Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical Studies
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...
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MDPI AG
2021-03-01
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| Series: | International Journal of Molecular Sciences |
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| author | Ruslan M. Sarimov Vladimir N. Binhi Tatiana A. Matveeva Nikita V. Penkov Sergey V. Gudkov |
| author_facet | Ruslan M. Sarimov Vladimir N. Binhi Tatiana A. Matveeva Nikita V. Penkov Sergey V. Gudkov |
| author_sort | Ruslan M. Sarimov |
| collection | DOAJ |
| description | 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 (Δ<i>n</i> ~ 4.5 × 10<sup>−5</sup>). 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. |
| first_indexed | 2024-03-09T05:04:03Z |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T05:04:03Z |
| publishDate | 2021-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-724472c3b1d84b7d8ad70c73e7f03cc52023-12-03T12:57:21ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-03-01225271010.3390/ijms22052710Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical StudiesRuslan M. Sarimov0Vladimir N. Binhi1Tatiana A. Matveeva2Nikita V. Penkov3Sergey V. Gudkov4Prokhorov General Physics Institute of the Russian Academy of Sciences, Vavilov St., 38, 119991 Moscow, RussiaProkhorov General Physics Institute of the Russian Academy of Sciences, Vavilov St., 38, 119991 Moscow, RussiaProkhorov General Physics Institute of the Russian Academy of Sciences, Vavilov St., 38, 119991 Moscow, RussiaInstitute of Cell Biophysics of the Russian Academy of Sciences, PSCBR RAS, Institutskaya St., 3, Pushchino, 142290 Moscow, RussiaProkhorov General Physics Institute of the Russian Academy of Sciences, Vavilov St., 38, 119991 Moscow, RussiaUsing 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 (Δ<i>n</i> ~ 4.5 × 10<sup>−5</sup>). 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.https://www.mdpi.com/1422-0067/22/5/2710protein hydration shellsoptical methods in biochemistryinterferometrydynamic light scattering (DLS)UV/visible absorbance spectroscopyanalysis of protein activity |
| spellingShingle | Ruslan M. Sarimov Vladimir N. Binhi Tatiana A. Matveeva Nikita V. Penkov Sergey V. Gudkov Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical Studies International Journal of Molecular Sciences protein hydration shells optical methods in biochemistry interferometry dynamic light scattering (DLS) UV/visible absorbance spectroscopy analysis of protein activity |
| title | Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical Studies |
| title_full | Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical Studies |
| title_fullStr | Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical Studies |
| title_full_unstemmed | Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical Studies |
| title_short | Unfolding and Aggregation of Lysozyme under the Combined Action of Dithiothreitol and Guanidine Hydrochloride: Optical Studies |
| title_sort | unfolding and aggregation of lysozyme under the combined action of dithiothreitol and guanidine hydrochloride optical studies |
| topic | protein hydration shells optical methods in biochemistry interferometry dynamic light scattering (DLS) UV/visible absorbance spectroscopy analysis of protein activity |
| url | https://www.mdpi.com/1422-0067/22/5/2710 |
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