Enzymatic characterization of a novel phospholipase A(2) from Crotalus durissus cascavella rattlesnake (Maracamboia) venom

The PLA(2) and crotapotin subunits of crotoxin from Crotalus durissus casca vella venom were purified by a combination of HPLC molecular exclusion (Pro tein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA(2) and crotapotins migrated as single bands with estima ted molecular masses of 15 and 9 kDa, respectively. The amino acid composit ion of the PLA(2) showed the presence of 14 half-cysteines and a high conte nt of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA(2) sho wed allosteric behavior, with maximal activity at pH 8.3 and 35-40 degreesC . The C. d, cascavella PLA(2) required C2+ for activity, but was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA(2) by acting as allosteric inhibitors.