Abstract
1. 1. The sedimentation behavior of clotting systems containing 4 g. fibrinogen/l. and 1 unit thrombin/ml. has been studied under various conditions of pH, ionic strength, and concentration of hexamethylene glycol. 2. 2. In glycol-free systems of pH below about 6.1, only one component is identified before the moment of clotting; its sedimentation constant is the same as that of fibrinogen and activated fibrinogen. At higher pH, a second faster component appears before the moment of clotting; its sedimentation constant is similar to that of the intermediate polymer previously identified in inhibited systems. The critical pH is increased slightly by increasing ionic strength. 3. 3. Below the critical pH, it is concluded that practically no polymerization of activated fibrinogen occurs before the moment of clotting. This has been confirmed at pH 5.68, ionic strength 1.72, by measurements of viscosity and light scattering. 4. 4. In the presence of hexamethylene glycol, either one (slow) or two (slow and fast) components are observed, depending on the pH and glycol concentration; their sedimentation constants are again the same as those of fibrinogen and the intermediate polymer, respectively. The critical pH for appearance of the fast component is at first lowered by addition of glycol, so that two components appear at pH 5.80, 0.3 M glycol, for example. Higher concentrations of glycol raise the critical pH, so that one component is observed, for example, at pH 6.25, 0.8 M glycol. 5. 5. The results are interpreted in terms of the effect of pH and ionic strength on the ionization of certain histidine residues which, it is postulated, must be uncharged to allow activated fibrinogen to form the intermediate polymer, and in terms of van der Waals' interaction of the glycol with the surface of the fibrinogen molecule, which at low concentrations impedes the aggregation of the intermediate polymer and at higher concentrations impedes the formation of the polymer itself.
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