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Purification and Characterization of a New Weak Hemorrhagic Metalloproteinase BmHF-1 from Bothrops marajoensis Snake Venom

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Abstract

BmHF-1, from the venom of Bothrops marajoensis, was purified by Sephadex G-75 and HPLC-RP on μ-Bondapak C-18 column chromatography. It presented a molecular mass of 27162.36 Da determined by MALDI-TOF MS. BmHF-1 had a sequence of 238 residues of amino acids. The multiple alignment of its amino acid sequence and those of other snake venom metalloproteinases showed high structural similarity, mainly among P–I class. The enzyme initially cleaves the Aα-chain of fibrinogen, followed by the Bβ-chain, and shows no effects on the γ-chain. BmHF-1 had, caseinolytic and weakly hemorrhagic activities, which were inhibited by EDTA. In contrast, PMSF did not affect these activities. The caseinolytic activity of BmHF-1 had a pH optimum of 8.0 and was stable in solution up to 40 °C; activity was completely lost at ≥70 °C. The proteolytic activity was also inhibited by sDa (opossum sera) and Da2-1, Da2-II, antihemorrhagic factors isolated from the opossum sera of Didelphis albiventris. BmHF-1 presents weak hemorrhagic activity, with a MHD of 41.14 μg and it induces dose-dependent edema. We could concluded that, despite its weak hemorrhagic activity, BmHF-1 contributes to local tissue damage by inducing edema, releasing pharmacologically active mediators from protein precursors due to its enzymatic action.

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Abbreviations

HPLC-FR:

High performance liquid chromatograph reverse phase

BmHF-1:

Metalloproteinase from Bothrops marajoensis snake venom

MHD:

The minimum hemorrhagic dose

EDTA:

Ethylenediamine tetraacetic acid

EGTA:

Ethyleneglycol tetraacetic acid

PMSF:

Phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride

DL-BapNA:

The synthetic substrate Na-Benzoyl-d,l-arginine 4-nitroanilide hydrochloride

DTT:

Dithiothreitol

MALDI-TOF MS:

Mass Spectrometry Matrix-assisted laser desorption/ionization (Type: Time of fligth)

RC-BmHF-1:

The reduced carboxymethylated BmHF-1

CK:

The plasma creatine kinase

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Acknowledgments

The authors thank Mr. Paulo A. Baldasso for technical assistance. This work was supported by the National Council for Scientific and Technological Development (CNPq) and FAPESP process 06/51566-0. It is part of Frank Denis Torres-Huaco Mg thesis.

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Authors and Affiliations

  1. Department of Biochemistry, Institute of Biology (IB), State University of Campinas (UNICAMP), P.O. Box 6109, Campinas, SP, 13083-970, Brazil

    Frank Denis Torres-Huaco, Luis Alberto Ponce-Soto, Daniel Martins-de-Souza & Sergio Marangoni

  2. Department of Pharmacology, Faculty of Medical Science (FCM), State University of Campinas (UNICAMP), Campinas, SP, Brazil

    Luis Alberto Ponce-Soto

  3. Max Planck Institute of Psychiatry, Munich, Germany

    Daniel Martins-de-Souza

Authors
  1. Frank Denis Torres-Huaco
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  2. Luis Alberto Ponce-Soto
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  3. Daniel Martins-de-Souza
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  4. Sergio Marangoni
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Correspondence to Luis Alberto Ponce-Soto.

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Torres-Huaco, F.D., Ponce-Soto, L.A., Martins-de-Souza, D. et al. Purification and Characterization of a New Weak Hemorrhagic Metalloproteinase BmHF-1 from Bothrops marajoensis Snake Venom. Protein J 29, 407–416 (2010). https://doi.org/10.1007/s10930-010-9267-z

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