In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by ∼2 kcal/mol (1 cal≡4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the ‘fast’ form. Finally, we have used hirudin fragment 1–47 as a molecular probe of the conformation of thrombin recognition sites in the fast and ‘slow’ form. From the effects of amino acid substitutions on the affinity of fragment 1–47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.
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Research Article|
August 23 2005
Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin
Vincenzo De Filippis;
Vincenzo De Filippis
1
1Department of Pharmaceutical Sciences and CRIBI Biotechnology Center, University of Padua, via F. Marzolo 5, I-35131 Padua, Italy
1To whom correspondence should be addressed (email vincenzo.defilippis@unipd.it).
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Elisa De Dea;
Elisa De Dea
1Department of Pharmaceutical Sciences and CRIBI Biotechnology Center, University of Padua, via F. Marzolo 5, I-35131 Padua, Italy
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Filippo Lucatello;
Filippo Lucatello
1Department of Pharmaceutical Sciences and CRIBI Biotechnology Center, University of Padua, via F. Marzolo 5, I-35131 Padua, Italy
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Roberta Frasson
Roberta Frasson
1Department of Pharmaceutical Sciences and CRIBI Biotechnology Center, University of Padua, via F. Marzolo 5, I-35131 Padua, Italy
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Publisher: Portland Press Ltd
Received:
February 09 2005
Revision Received:
June 17 2005
Accepted:
June 23 2005
Accepted Manuscript online:
June 23 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 390 (2): 485–492.
Article history
Received:
February 09 2005
Revision Received:
June 17 2005
Accepted:
June 23 2005
Accepted Manuscript online:
June 23 2005
Connected Content
A commentary has been published:
Slow thrombin in solution
Citation
Vincenzo De Filippis, Elisa De Dea, Filippo Lucatello, Roberta Frasson; Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin. Biochem J 1 September 2005; 390 (2): 485–492. doi: https://doi.org/10.1042/BJ20050252
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