Structural, enzymatic and biological properties of new PLA(2) isoform from Crotalus durissus terrificus venom

We isolated a new PLA(2) from the Crotalus durissus terrificus venom that designated F15, which showed allosteric behavior with a V(max) of 8.5nmol/min/mg and a K(m) of 38.5 mM. The incubated heparin salt of this isolated F15 act a positive allosteric effector by increasing the V(max) to 10.2 nmol/min/mg, with decreasing the V(max) value to 20.5 mM. The crotapotin, on the other hand acts as a negative allosteric effector by increasing the V(max) values to 58.4 mM. F15 also showed high calcium dependence for its catalysis similar to that found for other PLA(2) enzymes isolated from these snake venoms. The replacement of calcium by other divalent ions such Mg(2+), Mn(2+), Cd(2+), Sn(2+) and Cu(2+) resulted in lower enzymatic activity. The optimum pH and temperature for the enzyme was 8.5 and 18 degrees C, respectively. F15 alone showed moderate neurotoxic activity in isolated mouse phrenic nerve diaphragm in comparison to other strong myotoxic PLA(2) such as bothropstoxin-I (BThtx-I), but this activity was highly neurotoxic in a chick biventrer cervis preparation, whereas BthTx-I did not reveal this high neurotoxicity. This new protein showed a high bactericidal effect against both Gram-negative and Gram-positive bacterial strains. F15 contained 122 amino acid residues, with a primary structure of: HLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGQRRPKDATDRCCFVHDCCYGKLTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPKSRCRRPSETC. Its molecular mass and isoeletric point were 14.5 kDa and 8.85, both estimated by two dimensional electrophoresis. The amino acid sequence of the F15 revealed high sequence homology with F16 and F17. F15 and the other PLA(2)s revealed highly conserved amino acid sequences principally for calcium binding loop and active site helix. F15 also showed a high homology with the lysine-rich region of myotoxic PLA(2).