Studies of the shape and calcium binding properties of fibrinogen and its degradation product fragment D
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The main aims of this work were to develop and test methods for the analysis of the shape and calcium binding properties of fibrinogen and its degradation product fragment D. The techniques of photo-sensitized labelling and cross-linking were used to obtain information about the shape of these proteins and both revealed interesting information. Photosensitized labelling proved particularly useful and results obtained using this technique suggested that fragment D (Ca++) in solution and the fragment D domains of fibrinogen are conformationally very similar. These studies also suggested that the A chain of fibrinogen is highly exposed at the surface of the molecule and that it shields other portions, particularly the D-domains of the molecule. The use of chemical cross-linking reagents revealed a gross-conformational difference between fragment D (Ca++) and fragment D (EDTA) and it was further shown that this difference is a result of cleavage of the chain of fragment D (Ca++) rather than calcium removal. This suggests that the calcium ion bound by fragment D (Ca++) exercises a protective influence over a plasmin susceptible bond and that cleavage of this bond allows a gross conformational change. This is consistent with the results from photo-sensitized labelling which showed the C-terminal portion of the chain of fragment D to the surface exposed. It was also shown that the technique of photo-sensitized labelling could be modified to induce cross-linking yielding a plasmin digestable product. These studies confirmed results from labelling studies which suggested that the chain C-terminii protect the N-terminal portion of fibrinogen. Calcium binding studies revealed two classes of high affinity calcium binding site in fibrinogen. Two sites of high (Kd > 10-5m) were shown to exist in the D-domains of the molecule, a third site of even higher affinity (Kd > 10-7m) was also found although the position of this site was not identified. These results of the study may be used to reconcile some of the more extreme views of fibrinogen shape as they suggest that fibrinogen is a protected trinodular structure.
Thesis, PhD Doctor of Philosophy
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