Pressure assisted partial filling affinity capillary electrophoresis employed for determination of binding constants of human insulin hexamer complexes with serotonin, dopamine, arginine, and phenol

• Published in: Analytica chimica acta Vol. 1052; pp. 170 - 178
• Main Authors: Šolínová, Veronika, Žáková, Lenka, Jiráček, Jiří, Kašička, Václav
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 04.04.2019; Elsevier BV
• Subjects: Affinity; Affinity capillary electrophoresis; Amino Acid Sequence; Amino acids; Aqueous solutions; Arginine; Arginine - metabolism; Binding; Binding constant; Capillary electrophoresis; Capillary pressure; Cations; Coordination compounds; Dependence; Dopamine; Dopamine - metabolism; Electroosmosis; Electrophoresis; Electrophoresis, Capillary - methods; Expulsion; External pressure; Fused silica; Human insulin; Humans; Insulin; Insulin - chemistry; Insulin - metabolism; Insulin hexamer; Ligands; Molecular interactions; Neurotransmitters; Partial filling; pH effects; Phenol - metabolism; Phenolic compounds; Phenols; Pressure; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Serotonin; Serotonin - metabolism; Silica; Silicon dioxide; Sodium hydroxide; BGE; HI; Insulin hexamer; ACE; Partial filling; Binding constant; BI; Human insulin; AP; BSA; Neurotransmitters; PC; MD; Affinity capillary electrophoresis; PF-ACE
• ISSN: 0003-2670; 1873-4324; 1873-4324
• DOI: 10.1016/j.aca.2018.11.026

A new method, pressure assisted partial filling affinity capillary electrophoresis, has been developed to study noncovalent molecular interactions of the hexamer of human insulin (HI) with biologically relevant ligands, basic phenolic neurotransmitters serotonin and dopamine, basic amino acid arginine, and very weakly acidic phenol, in alkaline aqueous media. The apparent binding constants, Kb, of the HI-ligand complexes were determined from the dependence of the effective migration time changes of the above ligands on the variable zone lengths of HI hexamer dissolved in the background electrolyte (BGE) and hydrodynamically introduced into the bare fused silica capillary close to the UV detector. The strong cationic electroosmotic flow (EOF) in alkaline BGEs, 40/40 mM Tris/tricine, pH 8.1, and 25/34 mM NaOH/tricine, pH 8.5, with EOF mobilities 52.0 × 10−9 and 58.0 × 10−9 m2V−1s−1, respectively, was reduced by the hydrodynamic counter flow induced by external pressure at the outlet capillary end to avoid expulsion of HI zone out of the capillary and to allow HI interaction with both cationic and anionic ligands inside the capillary. The HI hexamer interactions with the above ligands were found to be weak to moderately strong, with Kb values in the range 385–1314 L mol−1, and decreasing in the order HI-phenol > HI-dopamine > HI-serotonin > HI-arginine. [Display omitted] •A new method, pressure-assisted partial filling ACE (PF-ACE), has been developed.•EOF velocity was regulated by pressure induced hydrodynamic counter flow.•PF-ACE was used to study human insulin (HI) complexes with low molecular mass ligands.•HI formed medium-strength complexes with serotonin, dopamine, arginine, and phenol.•Binding constants of the HI–ligand complexes were in the range 385–1314 Lmol−1.