Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: Part 3: Anions

Abstract

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.

Recent Advances in Affinity Capillary Electrophoresis for Binding Studies

“…Therefore the use of EOF neutral marker (e.g., acetanilide [6]) is necessary to avoid the measurement errors that are coming from EOF fluctuation [4,8]. Accordingly, it is more accurate to describe the migration behavior of receptor or ligand as mobility ratio which can be expressed as shown in Equation 1 [4,9]. future science group Review Albishri, El Deeb, AlGarabli et al…”

“…Despite the presence of many techniques to study affinity parameters such as MS [11], nuclear magnetic resonance (NMR) [12], spectometry [13], Stop-flow [14] and HPLC [15], ACE offers advantages over the other techniques as we will comprehensively discuss in the next sections. ACE only requires a very small sample amount, provides high speed of analysis [16,17] and high efficiency [2], in addition to good precision [9], high selectivity and low cost [16]. In particular, the possibility to directly inject impure samples [4,6] and the ability to study mixtures of analytes in the same solution [18] facilitate the analysis of biological samples.…”

“…The first mode is referred to as pre-equilibrium [9] (also called nonequilibrium electrophoresis of preequilibrated sample mixture [2]). In this mode, the two species receptor and ligand are premixed in the sample vial before electrophoresis [1,2].…”

“…The second ACE mode is referred to as dynamic equilibrium [1,2], also called mobility shift [9]. In this mode one of the analysis species is located in the sample solution and the other in the electrophoresis separation buffer.…”

“…In this mode, the two species receptor and ligand are premixed in the sample vial before electrophoresis [1,2]. This mode requires the kinetics data of interaction (association constant K on and dissociation constant K off ) [1,9] in order to be able to investigate complex formation. It is, for example, suitable for analysis of DNA-related interactions (DNA with protein or small molecules) [1].…”