Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

• Published in: Symmetry (Basel) 2019-02, Vol.11 (2), p.153
• Main Author: Vorob’ev, M.M.
• Format: Article
• Language: English
• Subjects: Algorithms; Amino acids; Casein; demasking kinetics; Enzymes; Fluorescence; Hydrolysis; Kinetics; Lactoglobulin; Peptides; Polypeptides; Proteins; proteolysis mechanisms; Rate constants; Substrates; Trypsin; Tryptophan; tryptophan fluorescence
• ISSN: 2073-8994; 2073-8994
• DOI: 10.3390/sym11020153

To distinguish differences in enzymatic hydrolysis of various proteins, we propose an algorithm using a dataset of fluorescence spectra obtained at different moments of hydrolysis t. This algorithm was demonstrated in the example of β-lactoglobulin (β-LG) proteolysis by trypsin. The procedure involved processing the spectra to obtain the wavelength of the maximum fluorescence λmax, which was found to be proportional to the fraction of tryptophanes in hydrated proteolysis products (demasked tryptophanes). Then, the dependence λmax(t) was fitted by biexponential function with two exponential terms, one of which was responsible for the fast part of the fluorescence change during proteolysis. The contribution of this term was quite different for various protein substrates—it was positive for β-LG and negative for β-casein. The observed differences in proteolysis of different substrates were explained by different demasking processes. Combining the fluorescence data with the degrees of hydrolysis of peptide bonds allowed us to analyze the hydrolysis of β-LG in the framework of the two-step proteolysis model and estimate the ratio of rate constants of demasking and hydrolysis and the percentages of initially masked and resistant peptide bonds. This model predicted the existence of a bimodal demasking process with a fast part at the beginning of proteolysis and lag-type kinetics of release for some peptides. Compared with monitoring proteolysis in terms of the degree of hydrolysis only, the fluorescence data are helpful for the recognition of proteolysis patterns.