Optimization of affinity capillary electrophoresis for routine investigations of protein–metal ion interactions

Abstract

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra- and inter-instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra-instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter-instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein-metal ion interactions (relative standard deviation of 0.16-0.89%, 15 series, 12 runs for each).

Data quality in drug discovery: the role of analytical performance in ligand binding assays

“…ACE can successfully be applied to study the interactions of metal ions with biomolecules, since the complex is typically differently charged as compared to the biomolecules alone [101][102][103][104]. This is particularly true for metal containing binding sites.…”

“…One ACE experiment typically needs \10 min, including rinsing steps to avoid carry-over effects. Typically percent relative standard deviations of migration data within one measurement series of \1 % are found, the variability of the binding constants probably being in the same range [101][102][103][104]. Comprehensive information about method development and successful examples has recently been reviewed [96][97][98][99].…”

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis (2013–2015)

“…ACE is a particularly promising approach for the examination of ligand–receptor interactions and the estimation of binding constants in free solution . Binding constant estimation by ACE is usually achieved by following the change in the electrophoretic mobility of an injected analyte ligand when the interacting molecule is added to the running buffer.…”

Binding Behavior of Few Lanthanides and Heavy Metal ions with Proteins by Fast and Reliable Affinity Capillary Electrophoresis Method

“…Similarly Zn 2+ is known to interact with a variety of enzymes and amino acids like cysteine, histidine (-N), aspartate (-O), and glutamate (-O) residues 32 and some hormones like Prolactine 33 . It also plays role as a cofactor for various enzymes to act properly.…”