-order kinetic law. Both the theoretical simulations and experiment are in accordance giving the similar dependences of the hydrolysis degree on the degree of peptide bond demasking.