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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)


Biotechnologia Acta  Т. 14, No. 6 , 2021
P. 37-43, Bibliography 32, Engl.
UDC: 577.112.7: 612.115


Ye. M. Stohnii, A. V. Rebriev, O. V. Hornytska, O. Yu. Slominskiy, O. P. Kostiuchenko,
K. P. Klymenko, V. O. Chernyshenko

Palladin Institute of Biochemistry of of the National Academy of Sciences of Ukraine, Kyiv

Aim. One of the approaches for studying structure and functions of proteins is their limited proteolysis. Proteolytic fragments of macromolecules can preserve the biological activity and can be used for the study of their structural and functional peculiarities. Thus, the characterization of new proteolytic enzymes and determination of the specificity of their action can be of interest for exploration. In the present work, we focused on the action of protease from the venom of Gloydius halys halys on fibrinogen, the crucial protein of blood coagulation system.

Methods. Products of fibrinogen hydrolysis by protease from the venom of G. halys halys were studied by SDS-PAGE electrophoresis and western-blot analysis using monoclonal antibodies ІІ-5 Сand 1-5A targeted to 20–78 and 549–610 fragments of fibrinogen Aα-chain. Molecular weights of hydrolytic products were determined using MALDI-TOF analysis on Voyager DE PRO (USA). Sequence of hydrolytic products were predicted by «Peptide Mass Calculator» soft ware.

Results. SDS-PAGE showed that protease from the venom of Gloydius halys halys initially cleaved Аα-chain of fibrinogen molecule. Western-blot analysis confirmed that this protease specifically cleaves off fragment of C-terminal parts of Аα-chain with apparent molecular weight of 22 kDa. Cleaved fragment was identified by MALDI-TOF analysis as the 21.1 kDa polypeptide. "Peptide Mass Calculator" predicted that such a fragment corresponded to Аα414-610 residue of fibrinogen molecule. Thus, we showed that studied protease cleaved peptide bond AαK413-L414 with the formation of stable partly hydrolyzed fibrinogen desAα414-610.

Conclusions. The use of protease from the venom of Gloydius halys halys would allow obtaining the unique partly hydrolyzed fibrinogen     des Aα414–610 that is suitable for the study of structure and functions of fibrinogen αС-regions.

Key words: fibrinogen, limited proteolysis, protease, fibrin polymerization, hemostasis.

© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2021