Sheep prothrombin: purification and partial characterization.

Abstract

A procedure for the preparation of highly purified sheep prothrombin is described. The purified zymogen, when subjected to disc gel electrophoresis in polyacrylamide, gave rise to one single band. Only alanine was found as N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. The isoelectric point, as determined by isoelectric focusing in polyacrylamide gel slab, was shown to be 4.9-5.0. Non-chromatographed, but not the purified zymogen, could be converted into active thrombin in half-saturated trisodium citrate seeded with thrombin. Pure sheep prothrombin showed 5.6% of neutral sugars and the following amino acid composition: Ala35, Arg44, Asx54-55, -Cys24, Glx72, Gly53-54, His8, Ile19, Leu45, Lys31, Met7, Phe23, Pro36, Ser34, Thr29-29, Trp16, Tyr19 and Val33, which accounts for a molecular weight of about 66 000 (amino acids only). The molecular weight as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after reduction by 2-mercaptoethanol, was shown to be 77 000 +/- 3000 (carbohydrates included).