New Fibrinogenases Isolated From Marine Hydrobiont Adamussium colbecki | Chapter 13 | New Insights on Chemical Research Vol. 1
Aims:
Enzymes that affect hemostasis have been isolated from different sources.
Fibrinogenolytic enzymes because of their role in dissolving of blood clots as
well as preventionof their formation have attracted special medical and
scientific attention. The main goal of current research was to analyze marine
hydrobionts (an example of the Antarctic scallop Adamussium colbecki) of the
Antarctic region on the presence of potential fibrinogenolytic enzymes.
Methodology: The fraction that
consists of fibrinogenases was obtained by affinity chromatography on Blue
Sepharose column. SDS-PAGE was applied for the determination of protein
composition in the obtained fraction. The proteolytic activity was monitored by
the zymographic technique. Fibrinogenolytic activity and activity toward
collagen were assessed by incubation of samples with fibrinogen or collagen,
respectively followed by SDS-PAGE analysis. To test for substrate specificity,
the fraction of fibrinogenases was incubated with p-nitroanilide chromogenic
peptides such as S-2366, S-2238, S-2251, S-2222. The fraction of
fibrinogenaseswas preincubated with protease inhibitors EDTA, PMSF, and
enzymatic activity was measured.
Results: The results clearly
indicated the presence of enzymes with activity toward fibrinogen in the
extract of A. colbecki. Zymography analysis detected the presence of active
enzymes in the region of 27-30 kDa. The
fibrinogen cleavage pattern was analyzed by SDS-PAGE under reducing conditions
has revealed time- and dose-dependent hydrolysis of fibrinogen. The
susceptibility of fibrinogen chains to proteolytic degradation by fibrinogenases
from A. colbecki was different. The enzymes preferentially hydrolyzed the
Aα-chain; they also cleaved the Bβ-chain but at a slower rate. Collagen was
found to be resistant to the action of the fibrinogenases under similar
experimental conditions. Fibrinogenases from A. colbecki hydrolyzed chromogenic
substrates as S-2238, S-2251, S-2222 and S-2366 but with different specificity.
Specific protease inhibitors PMSF and EDTA were used to identify the nature of
fibrinogenases present in the tissue of hydrobiont. On the basis of this
analysis, the fraction of fibrinogenases from A. colbecki consisted of serine
proteases as well as metalloproteases.
Author(s) Details
Nataliia Raksha
Educational and Scientific
Center “Institute of Biologyand Medicine”,Taras Shevchenko National University
of Kyiv, 64/13, Volodymyrska Str., Kyiv 01601, Ukraine.
Dmytro Gladun
Educational and Scientific
Center “Institute of Biologyand Medicine”,Taras Shevchenko National University
of Kyiv, 64/13, Volodymyrska Str., Kyiv 01601, Ukraine.
Tetyana Vovk
Educational and Scientific
Center “Institute of Biologyand Medicine”,Taras Shevchenko National University
of Kyiv, 64/13, Volodymyrska Str., Kyiv 01601, Ukraine.
Tetyana Galenova
Educational and Scientific
Center “Institute of Biologyand Medicine”,Taras Shevchenko National University
of Kyiv, 64/13, Volodymyrska Str., Kyiv 01601, Ukraine.
Oleksii Savchuk
Educational and Scientific
Center “Institute of Biologyand Medicine”,Taras Shevchenko National University of Kyiv, 64/13,
Volodymyrska Str., Kyiv 01601, Ukraine.
Lydmila Ostapchenko
Educational and Scientific
Center “Institute of Biologyand Medicine”,Taras Shevchenko National University
of Kyiv, 64/13, Volodymyrska Str., Kyiv 01601, Ukraine.
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