Chemistry and Biology of Proteins - F. Haurowitz 1953

The Role of Proteins in Immune Reactions
Complement

When an animal is immunized with foreign erythrocytes, Antibodies Are Formed that possess The ability to agglutinate erythrocytes similar to those used for immunization. The Antigens responsible for inducing these agglutinins are the so-called Blood group-specific substances that we mentioned earlier in Chapter XI. Agglutinins and other antibodies are not destroyed by heating to 56°, so agglutination is not disrupted when immune serum is incubated for 30 minutes at 56°. However, a significant difference exists between heated and unheated immune serum: when unheated serum is used, hemolysis is observed In addition to erythrocyte agglutination, whereas heated serum lacks any hemolytic activity. Hemolysis is caused by a thermolabile complex present in the serum, known as complement. A common source of complement is guinea pig serum, which contains a considerable amount of this complex.

Unlike antibodies, complement is not a substance specific to a particular antigen. The addition of guinea pig serum complement to an antiserum containing specific antibodies, but devoid of complement, results in the lysis of both various animal species' erythrocytes and Bacteria. The system in which lysis occurs comprises the following components in all such cases: 1) antigenic Cells or bacteria undergoing lysis; 2) specific antibodies produced by immunizing an animal, such as a rabbit or a horse, with the corresponding antigenic cells or bacteria; and 3) complement.

Complement is a complex of several Proteins possessing the specific ability to lyse cellular antigens in the presence of corresponding antibodies. It has been shown that complement consists of at least four components: the mid-piece, the end-piece, the third component, and the fourth component. The following abbreviated designations have been proposed for these components: C'1, C'2, C'3, and C'4. These components can be separated from one another by means of ammonium sulfate fractionation [126].

C'1 (the mid-piece) is a globulin-like substance that is salted out by 1.39 M ammonium sulfate. It constitutes approximately 0.6% of the Serum proteins and is an euglobulin with an isoelectric point near pH 5.1 [126]. C'1 contains 16.3% nitrogen, 0.1% phosphorus, and 2.7% carbohydrate; it is destroyed by heating. C'2 (the end-piece) is an albumin-like protein salted out by 2.0–2.2 M ammonium sulfate. By combining with C'4, C'2 forms a complex possessing euglobulin properties. This complex contains 14.2% nitrogen and 10.3% CARBOHYDRATES, and has an isoelectric point near pH 6.3–6.4; it accounts for approximately 0.18% of the serum proteins [126]. C'2 is destroyed by acids [126]. C'3 is inactivated by Yeast, and C'4 by ammonia.

It was discovered long ago that complement binds to the antigen-antibody complex. Due to the hemolysis that occurs in the process, minute amounts of complement can be identified. This assay represents the most sensitive method among all those used for detecting the Combination of an antigen with an antibody. Erythrocytes subjected to the action of the corresponding agglutinin combine first with C'1, C'2, and C'4, and only subsequently with C'3 [127]. Quantitative determination of each of these four components presents great difficulties because any one of them can only be detected in the presence of the other three components, and also because their concentration in serum is very low.

Heidelberger and his coworkers, by adsorbing complement with an antigen-antibody precipitate and determining the nitrogen content of the precipitate before and after complement adsorption, found that the total nitrogen content of complement amounts to 24–32 у per 1 ml of serum [128]. Using a gravimetric method, the author found that 1 ml of serum contains 150–200 у of complement [129]. It remains unclear why complement binds exclusively to the antigen-antibody complex rather than to the antigen or antibody separately, and why it combines with the antigen-antibody complex derived from rabbit, rat, pig, or sheep blood, yet fails to combine with an antigen-antibody complex containing horse, dog, or human antibodies [130]. As emphasized above, complement lacks species Specificity; human complement and guinea pig complement are mutually interchangeable [131].

The Mechanism of complement action has not yet been fully elucidated. The lability of complement, as well as the fact that Cell and bacterial lysis occurs in its presence, provides grounds to suggest that complement is enzymatic in nature [127, 129]. This is further supported by the observation that the hemolysis of a single erythrocyte requires only 6 ∙ 10⁴ molecules of complement, whereas the hemolysis of one erythrocyte by oleic acid salts necessitates 10¹⁰ molecules [129]. A single molecule of complement thus proves to be 100,000 times more active than oleic acid molecules.