Monitoring of Demasking of Peptide Bonds During Proteolysis by Analysis of the Apparent Spectral Shift of Intrinsic Protein Fluorescence

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.