Cohn, The F I And F I + Iii Fibrinogen Purification And Identification

Fibrinogen (Fg), also called Coagulation factorⅠ, is the biggest proportation ofcoagulation factors in human plasma. Thrombin was generated in the last step ofblood clotting, which converts Fg to fibrin monomer. Fg is the major component offibrin sealant used in surgery hemostasis.Currently, it brings a great deal of fractionⅠand fractionⅠ+Ⅲprecipitation during the Cohn Process. So it has very importantsignificance for the integrated utilization of plasma in theoretical research and actualapplication.Using a combination of Cohn ethanol fractionation, glycine precipitation andchromatography obtained highly purified fibrinogen with both biological andimmunological activity. Firstly, FractionⅠprecipitation or FractionⅠ+Ⅲprecipitation was extracted at a ratio of 3～4g:30ml extraction buffer and themixture was incubated with stirring for 90min at 4°C. The pH and ConcNaCl range ofthe storage buffer is 6.8～7.2, 0.6～0.8M and the buffer also comprisedNa2HPO4-acid citrate at a concentration of about 0.02M, after the Glycineprecipitation resolubilised, appearance of fragments was observed by SDS-PAGE andlow coagulation activity was detected by Jacobsson method in few hours. The storagebuffer could slow down that in 24 hours. The reseach indicated that after thepreparation of RGPS was completed, to maintain the activity of Fibrinogen, the nextprocess should be carried out. Seconlly, The further purification of Fg was carried outby ion exchange chromatography and hydrophobic interaction chromatography,respectively. The ion exchange matrixes were Q Sepharose High Performance (GE)and Q Sepharose XL (GE). The results indicated that the two matrixes were notsuitable for the purification of fibrinogen. The hydrophobic interactionchromatography (HIC) matrix was Butyl Sepharose 4FF (GE). The fibrinogen purifiedfrom Cohn FractionⅠprecipitation contained 85.6% clottable protein, the averageclotting time is 46s and the recovery rate was 78.5%, however, from Cohn FractionⅠ+Ⅲprecipitation, the purity was 67%, the average clotting time is 177s and therecovery rate was 60.6%. Finally, no degradation was observed after urokinase treatment by SDS-PAGE. The SDS-gel electropherogram showed Fg preparation onreduced and non-reduced conditions. On non-reduced condition, a broad bandappeared at the molecular size of 300～340kDa, and on reduced conditions the threesubunits Aα(68kDa), Bβ(54kDa) andγ(48kDa) appeared.