The C3b INA-dependent breakdown of fluid phase C3b has been shown to have an absolute requirement for a second factor. This factor is contained in catalytic amounts (with respect to C3b) of highly purified β1H. β1H alone does not cause proteolysis of C3b. In the presence of C3b INA and β1H, proteolysis of the larger‡ polypeptide chain (116K) of C3b occurs. Initially, a single scission gives two chains of 68K and 46K. A subsequent split of the 46K chain yields a smaller product of 43K. All of these chains remain covalently bonded to the β chain of C3b. These initial reactions are the same whether purified components or whether low concentrations of serum as a source of C3b INA and β1H are used. Pre-treatment of C3b, C3b INA and β1H with DFP has no effect on these events.

Further protelysis of the 68K. chain requires a DFP-sensitive protease and leads to the formation of an additional high molecular weight breakdown product of C3. This has the same polypeptide chain composition as the high molecular weight breakdown product of C3 purified from aged serum, and is therefore identified as C3c.