Proteolysis of Micellar β-Casein by Trypsin: Secondary Structure Characterization and Kinetic Modeling at Different Enzyme Concentrations

被引:4
|
作者
Vorob'ev, Mikhail M. [1 ]
Acikgoez, Burcin Dersu [2 ]
Guler, Gunnur [3 ,4 ]
Golovanov, Andrey V. [1 ]
Sinitsyna, Olga V. [1 ]
机构
[1] RAS, AN Nesmeyanov Inst Organoelement Cpds, 28 ul Vavilova, Moscow 119991, Russia
[2] Izmir Univ Econ, Grad Sch, Div Bioengn, TR-35330 Izmir, Turkiye
[3] Izmir Inst Technol, Dept Phys, Biophys Lab, TR-35430 Izmir, Turkiye
[4] Izmir Univ Econ, Biomed Bioengn, Sakarya Cad, TR-35330 Izmir, Turkiye
关键词
proteolysis kinetics; Fourier-transform infrared spectroscopy; atomic force microscopy; beta-casein; trypsin; INFRARED FT-IR; TRYPTIC HYDROLYSIS; SPECTROSCOPY; DEMASKING; PROTEASE; MILK; SITE;
D O I
10.3390/ijms24043874
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Tryptic proteolysis of protein micelles was studied using beta-casein (beta-CN) as an example. Hydrolysis of specific peptide bonds in beta-CN leads to the degradation and rearrangement of the original micelles and the formation of new nanoparticles from their fragments. Samples of these nanoparticles dried on a mica surface were characterized by atomic force microscopy (AFM) when the proteolytic reaction had been stopped by tryptic inhibitor or by heating. The changes in the content of beta-sheets, alpha-helices, and hydrolysis products during proteolysis were estimated by using Fourier-transform infrared (FTIR) spectroscopy. In the current study, a simple kinetic model with three successive stages is 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. 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 beta-CN at different concentrations of the enzyme.
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页数:16
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