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Biochemical, Pharmacological and Structural Characterization of a New PLA2 from Crotalus durissus terrificus (South American Rattlesnake) Venom

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Abstract

A new PLA2 (F16) was purified from Crotalus durissus terrificus venom by molecular exclusion chromatography followed by analytical reverse phase HPLC. The PLA2 (14.86 kDa by MALDI-TOF mass spectrometry) had an amino acid sequence of SLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGRRRPKDATDRCCFVHDCCYEKVTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNGYMFYPDSRCRGPSETC, and showed highly conserved Ca2+-binding and catalytic sites. F16 showed allosteric behavior with 10 mM Ca2+ and had temperature and pH optima of 25°C and 7.9, respectively. F16 (10 μg/ml) produced neuromuscular blockade in chick biventer cervicis preparations in the absence and presence of crotapotin, indicating that crotapotin was not essential for neuromuscular action in this preparation. In contrast, in mouse phrenic nerve-diaphragm preparations, the neuromuscular blockade produced by the same concentration of toxin was dependent on crotapotin. Pre-incubation with heparin markedly reduced the neurotoxicity of F16. These results show that the biochemical and structural properties of F16 are similar to those of the PLA2 isoforms F15 and F17, but that the neurotoxicity and the requirement for crotapotin to form the crotoxin complex varies according to the neuromuscular preparation.

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Abbreviations

Clt:

clostripain

CNBr:

cyanogen bromide

DTT:

dithiothreitol

MALDI-TOF:

matrix-assisted laser desorption/ionization time-of-flight

RC:

reduced and carboxymethylated

RP-HPLC:

reversed-phase high performance liquid chromatography

SV8:

Staphylococcus aureus protease V8

TFA:

trifluoroacetic acid.

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Authors and Affiliations

  1. Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil

    Saraguaci Hernandez-Oliveira, Sergio Marangoni, Stephen Hyslop & Léa Rodrigues-Simioni

  2. Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil

    Marcos Hikari Toyama & Daniela Oliveira Toyama

Authors
  1. Saraguaci Hernandez-Oliveira
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  2. Marcos Hikari Toyama
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  3. Daniela Oliveira Toyama
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  4. Sergio Marangoni
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  5. Stephen Hyslop
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  6. Léa Rodrigues-Simioni
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Correspondence to Léa Rodrigues-Simioni.

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Hernandez-Oliveira, S., Toyama, M.H., Toyama, D.O. et al. Biochemical, Pharmacological and Structural Characterization of a New PLA2 from Crotalus durissus terrificus (South American Rattlesnake) Venom. Protein J 24, 233–242 (2005). https://doi.org/10.1007/s10930-005-6718-z

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