期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 15, 页码 -出版社
MDPI
DOI: 10.3390/ijms23158089
关键词
protein degradation; proteolysis mechanisms; trypsin; tryptophan fluorescence; demasking kinetics
资金
- Russian Foundation for Basic Research [20-53-46006]
- Ministry of Science and Higher Education of the Russian Federation [075-00697-22-00]
This study investigated the demasking effect during the degradation of beta-lactoglobulin and beta-casein by trypsin. It was found that the secondary masking of intermediate polypeptides restricted the opening of the substrate. Additionally, demasking and secondary masking were shown to compete with each other.
The opening of protein substrates during degradation by proteases and the corresponding exposure of their internal peptide bonds for a successful enzymatic attack, the so-called demasking effect, was studied for beta-lactoglobulin (beta-LG) and beta-casein (beta-CN) hydrolyzed by trypsin. Demasking was estimated by monitoring the redshift in intrinsic tryptophan fluorescence, characterizing the accessibility of polypeptide chains to aqueous medium. The secondary masking of intermediate polypeptides, giving an inverse effect to demasking, caused a restriction of the substrate opening. This led to the limitations in the red shift of fluorescence and the degree of hydrolysis with a long time of hydrolysis of beta-LG and beta-CN at a constant substrate concentration and reduced trypsin concentrations. The proposed proteolysis model included demasking of initially masked bonds in the protein globule or micelle, secondary masking of intermediate polypeptides, and their subsequent slow demasking. The hydrolysis of peptide bonds was modeled taking into account different hydrolysis rate constants for different peptide bonds. It was demonstrated that demasking competes with secondary masking, which is less noticeable at high trypsin concentrations. Modeling of proteolysis taking into account two demasking processes and secondary masking made it possible to simulate kinetic curves consistent with the experimental data.
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