Formation and Characteristics of an Intermediate Complex EAC 1,4,2 in Immune Hemolysis by Carp Complement*¹

A hemolytic intermediate complex EAC1,4, 2 was prepared by incubating carp EAC1, 4 with hydrazine-treated carp serum, which is deficient in C4 and C3, at 0°C for 20 min. The difference between EACl, 4, 2 and EACl, 4 was shown by tests with inactivated carp sera. The former was hemolyzed by carrageenan-treated serum (Cn), ammonia-treated serum (Am) and heat-treated serum (H), whereas the latter was hemolyzed by Cn and Am, but not by H. EACl, 4,2 was unstable. Its half-life was about 15 min at 20°C and about 2.5 h at 0°C. Addition of Ca²⁺ to the buffer was not effective in preventing the deterioration of this complex. The reaction between EACl, 4 and Cy’ (a mixture of complement components other than Cl and C4) proceeded only in the presence of Mg²⁺, whereas the reaction of EACl, 4,2 with Cy’ proceeded without Mg²⁺. This indicates that the action of C2 requires Mg²⁺ while C3 acts without Mg,^{2 +}.