Proteolytic action of Bothrops jararaca venom upon its own constituents.
Many of the components of Bothrops jararaca venom are proteolytic enzymes. In the present work, we investigated the proteolytic action of B. jararaca venom upon its own constituents. Crude venom was reconstituted and incubated at pH 5.0 or 8.5 for up to 48h at room temperature. Aliquots taken at 0, 24 and 48h of incubation were then tested for proteolytic activity and several biological activities, as well as electrophoretic migration pattern and antibody recognition. Rate of hydrolysis of azocasein by venom samples was not changed by the incubation, but hemagglutinating activity decreased by 93% after 24h of incubation at pH 8.5, with no detectable changes at pH 5.0. Incubation of venom samples caused a progressive increase in phospholipase A(2) and procoagulant activities that was more evident in samples incubated at pH 5.0. The electrophoretic migration pattern showed no significant change for venom samples incubated at pH 5.0, whereas in samples incubated at pH 8.5 bands in the region between 66 and 45kDa gradually disappeared. The addition of a mixture of protease inhibitors (EDTA, PMSF, PPACK and benzamidine) effectively protected against venom degradation at pH 8.5. The cocktail of inhibitors also reduced the changes in phospholipase A(2) activity found in venom samples incubated at pH 5.0. Recognition of venom samples by polyclonal antibodies raised against crude venom was progressively lost during incubation at both pH 5.0 and 8.5; again the addition of protease inhibitors protected against loss of antibody recognition. We conclude that prolonged manipulation of B. jararaca venom at an acidic or alkaline pH can produce significant changes in its biological properties.
Sousa
J R
JR
Departamento de Bioquímica Médica/ICB/CCS, Ilha Fundão, Cidade Universitária, Univ. Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.
Monteiro
R Q
RQ
Castro
H C
HC
Zingali
R B
RB
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D017837:Bothrops; D003435:Crotalid Venoms / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010741:Phospholipases A / Q000378:metabolism
D001798:Blood Proteins; D003435:Crotalid Venoms; D010741:Phospholipases A