Isolation and characterization of an active three-chain molecular species of bovine thrombin.
Partially purified bovine prothrombin was activated in half-saturated trisodium citrate seeded with thrombin, and the resulting thrombin was chromatographed on Amerblite IRC-50, followed by rechromatography on DEAE-Sephadex A-50. Five fractions, possessing both esterase and clotting activities, were partially isolated, but fraction VI was shown to be a pure three-chain active species with threonine, isoleucine and lysine, in 1:1:1 molar proportions as N-termini. The amino acid composition and C-termini of fraction VI were determinied. The molecular weights of the isolated chains, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 7300, 12000 and 19500 respectively. These data, when taken together with the amino acid sequence of the two-chain thrombin reported by Magnusson et al. (1975) [in Prothrombin and related Coagulation Factors, (Hember, H. C. & Veltkamp, J. J., eds.), pp. 25-46, Leiden University Press, Leiden], indicated that proteolysis occurred at the Arg(78)-Lys(79) peptide bond of the B chain of a precursor molecular species, thus converting this two-chain species into the three-chain active form described here.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D007532:Isoleucine / Q000032:analysis; D008239:Lysine / Q000032:analysis; D008970:Molecular Weight; D010443:Peptide Chain Termination, Translational; D011516:Prothrombin; D013912:Threonine / Q000032:analysis; D013917:Thrombin / Q000032:analysis / Q000302:isolation & purification*
D000596:Amino Acids; D007532:Isoleucine; D013912:Threonine; D011516:Prothrombin; D013917:Thrombin; D008239:Lysine