The Protein Journal

, Volume 26, Issue 4, pp 221–230

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA2 (F6a) from Crotalus durissus collilineatus Snake Venom

  • Luis Alberto Ponce-Soto
  • Bruno Lomonte
  • Lea Rodrigues-Simioni
  • José Camillo Novello
  • Sergio Marangoni
Article

Abstract

A new crotoxin B isoform PLA2 (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA2 (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA2 (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA2 (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA2s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA2s. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA2 (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA2 (F6a) was clearly cytotoxic to these cells.

Keywords

Biventer cervicis chick Crotalus durissus collilineatus crotoxin crotoxin B isoform PLA2 (F6a) mass spectrometry Maldi-Tof myoblasts myotubes venom neurotoxin 

Abbreviations

Crotoxin B

complex crotoxinB

HPLC-FR

High performance liquid chromatograph reverse phase

F3 and F4

crotapotins isoforms

F6 and F6a

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

RC-PLA2

reduced and carboxymethylated-PLA2

SV8

Staphylococcus aureus protease

TFA

trifluoroacetate

Cvx

convulxin

Gyr

gyroxin

MS spectral profiles

mass spectrometric spectral profiles.

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Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Luis Alberto Ponce-Soto
    • 1
  • Bruno Lomonte
    • 3
  • Lea Rodrigues-Simioni
    • 2
  • José Camillo Novello
    • 1
  • Sergio Marangoni
    • 1
  1. 1.Departamento de Bioquímica, Instituto de Biologia (IB)Universidade Estadual de Campinas (UNICAMP) CampinasBrazil
  2. 2.Departamento de Farmacologia, Faculdade de Ciências MédicasUniversidade Estadual de Campinas (UNICAMP)CampinasBrazil
  3. 3.Facultad de Microbiología, Instituto Clodomiro PicadoUniversidad de Costa RicaSan JoséCosta Rica

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