Saez C, Thielens N M, Bjes E S, Esser A F
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City 64110, USA.
Biochemistry. 1999 May 25;38(21):6807-16. doi: 10.1021/bi990040j.
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.
通过分析超速离心和多角度激光散射研究了末端补体蛋白的缔合情况。天然C8和C9在生理离子强度溶液中形成异二聚体，自由能变化ΔG°为 -8.3 kcal/mol，解离常数Kd为0.6 μM（20℃时），该常数依赖于离子强度和温度。Kd变化的范特霍夫图在10至37℃之间呈线性，得出ΔH° = -12.9 kcal/mol和ΔS° = -15.9 cal mol⁻¹ deg⁻¹的值，表明静电力在C8与C9的相互作用中起重要作用。天然C8还与C5形成异二聚体，低浓度的聚离子配体如鱼精蛋白和苏拉明可抑制这种相互作用。苏拉明诱导C8发生高亲和力三聚化（20℃时Kd = 0.10 μM）和C9二聚化（20℃时Kd = 0.86 μM）。苏拉明诱导的C8寡聚化可能是该药物预防补体介导的溶血能力的主要原因。对沉降平衡以及苏拉明与蛋白质结合时荧光增强的分析提供了证据，表明在寡聚体中每个C9上有两个苏拉明结合位点，每个C8上有三个。高浓度的苏拉明可使寡聚化逆转，但模拟半个苏拉明分子的8-氨基萘-1,3,6-三磺酸盐（ANTS2⁻）不能与苏拉明竞争结合和寡聚化，这表明该药物也以非离子方式与蛋白质结合。
