Use of affinity chromatography for isolating prethrombin 1
Strukova, S.M.; Mitroshina, T.N.; Umarova, V.A.; Kudriashov, B.A.
Voprosy Meditsinskoi Khimii 27(6): 840-844
1981
ISSN/ISBN: 0042-8809 PMID: 7336661 Document Number: 169470
A procedure is described for isolation of prethrombin I using biospecific chromatography of the thrombin hydrolysate of prothrombin complex on heparin-Sepharose. The prothrombin complex was activated in 0.05 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, ImM EDTA-Na salt at 37 degrees within 15-20 min; the ratio of the enzyme and substrate was equal to 1 : 1300 (evaluated by the enzymatic activity). Then prethrombin I was isolated by the affinity chromatography. Prethrombin I was specially adsorbed on the immobilized heparin and eluted with 0.45 M NaCl. The substance was free from factor X and alpha-thrombin, which were eluted with 0.64 M and 0.8-1.0 M NaCl, respectively. Fragment I of prothrombin was not adsorbed on heparin-Sepharose and was found in the free volume of column. Preparation of prethrombin I (molecular mass 67,000 +/- 3,000) did not exhibit the coagulating, esterase and prothrombin activities but produced thrombin in presence of factor Xa. The abilities of prethrombin I to interact with blood vessel chemoreceptors and to activate the anticoagulation system were shown, when physiological activity of prethrombin I was studied using perfusion of rabbit carotid sinus, which was insulated from humoral action but retained the nervous connections with a body.