Capillary electrophoresis versus differential scanning calorimetry for the analysis of free enzyme versus enzyme‐ligand complexes: In the search of the ligand‐free status of cholinesterases

Abstract

Cholinesterases (ChEs) are highly efficient biocatalysts whose active site is buried in a deep, narrow gorge. The talent of CE to discover inhibitors in the gorge of highly purified preparations has fairly altered the meaning of a ChE ligand-free status. To attempt at a description of this one, we investigated the stability of Bungarus fasciatus acetylcholinesterase (AChE), alone or complexed with different inhibitors. Determination of mid-transition temperature for thermal denaturation, using differential scanning calorimetry (DSC) and CE, provided conflicting results. Discrepancies strongly question the reality of a ligand-free AChE state. DSC allowed estimation of the stability of AChE-ligands complexes, and to rank the stabilizing effect of different inhibitors. CE acted as a detector of hidden ligands, provided that they were charged, reversibly bound, and thus dissociable upon action of electric fields. Then, CE allowed quantification of the stability of ligand-free AChE. CE and DSC providing each fractional and nonredundant information, cautious attention must be paid for actual estimation of the conformational stability of ChEs. Because inhibitors used in purification of ChEs by affinity chromatography are charged, CE remains a leading method to estimate enzyme stability and detect the presence of bound hidden ligands.