Simultaneous evaluation of ligand binding properties and protein size by electrophoresis and Taylor dispersion in capillaries.
The interplay between biophysical characteristics such as protein size and shape and protein function is difficult to ascertain using simple methods. Here, we present an approach for characterizing both protein-ligand binding as well as protein hydrodynamic radius in one operation combining electrophoresis and size measurement by dispersion using capillaries. The methodology is based on the integration of Taylor dispersion analysis and capillary electrophoresis and is here demonstrated using commercially available capillary electrophoresis instrumentation modified with a pixel sensor UV area imager, allowing two detection points along the capillary. Analytes are the human serum proteins alpha(1)-acid glycoprotein and albumin interacting with the drug propranolol in a frontal analysis mode. Upon introduction of the propranolol-protein sample, voltage is initially applied to facilitate electrophoretically mediated separation of ligand and protein and frontal analysis. Then a pressure mobilization step is used whereby Taylor dispersion can be characterized online based on the signal from the UV area imager. Estimates of ligand binding and values for hydrodynamic radii agree with values obtained by independent methods.
Østergaard
Jesper
J
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. joe@farma.ku.dk
Jensen
Henrik
H
D004058:Diffusion; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008024:Ligands; D008970:Molecular Weight; D009961:Orosomucoid / Q000737:chemistry / Q000378:metabolism; D011433:Propranolol / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D008024:Ligands; D009961:Orosomucoid; D011506:Proteins; D012709:Serum Albumin; D011433:Propranolol