Association of terminal complement proteins in solution and modulation by suramin.
The association of terminal complement proteins was investigated by analytical ultracentrifugation and multi-angle laser light scattering. Native C8 and C9 formed a heterodimer in solution of physiological ionic strength with a free-energy change DeltaG degrees of -8.3 kcal/mol and a dissociation constant Kd of 0.6 microM (at 20 degrees C) that was ionic strength- and temperature-dependent. A van't Hoff plot of the change in Kd was linear between 10 and 37 degrees C and yielded values of DeltaH degrees = -12.9 kcal/mol and DeltaS degrees = -15.9 cal mol-1 deg-1, suggesting that electrostatic 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 induced high-affinity trimerization of C8 (Kd = 0.10 microM at 20 degrees C) and dimerization of C9 (Kd = 0.86 microM at 20 degrees C). Suramin-induced C8 oligomerization may be the primary reason for the drug's ability to prevent complement-mediated hemolysis. Analysis of sedimentation equilibria and also of the fluorescence enhancement of suramin when bound to protein provided evidence for two suramin-binding sites on each C9 and three on each C8 in the oligomers. Oligomerization could be reversed by high suramin concentrations, but 8-aminonaphthalene-1,3,6- trisulfonate (ANTS2- ), which mimics half a suramin molecule, could not compete with suramin binding and oligomerization suggesting that the drug also binds nonionically to the proteins.
Saez
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Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City 64110, USA.
Thielens
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Bjes
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Esser
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D002152:Calorimetry, Differential Scanning; D002499:Centrifugation, Density Gradient; D003185:Complement C8 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D003186:Complement C9 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007834:Lasers; D008027:Light; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013498:Suramin / Q000737:chemistry* / Q000494:pharmacology; D013816:Thermodynamics
D003185:Complement C8; D003186:Complement C9; D003169:Complement Inactivator Proteins; D046911:Macromolecular Substances; D012996:Solutions; D013498:Suramin