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Isolation and Characterization of a Serine Protease, Ba III-4, from Peruvian Bothrops atrox venom

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Abstract

A serine protease from Bothrops atrox (Peruvian specimen’s venom) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. This protein was denominated Ba III-4 (33,080.265 Da determinated by MALDI-TOF mass spectrometry) and showed pI of 5.06, Km 0.2 × 10−1  M and the V máx 4.1 × 10−1 nmoles p-NA/lt/min on the synthetic substrate BapNA. Ba III-4 also showed ability to coagulate bovine fibrinogen. The serine protease was inhibited by soyben trypsin inhibitor and DA2II, which is an anti-hemorrhagic factor isolated from the opossum specie Didelphis albiventris. The primary structure of Ba III-4 showed the presence of His(44), Asp(94) and Ser(193) residues in the corresponding positions to the catalytic triad established in the serine proteases and Ser(193) are inhibited by phenylmethylsulfonylfluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro, as well as 12 half-cysteine residues. Ba III-4 contained 293 amino acid residues and the primary structure of VIGGDECDIN EHPFLAFMYY SPRYFCGMTL INQEWVLTAA HCRYFCGMTL IHLGVHRESE KANYDEVRRF PKEKYFIFCD NNFTDDEVDK DIMLIRLDKP VSNSEHIAPL SLPSNPPSVG SVCRIMGWGQ TTTSPIDVLS PDEPHCANIN LFDNTVCHTA HPQVANTRTS TDTLCAGDLQ GGRDTCNGDS GGPLICNEQL HGILSWGGDP CAQPNKPAFY TKVYYFDHPW IKSIIAGNKK TVNFTCPPLR SDAKDDSTTY INQEWDWVLT AEHCDRTHMR NSFYDYSSIN SDS. Titration experiments did not show the presence of free sulfhydryl groups after 4 h incubation, nor were differences found in relation to titration kinetics in the presence of nondenaturating buffer. The isolation of this protein, Ba III-4, is of potential interest for the understanding of the pathomechanism of the snake venom action and for the identification of new blood coagulation enzymes of natural sources.

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Abbreviations

RP-HPLC:

reverse phase high performance liquid chromatography

DTT:

dithiothreitol

BaIII-4:

serine protease from Bothrops atrox

MALDI-TOF:

matrix assisted laser desorption ionization time-of-flight

K m :

Michaelis constant

V máx :

maximum velocity

DA2II:

anti-hemorrhagic factor Didelphis albiventris

PMSF:

phenylmethylsulfonyl fluoride

BapNA:

N-benzoyl-l-arginine-p-nitroanilide

SBTI:

soybean trypsin

SDS-PAGE:

sodium dodecyl sulpate-polyacrilamide gel electrophoresis

DTNB:

dithiobis-(2-nitrobenzoate)

EDTA:

ethylenedinitro tetraacetic acid

RC-BaIII-4:

reduced carboxymethylated-serine protease from B. atrox

SV8:

staphylococcus aureus protease

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Acknowledgements

The authors thank Marino Forner and Paulo A. Baldasso for technical assistance. This work was supported by the Fundação de Amparo a Pesquisa, São Paulo (FAPESP).

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Authors and Affiliations

  1. Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), P.O. Box 6109, 13083-970, Campinas, SP, Brasil

    L. A. Ponce-Soto, V. L. Bonfim, J. C. Novello & S. Marangoni

  2. Laboratorio de Química Biológica, Universidad Nacional de San Agustín (UNSA), Arequipa, Peru

    R. Navarro Oviedo

  3. Laboratório de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos (UNMSM), Lima, Peru

    A. Yarlequé Chocas

Authors
  1. L. A. Ponce-Soto
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  2. V. L. Bonfim
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  3. J. C. Novello
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  4. R. Navarro Oviedo
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  5. A. Yarlequé Chocas
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  6. S. Marangoni
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Correspondence to L. A. Ponce-Soto.

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Ponce-Soto, L.A., Bonfim, V.L., Novello, J.C. et al. Isolation and Characterization of a Serine Protease, Ba III-4, from Peruvian Bothrops atrox venom. Protein J 26, 387–394 (2007). https://doi.org/10.1007/s10930-007-9078-z

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  • DOI: https://doi.org/10.1007/s10930-007-9078-z

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