The Protein Journal

, Volume 26, Issue 1, pp 39–49

Biochemical, Pharmacological and Structural Characterization of Two PLA2 Isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima Snake Venom

  • Luis Alberto Ponce-Soto
  • Paulo Aparecido Baldasso
  • Frey Francisco Romero-Vargas
  • Flávia V. Winck
  • José Camillo Novello
  • Sergio Marangoni
Article

DOI: 10.1007/s10930-006-9042-3

Cite this article as:
Ponce-Soto, L.A., Baldasso, P.A., Romero-Vargas, F.F. et al. Protein J (2007) 26: 39. doi:10.1007/s10930-006-9042-3

Abstract

Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on μ-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry .The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13

This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes.

Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively.

In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro.

Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins.

Keywords

Phospholipase A2 D49 snake venom Crotalus durissus ruruima neurotoxin myotoxin 

Abbreviations

HPLC-FR

high performance liquid chromatograph reverse phase

Cdr-12 and Cdr-13

isoforms of PLA2

MALDI-tof:

Matrix Assisted Laser Desorption Ionization-Time-of-flight. Mass Spectrometry

μ-Bondapack C-18

column HPLC with 18 carbons

PTC-aminoacids

phenylthiocarbamyl- aminoacids

EDTA

ethylenediaminetetraacetic

DTT

dithiothreitol

Da

dalton

CK

the plasma creatine kinase

C2C12

myoblasts and myotubes (ATCC CRL-1772)

PBS

phosphate basic sodium buffer

RC-PLA2

reduced and carboxymethylated-PLA2

SV8

Staphylococcus aureus protease

Clt

clostripain endoprotease of Clostridum perfringes

TFA

trifluoroacetate

MS spectral profiles

mass spectrometric spectral profiles

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Luis Alberto Ponce-Soto
    • 1
  • Paulo Aparecido Baldasso
    • 1
  • Frey Francisco Romero-Vargas
    • 1
  • Flávia V. Winck
    • 1
  • José Camillo Novello
    • 1
  • Sergio Marangoni
    • 1
  1. 1.Department of Biochemistry, Institute of BiologyState University of Campinas (UNICAMP)CampinasBrazil

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