Factor Xa with the active site irreversibly blocked by the fluorescent inhibitor, Dansyl-EGRck. Several modified forms of factor Xa are also available including: A) active-site blocked factor Xa containing the tripeptide chloromethyl ketone inhibitor EGRck, and B) human Gla-domainless b-factor Xa. Participation of Factor Xa in Prothrombinase: Membrane bound factor Xa binds to membrane bound factor Va to form the prothrombinase complex. This complex effectively converts the zymogen prothrombin (II) to the active serine protease thrombin (IIa) by proteolytic removal of the fragment 1.2 (F1.2) portion of prothrombin. Activation of the zymogen, factor X, by either the intrinsic or extrinsic factor Xase complexes produces the active serine protease factor Xa (1,2). The activation of factor X requires proteolytic cleavage of the heavy chain, resulting in the release of an activation glycopeptide. The heavy chain region in factor Xa contains the serine protease catalytic domain, while the light chain, as in the zymogen, contains the membrane binding domain. Factor Xa participates in the prothrombinase complex, which catalyzes the rapid conversion of prothrombin to thrombin. Prothrombinase is an enzyme complex composed of factor Xa (enzyme) and factor Va (cofactor) assembled on a cellular surface in the presence of calcium ions. Although factor Xa can independently catalyze the activation of prothrombin, the rate at which this reaction occurs is increased nearly 300,000-fold with complete assembly of the prothrombinase complex. The clotting activity of factor Xa in vivo is terminated by either inactivation of the cofactor, factor Va, or by direct inhibition of factor Xa by inhibitors, such as ATIII, after disassembly of the prothrombinase complex. In recent years, molecular biologists have utilized factor Xa for site specific cleavage of fusion proteins expressed in bacteria (9-12). A factor Xa-sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with factor Xa. The factor Xa can then be easily removed by affinity chromatography. 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. Additional Specifications: Localization: Plasma Mode of Action: Enzyme component of the prothrombinase complex Extinction Coefficient: E1%1cm, 280nm=12.4 (7) Specific Activity: ~1000 units/mg Structure: Two subunits, Mr=16,500 and 28,800 (6), NH2-terminal gla domain, two EGF domains Percent Carbohydrate: 2.1 % (8) Post-translational Modifications: Eleven gla residues, one b-hydroxyaspartate
Molekulargewicht:
45300
Reinheit:
95%. Factor Xa is prepared by activating purified factor X with the factor X activator isolated from Russells viper venom. Factor Xa is purified from the activation mixture by chromatography over benzamidine-Sepharose followed by gel filtration (1,3). Purity is determined by SDS-PAGE analysis and activity is measured in a factor Xa clotting assay and/or chromogenic substrate assay.
Formulierung:
The enzyme is supplied in 50% glycerol/H2O. Another modified form of factor Xa is also available: Active site blocked Factor Xa containing the tripeptide chloromethyl ketone inhibitor EGRck.
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