Two clotting
serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis
snake venom. The
enzymes were purified using two chromatographic steps: molecular exclusion on
Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the
proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic,
coagulant and toxic properties of the two
enzymes. The
serine proteinases hydrolyzed specific
chromogenic substrate (
BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of
snake venom thrombin-like
enzymes. These
serine proteinases differed in their clotting activity on human plasma, showing a minimum
coagulant dose of 25 μg and 0.571 μg for Cdc SI and Cdc SII, respectively.
Enzymes also showed
coagulant activity on bovine
fibrinogen and degraded chain α of this
protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(
ogen)ation, moderate
edema, and an increase in vascular permeability. These
serine proteinases may contribute indirectly to the local
hemorrhage induced by
metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.