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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...

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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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