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Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability
Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these d...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8869816/ https://www.ncbi.nlm.nih.gov/pubmed/35200354 http://dx.doi.org/10.3390/bios12020094 |
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author | Santos, João H. P. M. Feitosa, Valker A. Meneguetti, Giovanna P. Carretero, Gustavo Coutinho, João A. P. Ventura, Sónia P. M. Rangel-Yagui, Carlota O. |
author_facet | Santos, João H. P. M. Feitosa, Valker A. Meneguetti, Giovanna P. Carretero, Gustavo Coutinho, João A. P. Ventura, Sónia P. M. Rangel-Yagui, Carlota O. |
author_sort | Santos, João H. P. M. |
collection | PubMed |
description | Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30–40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C. |
format | Online Article Text |
id | pubmed-8869816 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-88698162022-02-25 Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability Santos, João H. P. M. Feitosa, Valker A. Meneguetti, Giovanna P. Carretero, Gustavo Coutinho, João A. P. Ventura, Sónia P. M. Rangel-Yagui, Carlota O. Biosensors (Basel) Article Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30–40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C. MDPI 2022-02-04 /pmc/articles/PMC8869816/ /pubmed/35200354 http://dx.doi.org/10.3390/bios12020094 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Santos, João H. P. M. Feitosa, Valker A. Meneguetti, Giovanna P. Carretero, Gustavo Coutinho, João A. P. Ventura, Sónia P. M. Rangel-Yagui, Carlota O. Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability |
title | Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability |
title_full | Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability |
title_fullStr | Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability |
title_full_unstemmed | Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability |
title_short | Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability |
title_sort | lysine-pegylated cytochrome c with enhanced shelf-life stability |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8869816/ https://www.ncbi.nlm.nih.gov/pubmed/35200354 http://dx.doi.org/10.3390/bios12020094 |
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