The elution characteristics from DEAE cellulose are presented for four components of guinea pig serum, which are capable of interacting sequentially with sheep erythrocytes sensitized with antibody and the first, fourth, and second components of complement (EAC'1,4,2) to cause immune hemolysis, and information is given regarding some of the properties of these components, termed C'3c, C'3b, C'3a, and C'3d. All can react in the presence of ethylene-diaminetetraacetate, and are non-dialyzable. C'3c is quite stable at 56°C, but is rapidly inactivated at low pH or by contact with hydrazine or ammonium hydroxide. C'3b is moderately heat-stable, quite susceptible to low pH, and less readily destroyed by hydrazine. C'3a is very heat-labile, but relatively stable at low pH, while C'3d is heat-labile, sensitive to low pH, and insensitive to bydrazine.

EAC'1,4,2 reacts with C'3c to form EAC'1,4,2,3c, which reacts then with C'3b to give the intermediate, EAC'1,4,2,3cb. The following reaction with C'3a yields EAC'1,4,2,3cba, which reacts finally with C'3d to give EAC'1,4, 2,3cbad (E*). The first and last reactions proceed moderately well at 0°C, but more rapidly at 30–37°C. The reaction with C'3b is almost completely inhibited at 0°C, while that involving C'3a proceeds almost as rapidly at 0°C as at higher temperatures. EAC'1,4,2,3cba cells have an increased fragility as compared with the other intermediate forms.

Depletion studies with purified fractions and appropriate intermediate complexes showed a high degree of depletion of C'3c, somewhat less of C'3b, and little or no depletion of C'3a from the fluid phase.

Examination of a beta1C globulin prepared from fresh human serum revealed high C'3c and C'3b activity, and very little C'3a or C'3d.

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