Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S

Abstract

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and 2 subsequent chromatographic steps. Plasma C4b-binding protein .apprx. 80% was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a MW of 570,000, as determined by ultracentrifugation and was composed of .apprx. 8 subunits (MW .apprx. 70,000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citate adsorption. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis in nonreducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating 2 forms differing slightly from each other in MW and net charge. The protein band with the higher MW in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma and in a system with purified components, by an agarose-gl electrophoresis technique. Protein S was found to form a 1:1 complex with the higher MW form of C4b-binding protein, whereas the lower MW form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was .apprx. 0.9 .times. 10-7 M. Rates of association and dissociation at 37.degree. C were low, namely, .apprx. 1 .times. 103 M-1.cntdot.s-1 and 1.8 .times. 10-4-4.5 .times. 10-4 s-1, respectively. In human plasma, free protein S and free higher MW C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Both proteins (.apprx. 40%) existed as free proteins. A KD of .apprx. 0.7 .times. 10-7 M was calculated for the C4b-binding protein-protein S interaction.