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Limited Proteolysis of Fibrinogen by Fibrinogenase from Echis multisquamatis Venom

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Abstract

Previously we purified fibrinogenase from venom of Echis multisquamatis and showed that the enzyme predominantly cleaves BβArg42-Ala43 peptide bond of fibrinogen. A much slower hydrolysis of its Aα-chain was also shown. To evaluate the accessibility of the hydrolysis sites to fibrinogenase’s hydrolytic action, the pathway of cleavage of Aα- and Bβ-chains of fibrinogen, monomeric and polymeric fibrin desA and desAB has been investigated using western blot with monoclonal antibodies to Bβ 26–42 and Aα 20–78 of fibrinogen. The data indicated that the BβArg42-Ala43 peptide bond is available for cleavage in all forms of fibrin(ogen) with the exception of polymerized fibrin desAB. This is direct evidence of BβN-domain involvement in formation of protofibrils that makes it inaccessible to protease. The Aα-chain of fibrinogen remained intact after 3 min of incubation with fibrinogenase. Further incubation resulted in cleaving of the fibrin(ogen) αC-regions with the formation of two kinds of degradation products (~30 and ~60 kDa). In the case of monomeric fibrin desA or desAB we observed simultaneous hydrolysis of Aα and Bβ-chains and the cleavage of Aα-chain was more apparent for both forms of polymeric fibrin.

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Abbreviations

tPA:

Tissue plasminogen activator

SDS:

Sodium dodecyl sulphate

PAGE:

Polyacrylamide gel electrophoresis

TBS:

Tris-buffered saline

PBS:

Phosphate-buffered saline

HRP:

Horseradish peroxidase

TEMED:

Tetramethylethylenediamine

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Acknowledgments

Author gratefully acknowledges Dr Lyuba Mikhalovska for her help with N-terminal analysis of polypeptides and Prof. Eduard Lugovskoy for helpful advice on the choice of specific antibodies.

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  1. Protein Structure and Functions Department, Palladin Institute of Biochemistry, 9, Leontovych Str., Kiev, 01601, Ukraine

    V. O. Chernyshenko

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  1. V. O. Chernyshenko
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Correspondence to V. O. Chernyshenko.

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Chernyshenko, V.O. Limited Proteolysis of Fibrinogen by Fibrinogenase from Echis multisquamatis Venom. Protein J 34, 130–134 (2015). https://doi.org/10.1007/s10930-015-9605-2

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