PEGylation as an efficient tool to enhance cytochrome c thermostability: a kinetic and thermodynamic study

João H.P.M. Santos, Gustavo Carretero, Sónia P.M. Ventura, Attilio Converti, Carlota O. Rangel-Yagui*

*Corresponding author

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Abstract

Cytochrome-c from equine heart was kinetically and thermodynamically investigated either in its native (Cyt-c) or PEGylated forms with different PEGylation degrees (Cyt-c-PEG-4 and Cyt-c-PEG-8). Maximum activities were observed at 80 °C, and the irreversible deactivation was well described by first-order kinetics. The results of activity at different temperatures were used to estimate the activation energy of the catalysed Cyt-c reaction (E∗ = 10.22 ± 0.40, 7.51 ± 0.06 and 8.87 ± 0.29 kJ mol-1 for Cyt-c, Cyt-c-PEG-4 and Cyt-c-PEG-8) and the standard enthalpy variation of enzyme unfolding (= 33.82 ± 4.92, 109.4 ± 13.1 and 58.43 ± 3.11 kJ mol-1 for Cyt-c, Cyt-c-PEG-4 and Cyt-c-PEG-8, respectively). The results of residual activity tests allowed estimating the activation energy (Ed∗ = 50.51 ± 1.71, 72.63 ± 0.89 and 63.36 ± 1.66 kJ mol-1 for Cyt-c, Cyt-c-PEG-4 and Cyt-c-PEG-8), enthalpy (ΔH‡), entropy (ΔS‡) and Gibbs free energy (ΔG‡) of the enzyme irreversible denaturation. The higher enthalpic contributions of PEGylated conjugates and the increase in ΔG‡, compared to the native protein, indorsed the protective role of PEGylation. Negative values of ΔS‡ suggested the occurrence of an aggregation phenomenon by increasing the temperature, which was confirmed by circular dichroism. The estimated thermodynamic parameters suggest that PEGylated Cyt-c forms have enhanced thermostability, which would be of great significance for industrial biosensing applications.
OriginalsprogEngelsk
TidsskriftJournal of Materials Chemistry B
Vol/bind7
Udgave nummer28
Sider (fra-til)4432-4439
Antal sider8
ISSN2050-750X
DOI
StatusUdgivet - 2019
Udgivet eksterntJa

Bibliografisk note

Funding Information:
The authors are grateful for the financial support of the São Paulo Research Foundation – FAPESP (grant #2016/22065-5), the Brazilian National Council for Scientific and Technological Development and the Coordenação de Aperfeiçoamento de Pessoal de Ńıvel Superior – Brazil (CAPES, Finance Code 001). This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. Authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD fellowship of João H. P. M. Santos (SFRH/BD/ 102915/2014) and Sónia P. M. Ventura Investigador FCT 2015 contract (IF/00402/2015).

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