Thermostability is an important and desired feature of therapeutic proteins and is critical for the success or failure of protein drugs development. It can be increased by PEGylation-binding of poly(ethylene glycol) moieties-or glycosylation-post-translational modification to add glycans. Here, the thermostability and thermodynamic parameters of native, PEGylated, and glycosylated versions of the antileukemic enzyme crisantaspase were investigated. First-order kinetics was found to describe the irreversible deactivation process. Activation energy of the enzyme-catalyzed reaction (E*) was estimated for native, PEGylated, and glycosylated enzyme (10.2, 14.8, and 18.8 kJ mol(-1) respectively). Half-life decreased progressively with increasing temperature, and lon-ger half-life was observed for PEG-crisantaspase (87.74 min) at 50 degrees C compared to the native form (9.79 min). The activation energy of denaturation of PEG-crisantaspase (307.1 kJ mol(-1)) was higher than for crisantaspase (218.1 kJ mol(-1)) and Glyco-crisantaspase (120.0 kJ mol(-1)), which means that more energy is required to overcome the energy barrier of the unfolding process. According to our results, PEG-crisantaspase is more thermostable than its native form, while Glyco-crisantaspase is more thermosensitive.
PEGylation versus glycosylation: effect on the thermodynamics and thermostability of crisantaspase
Converti, Attilio;Pessoa, Adalberto;
2024-01-01
Abstract
Thermostability is an important and desired feature of therapeutic proteins and is critical for the success or failure of protein drugs development. It can be increased by PEGylation-binding of poly(ethylene glycol) moieties-or glycosylation-post-translational modification to add glycans. Here, the thermostability and thermodynamic parameters of native, PEGylated, and glycosylated versions of the antileukemic enzyme crisantaspase were investigated. First-order kinetics was found to describe the irreversible deactivation process. Activation energy of the enzyme-catalyzed reaction (E*) was estimated for native, PEGylated, and glycosylated enzyme (10.2, 14.8, and 18.8 kJ mol(-1) respectively). Half-life decreased progressively with increasing temperature, and lon-ger half-life was observed for PEG-crisantaspase (87.74 min) at 50 degrees C compared to the native form (9.79 min). The activation energy of denaturation of PEG-crisantaspase (307.1 kJ mol(-1)) was higher than for crisantaspase (218.1 kJ mol(-1)) and Glyco-crisantaspase (120.0 kJ mol(-1)), which means that more energy is required to overcome the energy barrier of the unfolding process. According to our results, PEG-crisantaspase is more thermostable than its native form, while Glyco-crisantaspase is more thermosensitive.
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