sepharose and 3-acrylamidophenylboronic-acid

The glycated albumin (G-alb) is a potential marker of hyperglycemia in diabetes and other neurodegenerative disorders in humans. G-alb's presence in the total human serum albumin (tHSA) is an important indicator in the timely diagnosis of disease. To identify G-alb content, it needs to be isolated from non-glycated albumin (NG-alb). Here, we present Capillary electrophoresis (CE) methods with 3-acrylamido phenylboronic acid (3-APBA) as an entrapped ligand in the agarose gel to develop agarose-3-APBA functional capillary and as an affinity ligand added to the buffer without agarose. 3-APBA was selected by computational virtual screening of several phenylboronic acid (PBA) compounds and other ligands to bind G-alb and separate from NG-alb selectively. The agarose-3-APBA functional capillary method involved agarose gel dilution approach coupled with injection pressure to obtain reduced viscosity and sufficient injection volume of protein samples. The method delivered separation in 9.7 min, with a resolution of 3.4, G-alb recovery up to 65%, and took 25 min to complete the entire process. The second method involved 3-APBA as an affinity ligand in the buffer and delivered separation in 4.2 min, with a resolution of 6.4, G-alb recovery up to 102% recovery, with relatively easy procedures. Therefore, it was further applied to determine G-alb content from tHSA in human serum and saliva. The G-alb found content in serum samples was in the range of 21. 1 ± ± 1.4% to 40.5  ± 1.6% out of tHSA and 25.1  ± 1.6% to 33.3 1.4% in saliva. The binding mechanisms were investigated by molecular dockings, which revealed hydrogen bonding, π-π, and van der walls interactions between 3-APBA and G-alb. The affinity was validated by affinity capillary electrophoresis (ACE), which revealed relatively strong interactions between 3-APBA and G-alb with the binding constant (K

Topics: Boronic Acids; Buffers; Electrophoresis, Capillary; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Hydrogen-Ion Concentration; Ligands; Molecular Docking Simulation; Reproducibility of Results; Saliva; Sepharose; Serum Albumin