期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 22, 期 5, 页码 -出版社
MDPI
DOI: 10.3390/ijms22052710
关键词
protein hydration shells; optical methods in biochemistry; interferometry; dynamic light scattering (DLS); UV; visible absorbance spectroscopy; analysis of protein activity
资金
- Ministry of Science and Higher Education of the Russian Federation [075-15-2020-912]
Optical techniques were used to investigate the denaturation process of HEWL, revealing that denaturation with GdnHCl led to complete loss of enzymatic activity and increased protein size, while denaturation without GdnHCl resulted in aggregation and preservation of some enzymatic activity.
Using a number of optical techniques (interferometry, dynamic light scattering, and spectroscopy), denaturation of hen egg white lysozyme (HEWL) by treatment with a combination of dithiothreitol (DTT) and guanidine hydrochloride (GdnHCl) has been investigated. The denaturing solutions were selected so that protein denaturation occurred with aggregation (Tris-HCl pH = 8.0, 50 mM, DTT 30 mM) or without aggregation (Tris-HCl pH = 8.0, 50 mM, DTT 30 mM, GdnHCl 6 M) and can be evaluated after 60 min of treatment. It has been found that denatured by solution with 6 M GdnHCl lysozyme completely loses its enzymatic activity after 30 min and the size of the protein molecule increases by 1.5 times, from 3.8 nm to 5.7 nm. Denaturation without of GdnHCl led to aggregation with preserving about 50% of its enzymatic activity. Denaturation of HEWL was examined using interferometry. Previously, it has been shown that protein denaturation that occurs without subsequent aggregation leads to an increase in the refractive index (Delta n similar to 4.5 x 10(-5)). This is most likely due to variations in the HEWL-solvent interface area. By applying modern optical techniques conjointly, it has been possible to obtain information on the nature of time-dependent changes that occur inside a protein and its hydration shell as it undergoes denaturation.
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