Guidelines in selecting ligand concentrations for the determination of binding constants by affinity capillary electrophoresis.
This study examined various factors that affect the selection of ligand concentrations when using affinity capillary electrophoresis (ACE) for the determination of equilibrium constants in free solution. Two groups of model systems were used in this work: the binding of nitrophenols to alpha- or beta-cyclodextrin and the binding of D- or L-tryptophan to human serum albumin (HSA). Both systems gave 1:1 binding behavior in the ACE studies and good fits to previous equations derived to describe the shift in analyte mobility that occurs as the ligand concentration of the running buffer is varied. Some practical factors limiting the range of ligand levels that could be used in such studies included the relative amount of injected analyte, ligand solubility and the ligand's background signal. More fundamental factors included the size of the equilibrium constant for the system being investigated, the relative range of mobilities over which the analyte peak might be observed, the precision of the mobility measurements and the number of analytes present in the sample. Equations and graphs were developed for illustrating each of these latter items and their role in determining the range of ligand concentrations that could be used in ACE binding constant measurements. The results predicted by these equations and graphs showed good agreement with those observed experimentally, and should prove useful in optimizing ACE conditions for other solutes and ligands.
Bose
S
S
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Yang
J
J
Hage
D S
DS
D003505:Cyclodextrins / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008024:Ligands; D009596:Nitrophenols / Q000378:metabolism; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism; D013237:Stereoisomerism; D014364:Tryptophan / Q000378:metabolism
D003505:Cyclodextrins; D008024:Ligands; D009596:Nitrophenols; D012709:Serum Albumin; D014364:Tryptophan