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Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom
Authors:Frey Francisco Romero-Vargas  Luis Alberto Ponce-Soto  Daniel Martins-de-Souza  Sergio Marangoni
Institution:1. Department of Biochemistry, Institute Biology CP 6109, State University of Campinas (UNICAMP), P.O. Box 6109, 13083-970, Campinas, SP, Brazil;2. Max Planck Institute of Psychiatry, Munich, Germany
Abstract:This work reports the purification, biological characterization and amino acid sequence of two new basic PLA2 isoforms, Cdc-9 and Cdc-10, purified from the Crotalus durissus cumanensis venom by one step analytical chromatography reverse phase HPLC. The molecular masses of the PLA2 were 14,175 ± 2.7 Da for Cdc-9 and 14,228 ± 3.5 Da for Cdc-10 both deduced by primary structure and confirmed by MALDI-TOF. The isoforms presented an amino acid sequence of 122 amino acid residues, being Cdc-9: SLVQFNKMIK FETRKSGLPF YAAYGCYCGW GGQRPKDATD RCCFVHDCCY GKVAKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLS TYKNEYMFYP DSRCREPPEY TC with pI value of 8.25 and Cdc-10: SLLQFNKMIK FETRKSGVPF YAAYGCYCGW GGRRPKDPTD RCCFVHDCCY GKLTKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLN TYKNEYMFYP DSRCRGPPEY TC with a pI value of 8.46, showing highly conserved Ca2+-binding and catalytic sites. The PLA2 activity decreased when the isoforms Cdc-9 and Cdc-10 were incubated with 4-bromophenacyl bromide (p-BPB), anhydrous acetic acid and p-nitrobenzene sulfonyl fluoride (NBSF) when compared with the activity of both native isoforms. In mice, the PLA2 isoforms Cdc-9 and Cdc-10 induced myonecrosis and edema. Myotoxic and edema activities were reduced after treatment of the isoforms with p-BPB; acetylation of the lysine residues and the treatment of PLA2 with NBSF have also induced edema reduction. However, p-BPB strongly diminishes the local and systemic myotoxic effects.
Keywords:Clt  Clostripain  SV8  Staphylococcus aureus protease V8  DTT  Dithiothreitol  MALDI-TOF  Matrix-assisted laser desorption/ionization time-of-flight  RC  Reduced and carboxymethylated  RP-HPLC  Reversed-phase high performance liquid chromatography  TFA  Trifluoroacetic acid  PDA  Photo diode array detector  EDTA  Ethylene diamine tetracetic acid  DMSO  Dimethyl sulfoxide  NBSF  2-Nitrobenzenesulphonyl fluoride  PTH  Phenylthiohydantoin  CK  Creatine kinase  µ  -Bondapack C-18  Column 18 high carbon load high activity silica  p-BPB  4-Bromophenacyl bromide  Amicom YM-3  Amicom membrane with pore of 3000 Da  MED  Minimum edema-forming dose  SDS-PAGE  Dodecyl sulfate polyacrylamide gel electrophoresis
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