Cytochrome C Binds Complement 3b

The purpose of this study was to investigate the ability of cytochrome c (CytC) to bind to complement 3b (C3b). Using an enzyme‐linked immunosorbent assay (ELISA), bovine and equine CytC bound C3b with high affinity (K D ~ 10 ‐10 M) in low ionic strength buffers. This affinity decreased with increasing ionic strength; however, CytC still bound C3b with a K D ~ 10 ‐7 M in physiologic strength buffers. Western blot analysis indicated that CytC preferentially bound the α' chain (compared to the β chain) of C3b. SDS‐PAGE gels were used to monitor the effect of CytC on C3b degradation. These studies suggested that soluble CytC neither altered the degradative capacity of Factor H and Factor I towards C3b, nor did CytC behave as a cofactor itself in C3b degradation. A competition ELISA demonstrated that soluble CytC was unable to effectively compete for C3b's binding to immobilized CytC. In conclusion, immobilized CytC binds C3b with higher affinity in low ionic‐strength buffers than in physiologic buffers. CytC binds preferentially to the α' chain of C3b. Soluble CytC does not appear to significantly affect the cofactor‐mediated degradation of C3b nor the binding of C3b to immobilized CytC. It may be of functional significance that soluble CytC interacts with C3b differently than immobilized and thus possibly partially denatured CytC, since CytC switches from its native form to a partially unfolded conformation during its role in apoptosis. Thus, the interaction of complement with apoptotic cells may be regulated partly by the binding of C3b to the partially unfolded CytC present during apoptosis, but not to the native CytC normally found in mitochondria. Funded by the PNWU Research Seed Program.