A catalytic role for heparin. Evidence for a ternary complex of heparin cofactor thrombin and heparin

  • Pomerantz M
  • Owen W
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Abstract

The interaction of heparin with chemically modified thrombin and heparin cofactor is studied. Amidinated heparin cofactor does not bind to heparin-agarose and the reaction rate of the amidinated inhibitor with unmodified thrombin is not affected by heparin. Likewise, thrombin modified with 1,2-cyclohexanedione does not bind to heparin agarose and the reaction rate of the modified enzyme with unmodified inhibitor is not affected by heparin. In the absence of heparin, the modified and unmodified proteins react at the same rate in all possible combinations. Affinity chromatography of diisopropylphosphoryl thrombin on heparin cofactor coupled to Sephadex G-50 is used to study the binding of heparin cofactor and thrombin to heparin. The thrombin for all experiments is tritium-labeled and then inactivated with diispropyl-fluorophosphate. Thrombin is not bound to heparin cofactor-Sephadex columns. However, after treatment of the columns with a heparin solution, thrombin binds tightly, and is eluted at high ionic strength. Bound thrombin can also be eluted with either excess non-radioactive thrombin or excess free heparin. Heparin-dependent binding of thrombin does not occur if the heparin cofactor-Sephadex is heat-denatured. The ability of heparin to couple solution-phase thrombin to solid-phase heparin cofactor indicates that a ternary complex is formed. Analysis of the binding of the proteins to heparin by a dye displacement method suggests that at least one site on heparin binds to thrombin but not to heparin cofactor. Further support for a catalytic role for heparin derives from the ability of catalytic concentrations of heparin to enhance the rate of hydrolysis of prothrombin by thrombin, another protein pair which bind mutually to heparin. © 1978.

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Authors

  • Mark W. Pomerantz

  • Whyte G. Owen

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