Two Activation Steps of Carp Complement Requiring Ca²⁺and Mg²⁺and an Intermediate Product in Immune Hemolvsis

The action of carp complement on sensitized cells (EA) proceeds in two steps requiring Ca²⁺and Mg²⁺, respectively. In order to decide which of these steps precedes in immune hemolysis, we carried out a kinetic experiment by use of a buffer and carp serum depleted of cations by passage through Amberlite IRC-50. In a reaction mixture composed of EA (sheep red blood cells sensitized with carp antibody), carp complement, optimal Ca²⁺and optimal Mg²⁺, the hemolysis took place after a lag period of about 10 min, but when Ca²⁺was present at zero-time and Mg²⁺was added 20 min later, there was almost no lag. On the contrary, when Mg²⁺was present at zero-time and Ca²⁺was added 20 min later, there was a lag of about 20 min. These observations indicate that the step requiring Ca²⁺precedes that in which Mg²⁺functions. A hemolytic intermediate complex, which was provisionally designated EACx’, was prepared by treating EA with carp complement in the presence of Ca²⁺, but with the absence of Mg²⁺at 20°C for 10 min. Furthermore, carp serum free from Cx’, which was provisionally designated Cy’, was also obtained by treating resin-passed carp serum with excessive EA in the presence of Ca²⁺but absence of Mg²⁺at 0°C for 3 h. It was confirmed that the cells in the state EACx’ were completely lysed when treated with a sufficient quantity of Cy’ and optimal Mg²⁺, and that Cy’ did not react with EA even in the presence of optimal Mg²⁺.