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Isolation and characterization of two new non-hemorrhagic metalloproteinases with fibrinogenolytic activity from the mapanare (Bothrops colombiensis) venom

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Abstract

Colombienases are acidic, low molecular weight metalloproteinases (Mr of 23,074.31 Da colombienase-1 and 23,078.80 Da colombienase-2; pI of 6.0 and 6.2, respectively) isolated from Bothrops colombiensis snake venom. The chromatographic profile in RP-HPLC and its partial sequence confirmed its high homogeneity. Both colombienases present fibrino(geno)lytic activity, but did not show any hemorrhagic, amidolytic, plasminogen activator or coagulant activities, and no effect on platelet aggregation induced by collagen or ADP. Both enzymes were strongly active on fibrinogen Aα chains followed by the Bβ chains, and colombienases-2, at high doses, also degraded the γ chains. This activity was stable at temperatures ranging between 4 and 37 °C, with a maximum activity at 25 °C, and at pHs between 7 and 9. The homology demonstrated by the comparison of sequences, with zinc-dependent metalloproteinases, as well as the metal chelant effects on, confirmed that the colombienases were metalloproteinases, particularly to α-fibrinogenases belonging to the P-I class of SVPMs (20–30 kDa), which contain only the single-domain proteins. The biological characteristics of the colombienases confer a therapeutic potential, since they contain a high fibrino(geno)lytic activity, devoid of hemorrhagic activity. These metalloproteinases might be explored as thrombolytic agents given that they dissolve fibrin clots or prevent their formation.

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Acknowledgments

The authors thank Zoila Carvajal, Amparo Gil, and Luis F. Navarrete for the technical assistance. Financial support was provided by the Science and Technology Foundation (FONACIT, Grant PG-2005000400), Venezuela; NCRR Viper Resource grant (P40 RR018300-09), Kingsville, TX, USA; and the Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

Conflict of interest

The authors have declared no conflict of interest.

Ethical statement

This research title was approved by the Instituto Venezolano de Investigaciones Científicas, Ethical Committee, and complies with the bioethical norms taken from the guide “Principles of laboratory animal care”. 1985. National Institute of Health of United States. Maryland USA, Publ. 85-23, 1–112.

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Authors and Affiliations

  1. Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico de la Universidad Central de Venezuela, Caracas, Venezuela

    María E. Girón & Alexis Rodríguez-Acosta

  2. Laboratorio de Fisiopatología, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 20632, Caracas, 1020A, Venezuela

    Ana María Salazar, Carlos Ibarra & Belsy Guerrero

  3. Department of Chemistry and the National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX, 78363, USA

    Elda E. Sánchez

  4. Department of Molecular Biology, Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC, H3S2C8, Canada

    Jacob Galán

  5. Laboratorio de Fisiopatología y Coagulación Sanguínea, Instituto Venezolano de Investigaciones Científicas, IVIC Apartado 20632, Caracas, 1020A, Venezuela

    Belsy Guerrero

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  1. María E. Girón
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Girón, M.E., Rodríguez-Acosta, A., Salazar, A.M. et al. Isolation and characterization of two new non-hemorrhagic metalloproteinases with fibrinogenolytic activity from the mapanare (Bothrops colombiensis) venom. Arch Toxicol 87, 197–208 (2013). https://doi.org/10.1007/s00204-012-0914-3

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