ИСТИНА

Proteolysis of b-CN by trypsin was analyzed using the two-step proteolysis model, which quantified the degree of hydrolysis of peptide bonds with the degree of masking (or degree of demasking) [1, 2]. In the two-step model, the proteolysis is considered as a simple two-step process with consecutive demasking and hydrolysis steps. The use of this model made it possible to determine the rate constant of demasking and the content of initially masked peptide bonds. The proteolysis of CN by chymotrypsin was studied with two-step proteolysis model using hydrolysis curves obtained at various substrate concentrations [3]. Trypsin proteolysis of the protein colloidal systems was studied using β-CN micelles as a substrate. The hydrolysis of specific peptide bonds in β-CN by trypsin leads to the degradation and rearrangement of the original micelles and the formation of new nanoparticles from their fragments. The samples of these nanoparticles dried on the surfaces of mica or graphite were characterized by atomic force microscopy (AFM). AFM analysis made it possible to estimate size, density, and structural heterogeneity of peptide nanoparticles [4, 5]. A simple kinetic model with three consecutive stages was proposed to predict the rearrangement of nanoparticles and the formation of proteolysis products, as well as changes in the secondary structure during proteolysis at various enzyme concentrations [5]. The model determines for which steps the rate constants are proportional to the enzyme concentration and in which intermediate nano-components the protein secondary structure is retained and in which it is reduced. The model predictions were in agreement with the FTIR results for tryptic hydrolysis of β-CN at different concentrations of the enzyme. Acknowledgments: This work was supported by RFBR (№ 20-53-46006). References: 1. Vorob’ev, M.M. Kinetics of peptide bond demasking in enzymatic hydrolysis of casein substrates. J. Mol. Catal. B 2009, 58, 146–152, doi:10.1016/j.molcatb.2008.12.007. 2. Vorob’ev, M.M.; Vogel, V.; Güler, G.; Mäntele, W. Monitoring of demasking of peptide bonds during proteolysis by analysis of the apparent spectral shift of intrinsic protein fluorescence. Food Biophys. 2011, 6, 519–526, doi:10.1007/s11483-011-9234-z. 3. Vorob’ev, M.M. Quantification of two-step proteolysis model with consecutive demasking and hydrolysis of peptide bonds using casein hydrolysis by chymotrypsin. Biochem. Eng. J. 2013, 74, 60–68, doi:10.1016/j.bej.2013.02.020. 4. Sinitsyna, O.V; Vorob'ev, M.M. Atomic force microscopy characterization of b-casein nanoparticles on mica and graphite. Mendeleev Commun. 2021, 31, 88-90, doi:10.1016/j.mencom.2021.01.027. 5. Vorob'ev, M.M.; Açıkgöz, B.D.; Güler, G; Golovanov, A.V.; Sinitsyna, O.V. Proteolysis of micellar β-casein by trypsin: secondary structure characterization and kinetic modeling at different enzyme concentrations. Int. J. Mol. Sci. 2023, 24, 3874, doi:10.3390/ijms24043874.