Heterogeneity of clottable fibrinogen isolated from plasma by affinity chromatography.
Human fibrinogen was adsorbed on thrombin-activated fibrinogen which had been immobilized by covalent coupling with Sepharose-6B (Fibrin-Sepharose). Subsequent desorption with a buffer containing 1 M KBr yielded a protein which, after removal of KBr, showed a clottability of 83%. If the same procedure was applied to plasma, a fibrinogen-containing fraction with a clottability of 90-95% was obtained. In addition to fibrinogen, it comprised substances of higher and lower molecular weight, as shown by gel electrophoresis. Following adsorption on DEAE-cellulose at pH 8.8, several fractions were obtained by a stepwise elution technique with buffers of increasing molarity and decreasing pH. The first contained fibrinogen with partially degraded Aalpha-chains. It was followed by unaffected fibrinogen. In subsequent fractions, fibrinogen was associated with another protein which, in dodecylsulfate gel electrophoresis, migrated with a rate similar to that of the gamma-chains. The last fraction contained high molecular weight substances which, by reduction, yielded a relatively high molecular weight cleavage product and some subunits of lower molecular weight. Finally, a stepwise elution from Fibrin-Sepharose was elaborated to fractionate adsorbed plasma proteins. A fraction giving only a slight reaction with antifibrinogen was eluted with KBr-free buffer at 37 degrees C. Subsequent desorption with a buffer containing 1 M KBr removed fibrinogen of 83% clottability with only minute amounts of accompanying proteins.
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D005337:Fibrin; D005340:Fibrinogen / Q000302:isolation & purification; D006801:Humans; D005779:Immunodiffusion; D008970:Molecular Weight; D012685:Sepharose
D005337:Fibrin; D005340:Fibrinogen; D012685:Sepharose