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.