A CAPILLARY ELECTROPHORESIS-BASED ASSAY FOR THE BINDING OF CA2-REACTIVE PROTEIN( AND PHOSPHORYLCHOLINE TO HUMAN C)

Affinity capillary electrophoresis was performed to quantitate the bin ding 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 co uld be examined under conditions of physiological ionic strength and p H. The values for the dissociation constants obtained here (K-D = 59 m u M for Ca2+-CRP and 18 mu M for the phosphorylcholine-CRP interaction ) were in close agreement with previous studies using gel filtration a nd equilibrium dialysis. As long as one of the reactants can be detect ed and recovered quantitatively in the capillary electrophoresis syste m, the method is generally useful to study interactions where complexe d molecules display an electrophoretic mobility that is different from that of unbound molecules and where the rates of association and diss ociation are sufficiently fast.