Purification of rat fibrinogen and its constituent chains.
Rat fibrinogen was purified from rat plasma by using lysine-Sepharose chromatography, repeated precipitation with 25%-satd. (NH(4))(2)SO(4) and gel chromatography on Sepharose 6B. To minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding and the collected blood was treated with Trasylol and di-isopropyl phosphorofluoridate. A 95%-clottable preparation was obtained in 70-75% yield; it proved to be free of factor XIII and plasminogen. It showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and on disc electrophoresis in 8m-urea. Alanine was the only detectable N-terminal amino acid. After reduction and modification of the thiol groups, the material could be separated into three distinct chains (Aalpha, Bbeta and gamma) by pore-limit polyacrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulphate. The amino acid compositions of the whole fibrinogen and of the separated modified chains were determined. The molecular weights were 61000, 58000 and 51000 for Aalpha-, Bbeta- and gamma-chains respectively. Our results for the chains are in contrast with previous reports on rat fibrinogen [Bouma & Fuller (1975) J. Biol. Chem.250, 4678-4683; Stemberger & Jilek (1976) Thromb. Res.9, 657-660], in which no separation between Aalpha- and Bbeta-chains was achieved on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for 3h. Evidence is presented that this is probably due to Aalpha-chain degradation as a result of incomplete inhibition of proteolytic enzymes during the purification. Complete inhibition of proteolytic activities is essential in all steps of the present purification procedure.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel; D005176:Factor XIII / Q000032:analysis; D005340:Fibrinogen / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D005779:Immunodiffusion; D008297:Male; D051381:Rats
D000596:Amino Acids; D005340:Fibrinogen; D005176:Factor XIII