Isolation And Characterization Of Fragments Of Human Fibrinogen Generated By Chemical Cleavage At Cysteine Residues

Human fibrinogen and its constituent S-sulfo chains were treated with 2-nitro-5-thiocyanobenzoic acid (NTCB) under conditions appropriate for cleaving on the amino-terminal sides of all cysteine residues. The fibrinogen or chains were first reduced with dithiothreitol (DTT) and the excess reducing agent removed by dialysis under nitrogen. The course of fragmentation was followed by SDS polyacrylamide gel electrophoresis and the purification of major fragments accomplished by gel filtration under a variety of solvent conditions. Our primary aim was to isolate and study the 112/113-residue polypeptides that exist between disulfide rings in the native molecule and that are thought to comprise the “coiled-coils” that connect the central and terminal domains. We also attempted to purify the other major fragments produced by NTCB-treatment, however, including 277-residue and 139-residue fragments from the α-chain, and a 108-residue β-chain polypeptide and a 144-residue fragment from the γ-chain. Of all of these, the 277-residue peptide from the α-chain is the easiest to isolate and was readily obtained in pure form. The 112/113-residue interdomainal connectors presented a number of technical problems with regard to their isolation, including a decided propensity to self-associate. Accordingly, we resorted to circular dichroism as a tool for following the residual α-helix that is an index of the presence of these segments, a technique we had planned to use to study the re-association of the separate segments. Preliminary evidence suggests that we were able to reform α-helical structures, presumably coiled- coils, to various degrees with different combinations of segments from the different chains.