Chemical and Immunological Properties of Reduced and Alkylated Polypeptide Chains of Bovine Fibrinogen

Abstract

The α, β and γ chains were isolated from reduced and carboxymethylated bovine fibrinogen by chromatography on CM‐cellulose. Electrophoretically pure polypeptide chains could be obtained as judged by three different methods. The chains were soluble in buffers at or above pH 8 but exhibited non‐covalent aggregation. The molecular weights of the α, β and γ chains were estimated in dodecylsulfate‐polyacrylamide gel electrophoresis as 62000, 58000 and 48000, respectively. Each chain differed considerably from the other in amino acid composition as well as in the fragment pattern obtained after CNBr treatment. The occurrence of unresolved α/β material suggested microheterogeneity of the individual chains. Sulfitolysis yielded, at least for the α chain, less pure products. Between 20% and 54% of the antibodies in rabbit antisera to native fibrinogen precipitated with the α and/or β chain. Absorption studies, gel precipitation and hemagglutination‐inhibition suggested a considerable antigenic homology of both chains. They could be clearly distinguished from the γ chain which reacted poorly in precipitation test but considerably better in passive hemagglutination. Cyanogen bromide cleavage did not destroy the serologic activity of the chains. These findings indicated that a considerable number of the antigenic determinants on fibrinogen are not dependent on intact conformation. However, antibodies specifically reacting with the native molecule occur as well.

Cyanogen bromide cleavage of bovine fibrinogen. Identification of a dimeric N‐terminal peptide and two other disulfide containing fragments

“…Bovine fibrinogen was purified by established procedures [7, 81 and was fmally lyophilized [6]. About 1 g was dissolved in 100 ml 70% formic acid, flushed with nitrogen and after addition of 3.2 g CNBr (sublimated before use) incubated for 4 hr at 30".…”

“…Ion-exchange chromatography was carried out at room temp. in the presence of 8 M or 6 M urea which was deionized prior to use [6]. For CM-cellulose a concave gradient (300 X 260 ml) from 0.005 to 0.1 M sodium acetate pH 5.3 was employed.…”

“…The amino acid composition was determined as described [6]. This method was used to quantitate the peptide content in the fractions obtained upon molecular sieve chromatography.…”

“…The disulfide content of this fragment, although very high, does not account for all the cysteine residues in the protein and suggests the existence of other large fragments in CNBr digests of fibrinogen which might have also a multiple chain structure. Several large CNBr peptides were at least indicated by studies on isolated polypeptide chains [6]. The interest in characterizing such fragments originates from our previous findings [6] that a considerable number of the antigenic determinants of fibrinogen require an intact disulfide structure.…”

“…Several large CNBr peptides were at least indicated by studies on isolated polypeptide chains [6]. The interest in characterizing such fragments originates from our previous findings [6] that a considerable number of the antigenic determinants of fibrinogen require an intact disulfide structure. For their better characterization respective CNBr peptides obtained from non-reduced bovine fibrinogen were isolated in the present study.…”