A capillary electrophoresis-based assay for the binding of Ca2+ and phosphorylcholine to human C-reactive protein.
Affinity capillary electrophoresis was performed to quantitate the binding of Ca2+ and phosphorylcholine to human C-reactive protein (CRP). The assay requires no modifications of any of the molecules involved, uses minuscule amounts of protein (8.5 x 10(-15) mol per analysis, i.e., less than 1 pmol for 15 triplicate data points), and the binding could be examined under conditions of physiological ionic strength and pH. The values for the dissociation constants obtained here (KD = 59 microM for Ca(2+)-CRP and 18 microM for the phosphorylcholine-CRP interaction) were in close agreement with previous studies using gel filtration and equilibrium dialysis. As long as one of the reactants can be detected and recovered quantitatively in the capillary electrophoresis system, the method is generally useful to study interactions where complexed molecules display an electrophoretic mobility that is different from that of unbound molecules and where the rates of association and dissociation are sufficiently fast.
Heegaard
N H
NH
Peptide and Immunochemistry Unit, National Institute of Dental Research, NIH, Bethesda, MD.
Robey
F A
FA
D001797:Blood Protein Electrophoresis / Q000379:methods*; D002097:C-Reactive Protein / Q000032:analysis / Q000378:metabolism*; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010767:Phosphorylcholine / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding
D010767:Phosphorylcholine; D002097:C-Reactive Protein; D002118:Calcium