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


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 degrees 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 (E-d* = 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 (Delta H-double dagger), entropy (Delta S-double dagger) and Gibbs free energy (Delta G(double dagger)) of the enzyme irreversible denaturation. The higher enthalpic contributions of PEGylated conjugates and the increase in Delta G(double dagger), compared to the native protein, indorsed the protective role of PEGylation. Negative values of Delta S-double dagger 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.




Materials Science


Santos, JHPM; Carretero, G; Ventura, SPM; Converti, A; Rangel-Yagui, CO

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The authors are grateful for the financial support of the SAo Paulo Research Foundation - FAPESP (grant #2016/22065-5), the Brazilian National Council for Scientific and Technological Development and the CoordenacAo de Aperfeicoamento de Pessoal de Nivel 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 JoAo H. P. M. Santos (SFRH/BD/102915/2014) and Sonia P. M. Ventura Investigador FCT 2015 contract (IF/00402/2015).

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