Analysis of reptilase and thrombin-induced changes in fibrinogen subunits by isoelectric focussing

Isoelectric focussing (IEF) in 6M urea/4% polyacrylamide gel of reduced human fibrinogen and the fibrins formed by Reptilase and thrombin gave a complex multibanded pattern. Similar results were obtained on IEF in urea/agarose gels containing excess mercapto-ethanol. To characterize these multiple bands on the basis of molecular weight secondary electrophoreses into a discontinuous SDS-polyacrylamide gel system were carried out perpendicular to the initial IEF's. At least five major Alpha chains ranging in pI from 7.14 to 7.60 were found in fibrinogen. These were shifted 0.40 pI units towards the cathode after the action of Reptilase or thrombin due to the release of electronegative fibrinopeptide A species. A similar change occurred with the three chain B beta chains with initial pI's of 6.81 to 7.13, but only after thrombin action. Gamma dimer showed no significant difference in pI from its component chains 6.05 to 6.17.