Solution-phase equilibrium binding interaction of human protein S with C4b-binding protein.
Solution-phase equilibrium binding studies of human protein S (HPS) and C4b-binding protein (C4BP) were undertaken using purified components. Free C4BP was measured in solutions at equilibrium by using HPS immobilized on a solid phase, coupled with an antibody detection system. Disruption of the solution-phase equilibrium was minimized by using a brief (15 min) exposure to the solid-phase HPS. These studies yielded an equilibrium dissociation constant (Kd) approximately 6 x 10(-10) M and a stoichiometry of approximately 1.7 molecules of HPS bound to each molecule of C4BP. This Kd is between 27-fold and 930-fold lower than previously published values obtained by using solid-phase and nonequilibrium methods. Equilibrium was achieved in solutions containing low nanomolar concentrations of both HPS and C4BP in less than or equal to 1 h at 37 degrees C, suggesting a rapid association rate constant for the interaction. Thrombin cleavage of HPS had no effect on the observed binding parameters. The binding interaction between HPS and C4BP appears to be partly calcium dependent, since in the presence of EDTA the Kd was increased to about 6 x 10(-9) M, with no change in the stoichiometry. This high-affinity binding interaction between HPS and C4BP, whose Kd is more than 500-fold lower than the proteins' plasma concentrations, heightens the apparent physiologic importance of complex formation.
Nelson
R M
RM
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
Long
G L
GL
D001798:Blood Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D015935:Complement C4b / Q000378:metabolism; D003169:Complement Inactivator Proteins; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017293:Protein S; D012996:Solutions; D013917:Thrombin / Q000378:metabolism
D001798:Blood Proteins; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017293:Protein S; D012996:Solutions; D015935:Complement C4b; D013917:Thrombin