Journal archive > 2006 > N 1 January-February


L. P. Shvachko, S. V. Litvinovich, V.?K.?Kibirev

Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kyiv;
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Secondary structure and enzymatic properties of human ?-thrombin and its ?-form (obtaining during autolysis of the native enzyme) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). According to DSC-data both ?-thrombin and ?-thrombin contained only one thermal transition peak at 58.5 and 53.3 °C, respectively. A comparison of these values suggested that ?-form is less stable than initial ?-thrombin. In contrast to that the thermogram of DIP-?-thrombin had two peaks (57.5 and 64.5?°C). CD spectra showed that conversion ?- to ?-thrombin influenced the secondary structure of the enzyme slightly. The study of the inhibitory effect of such polyanions as ATP and dextran sulfate (DS) upon thrombin-catalyzed cleavages of fibrinogen has shown that the growth of the negative charge of the polyanion molecule resulted in the increase of its inhibitory activity. The catalytically non-active DIP-?-thrombin, which retained the native anion-binding exosite 1, was shown to decrease the inhibitory power of the dextran sulfate. It was explained by competition of DS with the exosite 1 of both ?- and DIP-?-thrombin. In contrast to that DIP-?-thrombin having exosite 1 destroyed neither competed nor influenced the anticoagulant capacity of dextran sulfate toward the native ?-thrombin.
In accordance with our data thrombin consists of two rather strong interacting domains. It was shown further that its anion-binding exosite 1 may play a significant role in the interaction of the enzyme with dextran sulfate.

Key words: thrombin, inhibitors of thrombin, ?-thrombin, secondary structure, diffe­rential scanning calorimetry, circular dichroism.

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