Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest.
A comparative study of venoms from juvenile, sub-adult and adult wild Bothrops atrox specimens captured in Manaus region (Brazil) was performed. All venoms tested had acidic pH (5.5) and the human plasma coagulant activity was higher in venoms from juvenile and sub-adult specimens than in adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the most intense bands in adult venoms corresponded to polypeptides of 23 and 50kDa. The 23kDa protein was not detected in juvenile venoms. The 23 and 50kDa proteins were purified by two steps of reversed phase-HPLC followed by size exclusion HPLC. Partial amino acid sequence of the 23kDa protein showed homology to metalloproteinases from other snake venoms. Electrospray ionization mass spectrometric analysis (ESI-MS) showed that the 23kDa band contained at least three isoforms of 23030, 23300 and 23645Da. The 50kDa polypeptide was N-terminally blocked for Edman degradation and presented molecular masses ranging from 46.8 to 49.4kDa by ESI-MS. Both proteins were detected by anti-mutalysin II antibodies in immunoblotting assay indicating that they belong to the metalloproteinase family. Immunoblotting analysis also showed that the 23kDa band increased in intensity from juvenile to adult specimens.SDS-PAGE analysis of juvenile and adult venoms following autoproteolysis in pH 7.4 suggested that endogenous venom metalloproteinases can digest the 50kDa metalloproteinase, originating a new protein band of 27kDa. It was also demonstrated in juvenile venoms that the 23kDa band was not the result of proteolytic processing of the 50kDa metalloproteinase.
López-Lozano
Jorge Luis
JL
Laboratório de Bioquímica e Química de Proteínas, Departamento de Biologia Celular, Centro Brasileiro de Serviços e Pesquisas em Proteínas, IB, Universidade de Brasília, Brazil.
de Sousa
Marcelo Valle
MV
Ricart
Carlos André O
CA
Chávez-Olortegui
Carlos
C
Flores Sanchez
Eladio
E
Muniz
Emiro G
EG
Bührnheim
Paulo F
PF
Morhy
Lauro
L
D000375:Aging / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D017837:Bothrops / Q000254:growth & development*; D001938:Brazil; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011891:Rain; D021241:Spectrometry, Mass, Electrospray Ionization; D014197:Trees; D014329:Tropical Climate
D000925:Anticoagulants; D003435:Crotalid Venoms; D008666:Metalloendopeptidases