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.
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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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Acknowledgments
The authors thank Paulo A. Baldasso and Marino Forner for general technical help, Vera Lucia Bonfim for redacted help and FAPESP supported this work. We thank Dr J. M. Gutierrez to read this manuscript critically as well as for their valuable and important contributions. This work was supported by FAPESP and is part of PhD thesis by Luis Alberto Ponce-Soto.
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Ponce-Soto, L.A., Lomonte, B., Rodrigues-Simioni, L. et al. Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA2 (F6a) from Crotalus durissus collilineatus Snake Venom. Protein J 26, 221–230 (2007). https://doi.org/10.1007/s10930-006-9063-y
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DOI: https://doi.org/10.1007/s10930-006-9063-y