1/Amoleof all the phosphate monoestersproduced perceptibleinhibition and 10 emoles of these compounds produced practically totalinhibition of the precipitation reac-tion. The nonphosphorylated parent substances (glycerol, adenosine, and uridine) in equimolar amounts did not interfere with the precipitation of the CRP-C-polysaccharide complex. Cyclic 3', 5'-AMP, in which the phosphorus is not linked as a monoester, was not effective as an inhibitor. When the phosphate group was replaced by sulfate, eg, p-nitrophenyl sulfate, the compound was no longer inhibitory. Inasmuch as the reaction mixture contained an excess of calcium ion (18/imoles), it was unlikely that the inhibition produced by the phosphate monoester was due to chelation of the calcium ion necessary for the reaction of the pro-tein with the polysaccharide. As a further control, it was shown that the addition of 10, moles of EDTA had no inhibitory effect. Binding of phosphate monoesters and calcium ions by CRP: The above experi-ments did not establish whether the phosphate monoesters were interfering with the reaction by binding to the protein or to the polysaccharide. To explore this ques-tion further, the binding of phosphate monoesters to CRP was determined by gel filtration. Figure 1 shows the Scatchard plots of the data on the binding of UMP to CRP observed both at 50 and 25'at a calcium ion concentration of 0.010 Al1. A molecular weight of 24,000 for the subunit of CRP was used in the calculations. 4 Extrapolation to the abscissa yields a valence (n) of nearly one; ie, on the average, one mole of phosphate monoester can be bound per mole of subunit. Extrapola-tion to the ordinate yields nK, the product of the association constant and the valence for the reaction: