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 ( = 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 (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.