Employing sheep erythrocytes sensitized by antibody and the first and fourth components of complement (EAC'1,4), in such a manner as to prevent the development of immune adherence (I-A) reactivity during preparation, four separate substances required for the conversion of EAC'1,4,2 to the final damaged state (E*) were identified in whole guinea pig serum by cellulose chromatography, and tentatively termed C'3c, C'3b, C'3a, and C'3d.
I-A reactivity was induced in EAC'1,4,2 after interaction with only one of these four substances, C'3c. A detailed comparison of the effects of heat, hydrazine, low pH, freezing, absorption by immune complexes, and elution from cellulose columns indicated that this same substance which was capable of imparting I-A reactivity to EAC'1,4,2 was also essential for immune hemolysis. Other experiments showed that I-A-reactive cells prepared either by treating EA with different concentrations of whole C' at 0°C, or by treating EAC'1,4,2 with C'3c, underwent lysis by C'2 + C'3b + a + d in proportion to the amount of whole C' or of C'3c used to make the cells reactive in I-A. These data provide strong evidence that a single factor, C'3c, is required both for the conversion of EAC'1,4,2 to an I-A-reactive complex (EAC'1,4,2,3c) and for the lysis of EAC'1,4,2 by C'3b + a + d.
C'3c is the only one of the components studied which can induce I-A reactivity, and is the first to react with EAC'1,4,2. Formation of EAC'1,4,2,3c proceeds even at 0°C, but is much more rapid at elevated temperatures, showing a maximum in from 5 to 15 minutes at 37° or 30°C respectively. Prolonged incubation at these temperatures results in a decline in hemolytic reactivity without a noticeable effect on I-A. This loss was resolved into three phenomena: (a) a rapid loss of ability of SAC'1,4,2,3c to react with C'3b, presumably as a result of decay of the C'2 moiety in the complex, which is readily reversed by addition of fresh C'2; (b) a slow, irreversible spontaneous inactivation of SAC'1,4,2,3c; (c) a moderately rapid, irreversible inactivation of SAC'1,4,2,3c by some factor present in C'3c preparations.