Association of terminal complement proteins in solution and modulation by suramin

The association of terminal complement proteins was investigated by analyti cal ultracentrifugation and multi-angle laser light scattering. Native C8 a nd C9 formed a heterodimer in solution of physiological ionic strength with a free-energy change Delta G degrees of -8.3 kcal/mol and a dissociation c onstant K-d of 0.6 mu M (at 20 degrees C) that was ionic strength- and temp erature-dependent. A van't Hoff plot of the change in K-d was linear betwee n 10 and 37 degrees C and yielded values of Delta H degrees = -12.9 kcal/mo l and Delta S degrees = -15.9 cal mol(-1) deg(-1), suggesting that electros tatic forces play a prominent role in the interaction of C8 with C9. Native C8 also formed a heterodimer with C5, and low concentrations of polyionic ligands such as protamine and suramin inhibited the interaction. Suramin in duced high-affinity trimerization of C9 (K-d = 0.10 mu M at 20 degrees C) a nd dimerization of C9 (K-d = 0.86 mu M at 20 degrees C). Suramin-induced C8 oligomerization may be the primary reason for the drug's ability to preven t complement-mediated hemolysis. Analysis of sedimentation equilibria and a lso of the fluorescence enhancement of suramin when bound to protein provid ed evidence for two suramin-binding sites on each C9 and three on each C8 i n the oligomers. Oligomerization could be reversed by high suramin concentr ations, but 8-aminonaphthalene- 1,3,6-trisulfonate (ANTS(2-)), which mimics half a suramin molecule, could not compete with suramin binding and oligom erization suggesting that the drug also binds nonionically to the proteins.