The opening of protein globules and corresponding exposure of their internal peptide bonds, the so-called demasking effect, is required for successful hydrolysis of peptide bonds by proteases. Under the proteolytic action of trypsin on β-lactoglobulin (β-LG), the evolution of tryptophan fluorescence spectra showed that the demasking process consists of two stages with different demasking rate constants for each stage. It was found that the ratio of these constants depends on the concentration of trypsin and changes are approximately threefold when the concentration of trypsin changes in the range of 0.3–15 mg/L. Simulation of hydrolysis taking into account the demasking effect demonstrated how the apparent first-order rate constants obtained experimentally are related to the true hydrolysis rate constants and demasking parameters. The lag phase in the kinetic curves corresponding to the hydrolysis of various peptide bonds in β-LG was also analyzed. The increased lag times indicated sites that are hydrolyzed by a two-stage demasking mechanism.
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.
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