Characterization of the interaction of human C4b-binding protein with physiological ligands.

The binding of C4b to C4b-binding protein (C4BP) was demonstrated at physiological ionic strength by analytical ultracentrifugation. The sedimentation rate of C4BP gradually increased from 9.4 S to a maximum of 18.5 S with increasing C4b concentration. The stoichiometry of different C4BP X C4b complexes was calculated from the sedimentation-velocity data. A linear relationship was established between the number of C4b bound per C4BP and the sedimentation rate of the complex. In order to define further the C4BP-C4b interaction, sucrose density gradient ultracentrifugation was also used. Trace amounts of 125I-C4BP were centrifuged through 12 sucrose density gradients, each of which contained a different concentration of C4b throughout the gradient. The sedimentation rate of the C4BP increased with increasing C4b input to a maximum of 19.5 S. These binding data, in conjunction with the stoichiometry measurements determined in the analytical ultracentrifuge, were analyzed by the methods of Scatchard and Hill. At physiological ionic strength, C4BP exhibited four binding sites for C4b, each having an association constant of 1.2 X 10(7) M-1. A Hill coefficient of 1.1 was calculated, indicating that the four binding sites were independent. At reduced ionic strength, two additional sites were detected. The sedimentation coefficient of C4BP(C4b)6 was 24 S. The hydrodynamic data suggest that after four C4b molecules have bound to C4BP, the binding of additional C4b is sterically hindered. This interpretation implies that all six binding sites on C4BP are identical. C4BP also bound C4(H2O) (the product resulting from spontaneous hydrolysis of the thiol ester bond in native C4) and weakly bound C4c, but had no measurable affinity for native C4 or C4d at physiological ionic strength. A low-affinity interaction between C3b and C4BP was also demonstrated in the analytical ultracentrifuge. The C4BP X C3b complex was specific because C4BP mediated the cleavage of C3b by Factor I to C3bi with concomitant dissociation of the complex.