Detection of unwanted protein-bound ligands by capillary zone electrophoresis: The case of hidden ligands that stabilize cholinesterase conformation

Abstract

Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters. Likewise, ChE are characterized by the high catalytic efficiency of an active site positioned at the bottom of a deep gorge. The gorge can be partially or fully occupied by ligands, i.e., substrates and inhibitors that are currently used in affinity chromatography purification steps. Accordingly, a suitable method allowing to analyse the presence of unwanted ligands and its influence on the functional conformation and stability of these enzymes was essential. We have developed CZE approaches for that purpose. The factors causing discrepancies between data for thermal unfolding of ChE by electrophoretic and by calorimetric methods were investigated. The presence of unwanted hidden ligands bound to purified enzymes was first demonstrated. The incidence of these ligands was discussed. Altogether, our results raised several questions concerning the real conformation of the native state of enzymes. Finally, CZE was proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.

Subject Index To Volume 2

“…Rochu and coworkers have reported several studies on cholinesterases [21][22][23]26], which are enzymes that irreversibly unfold at high temperature. Rochu et al have used the Lumry-Eyring model for unfolding of globular proteins to describe this process [51].…”

“…In this circumstance, k 3 0 and the assumptions for a two-state reversible protein unfolding transition can be used to estimate thermodynamic constants. This assumption allowed Rochu and colleagues [21][22][23]26] to use the two-state unfolding model to evaluate thermal unfolding.…”

“…Protein unfolding studies by CE of phosphotriesterase (PTE) with various metal cations substituted into the active site [24,25] as well as different cho-linesterases [21][22][23] have been reviewed by Rochu and Masson [31]. The results obtained by thermal unfolding studies were validated using differential scanning calorimetry.…”

“…Although the co-factor was not required for refolding to occur, its presence allowed for a greater recovery of refolded protein, providing evidence that ligand addition enhanced stability of the native state of protein. Similarly, Rochu and co-workers demonstrated that decamethonium bound to the gorge of cholinesterases stabilizes the protein native state, which resulted in an increase in apparent T m by 9.5 o C [23]. The increase in protein stability was believed to be due to the additional non-covalent bonds formed with the bound-ligand.…”

“…CE was first introduced as a new method for examining protein conformational stability in 1991 by Rush et al [13] that offered several unique advantages over traditional platforms, such as classical slab gel electrophoresis. Since then, over 70 different CE studies have been reported for protein unfolding to date with major contributions made by the groups of Righetti [14][15][16][17][18] and Rochu [19][20][21][22][23][24][25][26]. However, it still remains a rather unrecognized technique for characterizing protein conformational stability in the wider biochemical community.…”