Incubation of fibrinogen with 0.5 mM dithiothreitol in the presence of .20 mM calcium chloride cleaved disulfide bonds located at: the N-terminal end of the Aα-chain (either Aα28-Aα28 or Aα45-γ23), the C-terminal end of the Aα-chain (Aα442-Aα472) and the N-terminal end of the γ-chain (either of the symmetrical γ8, γ9 disulfides or the Aα45-γ23 disulfide bond). In the absence of calcium ions two additional disulfides, γ326-γ339, and one in the N-terminal end of the γ-chain were reduced.Plasmin digestion of the reduced fibrinogens in buffers containing calcium chloride produced fragments D and E, except that smaller fragments of D were generated from the fibrinogen in which the γ326-γ339 disulfide bonds were reduced and alkylated. In these samples calcium did not protect the C-terminal end of the γ-chain from extensive digestion.Addition of thrombin to partially reduced and alkylated fibrinogen prepared in the presence of calcium gave a clotting time similar to control unreduced fibrinogen. However, when the γ326-γ339 disulfide bonds and another γ-chain disulfide bond most likely in the N-terminal region were cleaved in reduced fibrinogen prepared in the absence of calcium, the thrombin clotting time was extremely prolonged. Apparently the disulfide bonded structure supported by γ326-γ339 was important both for binding of calcium and also for normal clotting.
Essential Role of B-helix Calcium Binding Sites in Annexin V-Membrane Binding