Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S.
C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein 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 molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight 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-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight 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 approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.
D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D004587:Electrophoresis, Agar Gel; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D017293:Protein S; D013696:Temperature
D000596:Amino Acids; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D046911:Macromolecular Substances; D017293:Protein S