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In the present work a scheme is presented for the isolation of multiple components of human complement in a functionally and biochemically pure state and with full hemolytic activity. These preparative procedures allow high recovery of milligram and gram quantities of particular complement components from a large pool (2-11 liters) of fresh EDTA plasma in no more than four chromatographic steps. Many components (C3bINA, C5, C3, C1EI, C4, and C9) are recovered functionally pure or highly purified following the first chromatographic step employing DEAE-Sephacel and may be utilized as reagents with no further purification. Prior to anion exchange, individual units of plasma are treated with inhibitors of complement activation and serum proteases, the pooled plasma is fractionated with polyethylene glycol, depleted of plasminogen on Sepharose-lysine, and rapidly ultrafiltered to low ionic strength and high protein concentration. The high degree of resolution of the components on DEAE-Sephacel subsequently obtained is demonstrated by the functional recovery and purification in a representative experiment as indicated (in their order of elution) for the following proteins: C3bINA (24%, 18-fold), C2 (74%, 12-fold), C7 (87%, 14-fold), factor B (55%, 8.7-fold), C8 (50%, 16-fold), C6 (82%, 25-fold), beta 1H (39%, 12-fold), C5 (62%, 111-fold), C3 (99%, 64-fold), C1EI (42%, 135-fold), C9 (80%, 297-fold), and c4 (78%, 164-fold). Other components separated by these procedures include C1q and C4 binding protein. Additional steps described, which demonstrate the utility and effectiveness of this preparative scheme, have allowed isolation of C3, C5, and C7 as pure components with full hemolytic activity as judged by functional, immunochemical, and physicochemical criteria. C8, also isolated as a homogeneous protein, was recovered with partial hemolytic activity. All these components were recovered in high yield and in the purification as indicated: C3 (61%, 103-fold), C5 (24% 1350-fold), C7 (19%, 2260-fold), and C8 (32%, 547-fold). Complement components C6, beta 1H, factor B, and C2 in addition to C3bINA, C1EI, C4, and C9 are recovered partially purified with good activity and are amenable to further purification.

The NH2-terminal structures of the intracellular precursor of the third component of guinea pig complement (pro-C3), synthesized in macrophage cultures, and beta chain of C3 from guinea pig plasma were determined. Six of the first 16 residues of pro-C3 were identified by a microradiosequencing method. All six are nonpolar and are identical with the residues in beta chain of native plasma C3. This establishes beta chain as the NH2-terminal subunit in pro-C3. A comparison of primary structures of guinea pig and human C3 beta chains revealed identity of at least 8 residues within the NH2-terminal decapeptide. Incubation of plasmin with radiolabeled pro-C3 in macrophage lysate resulted in loss of pro-C3 and the generation of a two-chain molecule electrophoretically indistinguishable from native C3. These data indicate similarities between pro-C3 and the precursor of the fourth complement component, pro-C4, with respect to subunit organization, presence of phylogenetically conserved NH2-terminal regions, and the probable mechanism for generation of their respective native proteins.

C4, 96 C3, 97 and C5 98,99 are synthesized as single polypeptide-chain proforms that are processed into multiple-chain structures. C4 circulates in plasma as a 3-chain (αβγ) structure; C3 and C5 are comprised of 2 chains each, α and β, of unequal size. Individual chains are held together by an undetermined number of disulfide bonds and by ionic and hydrophobic forces. The biological activities of C4, C3, and C5 are expressed and subsequently regulated through limited proteolysis. Preparative procedures described in this chapter allow for the isolation of each protein in a state of high biochemical and functional purity. The native proteins, their constituent chains, and activation fragments have been subjected to primary sequence analysis. These studies have contributed to a fuller understanding of events germane to activation-inactivation pathways for each protein. The binding of nascent C3b and C4b to foreign cell surfaces, and subsequent interactions with specific receptors present on phagocytic cells provides for a positive control of infectious processes. In recent years, the nature and mechanism of the reaction responsible for the binding of C3b (and C4b) to cell surfaces has received considerable attention from investigators in several laboratories. It has been proposed that the bond between these two proteins and surface components on red cell membranes or bacterial cell wall products may be an oxygen ester. The mechanism of covalent attachment could therefore be one of transesterification. Support for this hypothesis has derived from the identification of an internal thiolester bond in native C3. In addition to this protein, data are available suggesting the presence of a thiolester in α 2 M and C4, and the absence of such a bond in C5. This site is proposed to ( a ) contribute to the stabilization of the native conformation prior to activation by the respective converting enzyme, ( b ) serve as the source of an activated acyl group for a transesterification reaction, and ( c ) sustain the DIC reaction of the C3α- and C4α-chains. Although there is considerable homology in size and structure between C5 and C3, including the existence of single polypeptide-chain proforms and similar activation mechanisms, there are substantial differences between these two proteins. Native C5 does not appear to contain an internal thiolester, and the association between C5b and cell-membrane structures is not covalent in nature.