Single-Chain C4 from Human Plasma

Serum C4 has been shown to be composed of three disulfide-linked nonidentical subunits. Recently, Hall and Colten (PNAS 74, 1707, 1977) achieved cell-free synthesis of guinea pig C4 and thereby demonstrated the existence of a single-chain form of C4 (Pro-C4). We now wish to report the isolation of Pro-C4 from human plasma. Plasma was obtained from fresh, unclotted blood and plasminogen removed by affinity chromatography. Crude total serum C4 was obtained by adsorption to and elution from anti-C4 Sepharose 4B. Gel filtration on Sepharose 6B in 6 M guanidine hydrochloride (GuHCl) and 0.6 M ε-aminocaproic acid removed contaminants. Subsequent filtration on Sepharose 6B in 6 M GuHCl and 15 mM Dithiothreitol (DTT) separated single-chain C4 from the three C4 subunits. Identification of Pro-C4 rests upon its similarity to three-chain C4 with respect to molecular size, amino acid composition and antigenicity. Specifically, comparison of the amino acid composition of both forms revealed them to be indistinguishable. Secondly, radiolabeled Pro-C4 migrates identically to labeled three-chain C4 upon SDS PAGE in 5% gels. Pro-C4 remained a single chain in SDS, both in the presence and absence of reducing agent, and could be visualized when stained for carbohydrate. Thirdly, γ-globulin (γG) fractions from anti-C4, or antisera produced to the α- or γ-chain of C4 quantitatively reacted with radiolabeled, renatured Pro-C4. Reactivity of Pro-C4 with antiserum to the β-chain has not yet been determined. Renaturation was brought about by dialysis of samples in 6 M GuHCl and DTT against 1 M GuHCl, followed by addition of Triton X-100 to 0.14% (v/v) and dialysis to remove residual GuHCl. Nonimmune γG was ineffective in bringing about Pro-C4 precipitation. Single-chain C4 was found to constitute 1-3% of the total serum C4 on the basis of yield. Isolation of Pro-C4 from plasma now allows resolution of questions as to the mechanism of its postsynthetic proteolytic processing and as to the physical order or α-, β-, and γ-chains in the single-chain Pro-C4 molecule.