Synthesis and use of new spin labeled derivatives of phosphorylcholine in a comparative study of human, dogfish, and Limulus C-reactive proteins.
New spin labeled derivatives of phosphorylcholine have been synthesized. The compounds cause reversible inhibition of the precipitation reactions between pneumococcal C-polysaccharide and the C-reactive proteins from humans, dogfish sharks (Mustelus canis), and horseshoe crabs (Limulus polyphemus). The spin labeled phosphorylcholine derivatives also rival phosphorylcholine as a ligand for the human, dogfish, and Limulus C-reactive proteins. The interactions of the purified C-reactive proteins with the spin labeled derivatives of phosphorylcholine have been studied using electron spin resonance spectrometry. The dramatic decrease in the ESR signal of some of the spin labels is due to immobilization of the label. Only the well known phosphate spin label, 4-phosphate-2,2,6,6-tetramethylpiperidine-1-oxyl could be used for binding studies on human and Limulus C-reactive proteins. Thus, by Scatchard analysis, the human C-reactive protein bound 1 mol of phosphate spin label per mol of protein with a Ka = 3.91 X 10(3) M-1, whereas the Limulus C-reactive protein bound only 0.5 mol of phosphate spin label per mol of protein with an overall Ka = 1.95 X 10(3) M-1. Inhibition studies using purified C-polysaccharide-induced inhibition of the phosphate spin label-human C-reactive protein interaction showed competitive inhibition with a KI of 4.78 X 10(-5) M at 18 degrees C. The phosphate spin label did not bind to dogfish C-reactive protein. However, one new phosphorylcholine spin label did bind and was used for Scatchard and Hill plot analyses. The dogfish C-reactive protein, which exists as a Mr = 50,000 dimer, bound 2 mol of the phosphorylcholine spin label per mol of protein, and this binding exhibited negative cooperativity as indicated by a Hill coefficient of 0.75.
D000818:Animals; D002097:C-Reactive Protein / Q000302:isolation & purification*; D002794:Choline / Q000031:analogs & derivatives; D004284:Dogfish; D004578:Electron Spin Resonance Spectroscopy; D006737:Horseshoe Crabs; D006801:Humans; D007700:Kinetics; D010767:Phosphorylcholine; D011485:Protein Binding; D013045:Species Specificity; D013113:Spin Labels
D013113:Spin Labels; D010767:Phosphorylcholine; D002097:C-Reactive Protein; D002794:Choline