A generic screening approach was performed to investigate the binding of various potential ligands to proteins in order to investigate how proteins interact with ions and the complete surrounding solution. This also allows to study binding behavior and its regulation and protein functionality in general in a native environment. The ACE technique affords an excellent precision by applying appropriate rinsing procedures using a pressure of 2.5 bar and a continuous flow concept. Confidence intervals were estimated to proof significance of the interactions. This enables to investigate smaller yet important interactions, which were not possible with less precise techniques before. The influence of various ions on ovalbumin, β-lactoglobulin, and BSA were screened by comparing the mobility ratios of the free protein and the influenced one. The analysis was performed using the metal ions Ba(2) +, Ca(2) +, Cu(2) +, Mg(2) +, Mn(2) +, Ni(2) +, the pharmaceutical cations ephedrine hydrochloride, ethambutol dihydrochloride, pilocarpine hydrochloride and pirenzepine dihydrochloride, and various anions, in particular phosphate, acetate, succinate, glutamate, and salicylate. For the anion influence study, myoglobin was also included to the screened proteins. The influence of these ions on the proteins was well diversified. The interactions could be distinguished with a fast and precise screening method since 90% of all mobility ratios had a RSD% below 2% and 79% had a RSD% lower than 1%. Hence, for more than 75% of the protein-ligand pairs significant interactions are observed with a very small confidence interval due to the very excellent precision of these used method.

No abstract

The influence of various cations as metal ions (barium, calcium, copper, magnesia, manganese, and nickel), pharmaceuticals (ephedrine, ethambutol, pilocarpine, and pirenzepine), arginine, and guanidine has been tested on BSA, β-lactoglobulin, and ovalbumin. Influences on proteins regarding changes in size, charge, or mass were of particular interest. ACE proved to be a suitable method to investigate these effects. ACE is able to observe slight but significant changes on proteins due to the excellent precision of the measurements. Therefore, some unexpected behaviors of protein-ligand interactions were found. The protein charge becomes more negative under metal ion influence and some pharmaceutical cations. Probably metal ions bound to the proteins form additional complexes with anions from the surrounding solution. Furthermore, already bound cations could be displaced at the protein surface. Both effects would change the overall charge of the ligand-protein complexes. In all studied cases, multiple-binding stoichiometries have been observed.

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).

The binding of physiologically anionic species or negatively charged drug molecules to proteins is of great importance in biochemistry and medicine. Since affinity capillary electrophoresis (ACE) has already proven to be a suitable analytical tool to study the influence of ions on proteins, this technique was applied here for comprehensively studying the influence of various anions on proteins of BSA, β-lactoglobulin, ovalbumin, myoglobin, and lysozyme. The analysis was performed using different selected anions of succinate, glutamate, phosphate, acetate, nitrate, iodide, thiocyanate, and pharmaceuticals (salicylic acid, aspirin, and ibuprofen) that exist in the anionic form at physiological pH 7.4. Due to the excellent repeatability and precision of the ACE measurements, not necessarily strong but significant influences of the anions on the proteins were found in many cases. Different influences in the observed bindings indicated change of charge, mass, or conformational changes of the proteins due to the binding with the studied anions. Combining the mobility-shift and pre-equilibrium ACE modes, rapidity and reversibility of the protein-anion bindings were discussed. Further, circular dichroism has been used as an orthogonal approach to characterize the interactions between the studied proteins and anions to confirm the ACE results. Since phosphate and various anions from amino acids and small organic acids such as succinate or acetate are present in very high concentrations in the cellular environment, even weak influences are certainly relevant as well.