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.

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Rabbit antibodies to bovine fibrinogen were used to study the antigenic activity of four cyanogen bromide peptides containing the disulfide regions of this molecule. In precipitation tests the highest activity was associated with the peptide F-CB3 which is exclusively derived from the a-chain. Reduction of the single disulfide bridge in peptide F-CB3 did not influence its serologic activity. Weaker reactions were observed with the N-terminal multichain peptide F-CB1. The antigenicity of peptide F-CB1 was not affected by removal of fibrinopeptides A and B but it was lost after reduction. The immunological activity of the multichain peptide F-CB2 was even less than that of peptide F-CB1 and the antigenic determinants were destroyed by reduction. A large fragment essentially composed of peptides F-CB1 and F-CB2 could be obtained by limited cyanogen bromide cleavage and showed considerably better immunological activity than peptides F-CB1 and F-CB2 together. Apparently no activity was associated with a mixture of small hydrophobic, disulfide-loop peptides tentatively called peptide F-CB4. The large loss of antigenic activity in cyanogen bromide digests of fibrinogen suggests that the disulfide-stabilized regions do not have an important role in maintaining conformational antigenic determinants of fibrinogen. Changes in noncovalently stabilized conformation requiring uncleaved chains is considered as a possible reason for the findings observed.Relatively little progress has been made towards a molecular understanding of the antigenic activity of the fibrinogen molecule, a protein which is among the most potent antigens in plasma. Recent investigations were mainly restricted to plasmin-derived fragments D and E and higher degradation products [l -71. A neoantigenic determinant could be defined on fragment D by Plow and Edgington [6,7] but is not found on the fibrinogen molecule itself. On the other hand antibodies to fibrinopeptides A and B [2,8] did not or only weakly cross-reacted with fibrinogen indicating large differences in their conformation relative to the original protein.In a different approach to the molecular antigenic properties, reduced and alkylated polypeptide chains of bovine fibrinogen were used [9]. Up to 50% of the antibodies in anti-fibrinogen sera reacted in precipitation tests with the a-and/or P-chain. Only a weak activity was associated with the y-chain. Since the reduced constituents did not carry all the immunological information of the entire molecule, the results suggested the presence of antigenic determinants depending on the native conformation.To examine the problem of conformational anL1-genic determinants in fibrinogen we considered the possibility that the major determinants may be associated with the disulfide regions of this protein. Cleavage of nonreduced fibrinogen by cyanogen bromide (CNBr) appeared as the method of choice to get appropriate fragments. By employing this technique a N-terminal disulfide knot was previously demonstrated in human fibrinogen by Blomback et al. [l...

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