Alpha-thrombin is a highly specific serine protease generated by proteolytic activation of the zymogen prothrombin (1). Purified forms of a-thrombin have been shown (2-4) to undergo autolysis upon long term storage to less active forms. Similar inactivation was observed upon limited digestion of a-thrombin with trypsin (5). These proteolyzed forms of a-thrombin have been termed b-thrombin and g-thrombin. b-thrombin is produced by the cleavage of the Arg-70 or the Arg-73 bond in the thrombin B-chain. Another form of proteolyzed thrombin, termed b-thrombin is formed by the single cleavage of thrombin at Arg-154. g-Thrombin is produced by proteolytic cleavage at both of these sites (Arg70/73 and Arg-154) in the B-chain. These cleavages cause release of peptides that are no longer covalently attached to the thrombin molecule, but remain associated through ion exchange and gel filtration chromatography. These proteolyzed forms of thrombin retain their ability to cleave small synthetic substrates (6,7) and some protein substrates such as factor XIII (8), antithrombin III (9) and prothrombin (10). Their ability to clot fibrinogen (11), cleave thrombospondin (12) or activate protein C (9) have been markedly decreased. Proteolyzed Forms of a-Thrombin: b-Thrombin is generated by cleavage at Arg-70/73 in the thrombin B-chain, giving rise to the B1 and B2 peptides. g-thrombin is generated from b-thrombin by the additional cleavage at Arg-154 of the B-chain, giving rise to the B4 and B5 peptides from the B2 peptide. Mode of Action: Proteolyzed forms of thrombin which retain activity toward small substrates, factor XIII and prothrombin, but have reduced activity toward fibrinogen, protein C activation and antithrombin III binding. Extinction Coefficient: E1%1cm, 280nm=18.3 Structure: Three chains (A, B1, B2), disulfide link between the A and the B2 chains. Storage and Stability: May be stored at 4C for short-term only. For long-term storage, store at -20C. Aliquots are stable for at least 6 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Molecular Weight:
35400
Purity:
95% by SDS-PAGE. Human b-thrombin is prepared from purified a-thrombin by limited proteolysis with TPCK-treated trypsin, essentially by the method of Braun et al. (5). Purity is assessed by SDS-PAGE and activity is assessed using a fibrinogen clotting assay.
Form:
The proteolyzed form of alpha thrombin is supplied as a liquid in 100mM sodium phosphate, pH 6.5.
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