Ukrainian Antarctic Journal

No 1 (2020): Ukrainian Antarctic Journal
Articles

Purification and biochemical characterization of fibrino(geno)lytic enzymes from tissues of Antarctic hydrobionts

PDF
  • N. Raksha,
  • I. Udovychenko,
  • T. Halenova,
  • T. Vovk,
  • O. Savchuk,
  • L. Ostapchenko
N. Raksha
Education and Scientific Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, Kyiv, 01601, Ukraine
I. Udovychenko
Education and Scientific Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, Kyiv, 01601, Ukraine
T. Halenova
Education and Scientific Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, Kyiv, 01601, Ukraine
T. Vovk
Education and Scientific Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, Kyiv, 01601, Ukraine
O. Savchuk
Education and Scientific Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, Kyiv, 01601, Ukraine
L. Ostapchenko
Education and Scientific Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, Kyiv, 01601, Ukraine
DOI: https://doi.org/10.33275/1727-7485.1.2020.380
Published July 7, 2020
Keywords
  • fibrino(geno)lytic enzymes,
  • hemostasis,
  • Antarctic hydrobionts
How to Cite
Raksha, N., Udovychenko, I., Halenova, T., Vovk, T., Savchuk, O., & Ostapchenko, L. (2020). Purification and biochemical characterization of fibrino(geno)lytic enzymes from tissues of Antarctic hydrobionts. Ukrainian Antarctic Journal, (1), 69-81. https://doi.org/10.33275/1727-7485.1.2020.380
Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Abstract

Considering the continuing increase of morbidity and mortality rates associated with cardiovascular diseases, the search for novel compounds able to affect the hemostasis system is among the current trends of modern science and pharmacology. Fibrino(geno)lytic enzymes because of their role in dissolving blood clots as well as prevention of their formation attract special attention. The main goal of the current research was to develop the methodological approaches to obtain fibrino(geno)lytic enzymes from Antarctic hydrobionts and study their effects on the functioning of the hemostasis system. A complex approach which included affinity chromatography and size-exclusion chromatography was applied to isolate the fibrino(geno)lytic enzymes from the tissue of Antarctic nemertea (Parborlasia corrugatus), Antarctic sea urchin (Sterechinus neumayeri), and Antarctic sea star (Odontaster validus). The presence of proteolytic activity was monitored by zymographic technique. Fibrin(ogen)olytic activity was assessed by incubation of the samples with fibrinogen followed by 10% SDS-PAGE analysis. To test the substrate specificity of the enzymes, the chromogenic substrates such as H-D-Phe-Pip-Arg-pNA, pyroGlu-Pro-Arg-pNA, H-D-Val-Leu-Lys-pNA and Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA were used. The influence of fib rino(geno)lytic enzymes on platelet aggregation was assessed in platelet-rich plasma. To analyze the effect of the fibrino(geno)lytic enzymes on coagulation the blood coagulation time was assessed. The obtained results clearly indicated the presence of enzymes with activity toward fibrinogen in the tissues of tested hydrobionts. Based on the results of SDS-PAGE and zymography the molecular weight of the fibrino(geno)lytic enzymes was in the range of 26–34 kDa. The fibrinogen cleavage pattern analyzed by SDS-PAGE revealed the susceptibility of fibrinogen chains to degradation by enzymes from tissues of Antarctic hydrobionts. The fibrino(geno)lytic enzymes from all tested hydrobionts cleaved preferentially the Aα-chain and more slowly the Bβ-chain of fibrinogen. The fibrino(geno)lytic enzymes mediated the significant prolongation of blood clotting time in chronometric tests and inhibition of ADP-induced platelet aggregation. The enzymes exhibit activity against chromogenic substrates, which was more expressed in case of pyroGlu-Pro-Arg-pNA — a specific synthetic substrate for activated protein C and factor XIa. The enzymes isolated from the tissues of Antarctic marine hydrobionts possess a fibrin(ogen)olytic activity and can be of medical interest as therapeutic agents in the treatment and prevention of thrombotic disorders.

PDF

References

  1. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry, 72 (1-2), 248-254, 1976. https://doi.org/10.1016/0003-2697(76)90527-3
  2. Carson, M.A., Clarke, S.A.: Bioactive compounds from marine organisms: potential for bone growth and healing Marine Drugs, 16 (9), 340, 2018. https://doi.org/10.3390/md16090340
  3. Cortelazzo, A., Guerranti, R., Bini, L., Hope-Onyekwere, N., Muzzi, C., Leoncini, R., Pagani, R.: Effects of snake venom proteases on human fibrinogen chains, Blood Transfusion, 8 (3), 120-125, 2010.
  4. Fořtová, H.J., Dyr, J.E., Suttnar, J.: Isolation of a fibrinogen-converting enzyme ficozyme from the venom of Bothrops asper by one-step affinity chromatography on Blue Sepharose, Journal of Chromatography A, 523, 312-316, 1990. https://doi.org/10.1016/0021-9673(90)85035-T
  5. Gardiner, E.E., Andrews, R.K.: The cut of the clot(h): snake venom fibrinogenases as therapeutic agents, Journal of Thrombosis and Haemostasis, 6 (8), 1360-1362, 2008. https://doi.org/10.1111/j.1538-7836.2008.03057.x
  6. Grienke, U., Silke, J., Tasdemir, D.: Bioactive compounds from marine mussels and their effects on human health, Food Chemistry, 142 (1), 48-60, 2014. https://doi.org/10.1016/j.foodchem.2013.07.027
  7. Jo, H., Jung, W., Kim, S.: Purification and characterization of a novel anticoagulant peptide from marine echiuroid worm, Urechis unicinctus, Process Biochemistry, 43 (2), 179-184, 2008. https://doi.org/10.1016/j.procbio.2007.11.011
  8. Jung, W., Kim, S.: Isolation and characterisation of an anticoagulant oligopeptide from blue mussel, Mytilus edulis, Food chemistry, 117 (4), 687-692, 2009. https://doi.org/10.1016/j.foodchem.2009.04.077
  9. Kong, Y., Huang, S.L., Shao, Y., Li, S., Wei, J.F.: Purification and characterization of a novel antithrombotic peptide from Scolopendra subspinipes mutilans, Journal of Ethnopharmacology, 145 (1), 182-186, 2013. https://doi.org/10.1016/j.jep.2012.10.048
  10. Laemmli, K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1), 680-685, 1970. https://doi.org/10.1038/227680a0
  11. Laraba-Djebari, F., Chérifi, F.: Pathophysiological and pharmacological effects of snake venom components: molecular targets, Journal of Clinical Toxicology, 4 (2), 2014.
  12. Lindequist, U.: Marine-derived pharmaceuticals - challenges and opportunities, Biomolecules and Therapeutics (Seoul), 24 (6), 561-571, 2016. https://doi.org/10.4062/biomolther.2016.181
  13. Ostapchenko, L., Savchuk, O., Burlova-Vasilieva, N.: Enzyme electrophoresis method in analysis of active components of haemostasis system, Advances in Bioscience and Biotechnology, 2, 20-26, 2011. https://doi.org/10.4236/abb.2011.21004
  14. Raksha, N.G., Gladun, D.V., Vovk, T.B., Savchuk, O.M., Ostapchenko, L.I.: New fibrinogenases isolated from marine hydrobiont Adamussium colbecki, Journal of Biochemistry International, 3 (1), 9-18, 2016.
  15. Sanchez, E.F., Flores-Ortiz, R.J., Alvarenga, V.G., Eble, J.A. Direct fibrinolytic snake venom metalloproteinases affecting hemostasis: structural, biochemical features and therapeutic potential, Toxins, 9 (12), 392, 2017. https://doi.org/10.3390/toxins9120392
  16. Varetskaya, T.V.: Microheterogeneity of fibrinogen. Cryofibrinogen, Ukrainskii Biokhimicheskii Zhurnal, 32, 13-24, 1960 (in Russian)