Eighth component of guinea pig complement: Purification and characterization

Abstract

A method for purification of the eighth component of guinea pig complement (C8) was developed. Twelve milligrams of C8 was obtained from 750ml of serum by a seven-step procedure consisting of removal of Cl by precipitation at pH 7.5, μ = 0.04, 2.0 M ammonium sulfate precipitation, removal of C5 by precipitation at pH 5.6, μ = 0.1, and successive chromatographies on CM-cellulose, DEAE-cellulose, hydroxylapatite and Sephadex G-200 columns. Using this method combined with procedures for purification of C5 [Kinoshita et al. (1981 b) J. Immun. 126, 2414–2418] and C9 [Tamura & Shimada (1971) Immunology 20, 415–425; Kinoshita et al. (1979) J. Immun. 123, 1989–1995], milligram amounts of these late-acting components could be obtained from a single batch of guinea pig serum. Purified C8 gave one protein band on disc polyacrylamide gel electrophoresis and produced monospecific antiserum in rabbits, showing γ 1 mobility on immunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction showed that C8 has a 3-polypeptide chain structure, the α-, β- and γ-chains having mol. wts of 60,000, 60,000 and 24,000, respectively. The α- and γ-chains are bound by disulfide bond(s) to form an α−γ subunit, which is linked noncovalently to the β-chain. The amino-terminal amino acids of the α- and β-chains are alanine and serine, respectively, but that of the γ-chain was not detectable by the dansyl method. C8 has a high content of cysteine (4.73 mole %). The α- and β-chains have the same mol. wt and similar amino acid compositions. These properties of guinea pig C8 were compared with those of human C8.