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Journal archive > 2006 > N 6 November-December


M. V. Kolodzeiskaya, V. A. Sokolovsky, G. L. Volkov

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
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Stabilization of enzymes is a key factor when using biocatalysis in practice. Each enzyme stabili­ty depends both on the structure of its molecule and on the effect of various environmental factors, thus, one of the methods of the enzyme stability preservation is the formation of optimal macromedium. Thus, water structure and enzyme hydration change in the presence of solvable additives that affects its stability and catalytic properties. The paper deals with a new method of stabilization of human thrombin developed by the authors. It is proposed to use some known organic ligands which have ion group and different nonpolar hydrophobic groups instead of traditional additives (salts, aminoacids, polyols, polyethylene glycols etc.). Thrombin stabilization proceeds in the conditions something changed compared with traditional ones. Processes of thrombin stabilization by the above compound have been investigated, enzyme stability at different temperatures and long-term storage of diluted solutions of the preparation in different conditions have been studied. It has been established that rosselin and orange II are the most efficient ligands. Optimal finite concentrations of stabilizing agents make ~0.0012-0.0014 M which are rather low in the system thrombin–ligand. It has been found that diluted solutions of thrombin are more stable, than  concentrated ones. In the latter case the process of autolysis is included that affects negatively the catalytic effect of the enzyme, as far as there occurs the change of thrombin molecule structure, especially of thrombin ?-chain sections, evoking conformational changes of some sites of its active centre. The experiments directed to increasing thrombin intensity in the presence of organic ligands­ rosselin and orange II are discussed in details. Special attention is given to autolytic method of thrombin inactivation. It is admitted on the basis of already obtained data that thrombin binding with organic ligands proceeds at the expense of anionic area of ?-domain of thrombin active centre where basic aminoacids arginin and lysine (Lys 68, Arg 78, Arg 77, Arg 66 etc.) were found. Under these conditions the hydrophobic interaction is provided at the expense of apolar binding of thrombin active centre area.

Key words: thrombin, denaturation, stability, stabilization, organic ligands.

The original article in Russian is available for download in PDF format.

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