The purification and characterization of subcomponent C1s of the first component of bovine complement

Abstract

Bovine C1s, a subcomponent of the first component of complement, was purified in good yield by a combination of euglobulin precipitation and ion-exchange and molecular-sieve chromatography. Approx. 10 mg can be obtained from 3 litres of serum, representing a yield of 11%. The C1s is obtained in zymogen form, with a mol.wt. of 85000-88000, determined by gel filtration and SDS/polyacrylamide-gel electrophoresis. It is haemolytically active when tested with human C1q and C1r. Activation can be achieved by incubation with human C1r, resulting in cleavage of the C1s chain into two chains of 65000 and 27000 mol.wt. and the generation of an isoleucine N-terminal residue on the smaller chain. Active C1s binds an equimolar amount of di-isopropyl phosphorfluoridate to the smaller chain, which is the C-terminal part in the zymogen. The chains can be separated by ion-exchange in 8 M-urea. All of these characteristics show that bovine C1s is very similar to its human counterpart.

The serine proteinase chain of human complement component C̅1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments

“…The reduced carboxymethylated chains of iPr2P-F-treated Cis were separated on DEAE-Sepharose CL-6B. The light chain was eluted with the starting buffer, and the heavy chain was elut-ed about half way up the gradient, in a manner very similar to that observed with bovine Clis (Campbell et al, 1979b). Both heavy chain and light chain were found to be labelled with 14C, but only the light chain contained 3H.…”

“…A combination of euglobulin precipitation and chromatography on DEAE-cellulose, Ultrogel AcA-34 and DEAE-Sephadex was used to purify Cls (Barkas et al, 1973;Campbell et al, 1979b;Ziccardi & Cooper, 1976). The purification schedule is shown in Scheme 1.…”

“…The Clr was added in four equal amounts at intervals of 30min to give a final CIr:CIs ratio of 1:25 (w/w). The degree of activation was checked by SDS/ polyacrylamide-gel electrophoresis under reducing conditions (Campbell et al, 1979b).…”

Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence

“…The sources of other chemicals have been described previously (Booth et al, 1979a,b;Campbell et al, 1979a,b;Nisbet et al, 1981). for 2 h. Conditions similar to these are known to cause activation of bovine C4 by C lr (Booth et al, 1979a) and to cause activation of human (Ziccardi & Cooper, 1976) and bovine Clr (Campbell et al, 1979b). Activated plasma was then frozen by storage at -200C for 4h, and allowed to thaw overnight at 40C.…”

“…The addition of 6aminohexanoic acid (1 M), a plasma carboxypeptidase B inhibitor (Vallota & Miiller-Eberhard, 1973) at the activation and following steps was not sufficient to prepare material containing the terminal arginine. This was achieved by purifying whole C4 (Booth et al, 1979a) followed by activation using Cls (Campbell et al, 1979b) in the presence of 1 M-6-aminohexanoic acid.…”