Purification and structural analysis of the fourth component of human complement

Publisher Website
Google Scholar
Abstract
The 4th component of human complement (C4) was purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which was depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on DEAE cellulose, QAE[quaternary ammonium ethyl]-Sephadex and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieved separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37.degree. C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A 3-chain structure for C4 was confirmed, and MW estimates of 93,000 .+-. 9300, 75,000 .+-. 7500 and 30,000 .+-. 3000 determined for the .alpha.,.beta. and .gamma. chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by Cl.hivin.s [activated S fragment of C1] and trypsin were accompanied by the fragmentation of the .alpha. chain. Inactivation of C4 by hydrazine produced no detectable change in chain size. Separation of the chains was acommplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains were performed, and N-terminal sequences of the latter established by automated Edman degradation.