Issue 28, 2019

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

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 (Image ID:c9tb00590k-t1.gif = 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.

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

Supplementary files

Article information

Article type
Paper
Submitted
25 mar 2019
Accepted
17 jún 2019
First published
18 jún 2019

J. Mater. Chem. B, 2019,7, 4432-4439

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

J. H. P. M. Santos, G. Carretero, S. P. M. Ventura, A. Converti and C. O. Rangel-Yagui, J. Mater. Chem. B, 2019, 7, 4432 DOI: 10.1039/C9TB00590K

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