Rat-Fbg was purified from rat plasma using Sepharose-1ysi ne chromatography, repeated ammonium sulphate precipitation (35% saturation) and gel chromatography on Sepharose 6B.In order to minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding, and the collected blood was treated with Trasylol and DFP.A preparation was obtained, which was 95% clottable and showed a single band on SDS-poly-acrylamide gel electrophoresis. Alanine was the only detectable am i no-term i na 1 amino acid.After reduction and modification of the SH groups the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit Polyacrylamide slab-gel electrophoresis in sod i urn dodecyl sulphate. The amino acid composition of the whole Fbg and of the separated modified chains were determined. The molecular weights were 61,000, 58,000 and 51,000 for Aα, Bβ and γ-chains, respectively.In as far as the chains are concerned, our results are in contrast with the findings of Bouma et al. (J. Biol. Chem. 250(1975) 4678), who could not discriminate between Aα- and Bβ-chains in SDS-polyacry1 am i de gel electrophoresis. Evidence will be presented that this can be due to Aa-chain degradation caused by incomplete inhibition of proteolytic enzymes during the purification.It is concluded, that complete inhibition of proteolytic activities in all purification steps is essential to obtain native fibrinogen. Moreover, in contrast to the conclusions of Bouma rat-Fbg does not differ essentially from Fbg from other mammalian species.
THE PURIFICATION AND PARTIAL CHARACTERIZATION OF SEVERAL FORMS OF HOG RENIN SUBSTRATE