STRUCTURE OF HUMAN THROMBIN IN RELATION TO AUTOLYTIC DEGRADATION

Human thrombin [EC 3.4.21.S] was obtained by activation of human prothrombin with venom of the Australian Taipan (Oxyuranus scutellatus scutellatus). This thrombin was precipitated with ammonium sulfate (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 study of 2340 Iowa units per absorbance unit .**GRAPHIC**. Thrombin proteins focused between 5 and 7, while prothrombin proteins focused to pH values less than 5. SDS-acrylamide gel electrophoresis indicated MW 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 phenylmethanesulfonyl 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 located 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. Human thrombin can apparently exist in at least 2 secondary structural forms, of different MW, probably due to autolytic degradation of the largest (39,000-dalton) form.