The structure of human thrombin in relation to autolytic degradation.
Human thrombin was obtained by activation of human prothrombin with venom of the Australian Taipan (Oxyuranus scutellatus scutellatus). This thrombin was precipitated with ammonium sulphate (75% saturation) and subsequently purified by gel-filtration (Sephadex G-75), ion-exchange (CM-Sephadex C-50) and affinity (aminobenzamidine-CH-Sepharose) chromatography. The final preparation (affinity thrombin) had a specific activity of 2340 Iowa units per absorbance unit (A1cm280). Thrombin proteins focused between 5 and 7, while prothrombin proteins focused to pH values less than 5. SDS-acrylamide gel electrophoresis indicated molecular weights of greater than 70 000 for prothrombin and 39 000, 28 000, 25 000-23 000 and 15 000-13 000 for affinity thrombin proteins. The 39 000-dalton species predominated (greater than 90%) when the enzyme was inhibited with phenylmethanesulphonyl fluoride prior to dialysis for SDS electrophoresis. Lack of such inhibition reduced the amount of the 39 000-dalton species to less than 60% with concomitant increase of the smaller species. Peptide mapping studies indicated that the smaller species were structurally related to the 39 000-dalton species. The amino acid compositions of the histidine and/or tyrosine containing peptides indicated a high degree of homology with bovine thrombin. It has been established that human thrombin can exist in at least two secondary structural forms, of different molecular weights, probably due to autolytic degradation of the largest (39 000-dalton) form.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D006801:Humans; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011516:Prothrombin; D013045:Species Specificity; D013917:Thrombin / Q000302:isolation & purification
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011516:Prothrombin; D013917:Thrombin