Rotraut Gollwitzer

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

Considerable efforts have been made in the last ten years to characterize the individual polypeptide chains of fibrinogen obtained upon cleavage of disulfide bonds (reviewed in [I] ). Other chemical methods like cyanogen bromide (CNBr) treatment were employed far less frequently. Such an approach led to the discovery of a N-terminal disulfide knot in human fibrinogen by Blombick et al. [2]. The amino acid sequence of the three constituent chains was elucidated recently [3-S]. 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. For their better characterization respective CNBr peptides obtained from non-reduced bovine fibrinogen were isolated in the present study.

Isolation and immunological characterization of a disulfide loop region in human fibrinogen α chain

A large portion of the (Y chain could be isolated from cyanogen bromide (CNBr) cleaved bovine fibrinogen in the peptide F-CB3 which contains a single disulfide bridge [l] . Biochemical [2,3] and immunological [4,5] evidence indicated that large parts of the F-CB3 moiety should be located on the surface of the intact fibrinogen molecule. This agrees with previous observations (re, viewed in [6] ) on a particular lability of the cy chain during degradation of fibrinogen by plasmin. Characterization of structural and immunological prbperties of F-CB3 may therefore aid in a better understanding of the process of fibrinolysis. The present study describes the isolation of peptide F-CB3 from human fibrinogen which is similar in structure but not in antigenicity to the previously reported bovine counterpart. A radioimmune assay and other serological methods demonstrated lack of cross-reaction with fragments D and E known to be the stable end products of plasmic degradation.

LASER DIFFRACTION OF ORIENTED FIBRINOGEN MOLECULES

Fibrinogen is an extended protein, organized in a few characteristic domains, which possesses various sites for precise and strong interactions with enzymes, metals and neighboring fibrin( ogen) molecules. The overwhelming information accumulated in recent years about the chemical and immunological characteristics of the molecule and the elucidation of the complete sequence of human fibrinogen provided a more detailed understanding of the fibrinogenfibrin conversion and the accommodation of the different domains in the aggregation process. However, the overall structure of fibrinogen is still a controversial subject and several contradictory models have been discussed. The three-nodular structure proposed by Hall and Slayter in 1959 and confirmed in recent publications 2 s reflects best our knowledge of fragments obtained by enzymatic or chemical cleavage and of the refined primary structure. X-ray crystallographic analysis is unquestionably the method of choice to obtain a detailed three-dimensional structure. Cohen and coworkers 4, succeeded in producing crystals of partially digested bovine and rabbit fibrinogen. An X-ray structure analysis of these crystals has not yet been published. Electron microscopy has been shown to be a reasonable approach to gain information about the gross structure and especially the alignment of the single molecules in fibrinogen aggregates and in fibrin fibers.6-8 In this paper we present electron microscopic studies on needle-like (para)crystals of native human fibrinogen and describe the evaluation and interpretation of these electron micrographs by laser diffraction and by autocorrelation.

Immunochemistry of bovine fibrinogen—I. Immunogenic activity and diversity of antigenic determinants of reduced and carboxymethylated α,β and γ chains

Abstract Antisera were produced in rabbits against carboxymethylated α, β and γ chains of bovine fibrinogen. Quantitative precipitation results indicated a comparable immunogenicity of α chains, β chains and fibronogen but γ chains were less immunogenic. Distinct antibody fractions could be prepared from each antiserum by successive immunoadsorption with different antigens. Antibodies bound onto and eluted from a fibronogen adsorbent showed in a hemagglutination-inhibition assay equal reactivity for fibronogen and the respective carboxymethylated or aminoethylated component chains. Subsequent adsorption on reduced and aminoethylated fibronogen demonstrated a second antibody fraction in the sera which reacted with carboxymethylated or aminoethylated chains but not with fibronogen. Final adsorption on reduced and carboxymethylated fibronogen and/or albumin gave rise to a third antibody fraction which reacted only with carboxymethylated chains. The results were interpreted as indicating three groups of antigenic determinants in the polypeptide chains of fibrinogen: (1) surface determinants of fibronogen; (2) chain determinants which are masked in fibrinogen; and (3) determinants probably located around the alkylated cysteine residues. Antibodies to determinants shared by fibronogen predominated in antisera to α and β chains. Antisera to γ chains contained mainly antibodies to the latter two types of determinants.

Isolation and chemical characterization of reduced and aminoethylated polypeptide chains of bovine fibrinogen.

Purification of the individual polypeptide chains of the fibrinogen molecule is a task still encountered with some difficulties. Suliitolysis and separation of the Ssulfo derivatives on CM-cellulose has been initially applied for this purpose [ 1,2] . Incomplete cleavage and/or reoxidation caused the occurence of polymerized a-chains which is considered as a serious handicap of this procedure [3,4]. Reduction of fibrinogen by thiol reagents followed by alkylation with iodoacetic acid furnished a complete cleavage to the constituent chains [4-61. Although chromatographic separation of the alkylated chains could be demonstrated on CMcellulose the presence of probably degraded o-chains caused problems in achieving a good and quantitative separation of oand P-chains [7] . This seems to be of particular importance for bovine fibrinogen [4]. Yet, the alkylation of methionine residues is a disadvantage of this method especially when it is intended to perform further structural studies. This dif~culty may be circumvented by the use of less than stoichiometric amounts of the alkylating agent [6]. Alkylation of cysteine residues in proteins can also be accomplished by ethylene imine [8,9]. Application of this technique to bovine fibrinogen proved to have several advantages in the present study. The polypeptide chains could be essentially purified by two chromatographic steps. Characterization of the chains by their amino acid composition indicated more reliable values than hitherto reported for cysteine and methionine in preparations of bovine origin. 2. Materials and methods

Identification of a large fragment after limited cyanogen bromide cleavage of fibrinogen

The disulfide regions of the fibrinogen molecule are becoming increasingly characterized by the aid of fragments obtained upon cyanogen bromide (CNBr) or plasmin cleavage [l-lo]. An interesting relation between two distinct CNBr peptides (called F-CBl and F-CB2 in case of bovine fibrinogen, [ref. 41) and plasmic fragments E and D, e.g. final products of the proteolytic treatment, could be substantiated by chemical and immunological evidence [2,3,6,7,9]. Initial stages of plasmin digestion give rise to large fragments such as fragment X being composed of two fragments D and one fragment E [5-8, lo]. In the present study a similar large fragment was obtained after limited CNBr cleavage in dilute hydrochloric acid.

Immunochemistry of bovine fibrinogen—II.: Analysis by immunoadsorption of the antibody response against three large cyanogen bromide peptides

Abstract The specificity of rabbit antisera against three large cyanogen bromide peptides which account for 80% of the disulfide-bonded regions in bovine fibrinogen were characterized by immunoadsorption and passive hemagglutination. Antibodies to the α chain derived peptide F-CB3 showed complete cross-reaction with fibrinogen. The antigenicity of F-CB3 was not changed after reduction of the single disulfide bridge. Antibodies to the N-terminal, multi-chain peptide F-CB1 either cross-reacted with fibrinogen or were directed to antigenic determinanrs unique to reduced, unfolded chain constituents of fibrinogen. However, part of the antibodies against determinants shared by native fibrinogen were also able to react with its component chains, particularly with β and γ chains. Two antibody fractions with a similar specificity as in antisera to F-CB1 could be detected in antisera to the multichain peptide F-CB2. The β chain constituent in F-CB2 was important for antigenic activity. Antisera to F-CB2 contained in addition antibodies which did not cross-react with native or reduced fibrinogen.

Immunochemistry of bovine fibrinogen--III. Localization of two antigenic determinants in fibrinopeptide B.

Abstract The antigenic structure of bovine fibrinopeptide B was investigated by determining the antibody response against peptide F-CB1s, the reduced and alkylated s-chain component of the N-terminal cyanogen bromide fragment F-CB1 of bovine fibrinogen. In a radioimmunoassay, 125I labelled F-CB1s showed binding to rabbit antisera againsts both native fibrinogen and F-CB1. The reaction could be completely inhibited by unlabelled F-CB1β, while the α- and γ-chain components of F-CB1 showed essentially no reaction. A appreciable cross-reaction was, however, observed with F-Cb1β from human fibrinogen. Thrombin treatment split F-CB1β into two fragments of which the smaller fragment, Th1, (fibrinopeptide B. 21 residues) inhibited in the assay to about 70% compared with uncleaved F-CB1β, while the larger fragment Th2 (122 residues) inhibited only 20%. An equimolar mixture it Th1 and Th2 reproduced the FCB1β inhibition pattern indicating that the inhibitory activities were additive. Further, each of the two fragments obtained by trypsin cleavage of fibrinopeptide B retained about 50% of its inhibitory capacity. The results indicate the presence of least two distinct antigenic determinants on fibrinopeptide B which are shared by native fibrinogen. One determinant is restricted to the C-terminal hexapeptide sequence (Val-Gly-Leu-Gly-Ala-Arg) and does not extend over the thrombin cleavage site. Antigenic determinants unique to unfolded fibrinogen were found to residue only in the larger thrombin fragment Th2.