At least 3 complement factors were found necessary for the conversion of the thermolabile intermediate complex EAC'1a,4,2a to a thermostable state. One of these factors is the earlier described ß1C-globulin. The second, a heretofore unrecorded serum protein, ß1F-globulin. The third factor has not yet been defined as a discrete serum protein entity. Kinetic experiments indicated that ß1C reacted prior to ß1F, which in turn seemed to precede the third factor in the reaction sequence. Therefore, the 3 components were tentatively designated the third (C'3), the fifth (C'5), and the sixth (C'6) components of complement, respectively. A procedure was developed allowing the isolation of highly purified ß1C-(C'3) and ß1F-globulin (C'5) and of partially purified C'6.
With respect to its function in immune hemolysis, ß1F-globulin or C'5 was found to be closely dependent on the simultaneous presence of C'6. The hypothesis that C'5 and C'6 form a functional unit was supported by the finding that both components interact with each other in solution resulting in the formation of a complex. A similar complex was also found in fresh human serum.