Isolation and characterization of three phospholipases A from the crotoxin complex.

1. Three phospholipases A (phosphatide acyl-hydrolase, EC 3.1.1.4) have been isolated from the crotoxin complex, the main toxic compound of the Crotalus durissus terrificus venom. 2. Two basic phospholipases A were highly purified from the crotoxin complex by single chromatography on carboxymethyl cellulose. The yields were 10% and 38% (w/w), respectively. They showed no differences with regard to isoelectric point, enzymatic activity, immunological properties, and toxicity. One acidic phospholipase A, purified to a final yield of 1-3% by chromatography on carboxymethyl cellulose, gel filtration on Sephadex G-50, and chromatography on DEAE-cellulose, was found to have one third of the specific enzymatic activity of the basic enzymes. The acidic phospholipase A was nontoxic and antigenically different from the basic enzymes. 3. Crotapotin, an acidic peptide of the crotoxin complex (31% yield, w/w), potentiated the toxicity and inhibited the enzymatic activity of the basic phospholipase A isoenzymes, but did not interact with the acidic phospholipase A. 4. The purified enzymes were homogeneous with respect to cellogel electrophoresis, polyacrylamide gel electrophoresis, dodecyl sulfate-gel electrophoresis, immunoelectrophoresis, and isoelectric focusing. 5. The molecular weights of the three phospholipases were found to be in the same range as determined by gel filtration in 6 M guanidine - HCl (14 500) and dodecyl sulfate-gel electrophoresis (15 800). The isoelectric points of these enzymes were at 9.7 and 4.8 for the first two and the third, respectivlar. The acidic enzyme contained more acidic instead of basic amino acid residues. The two methionine residues of each phospholipase were found to be positioned nearby the NH2 - and the C-terminal of the protein chains. A third methionine residue was demonstrated in the acidic phospholipase A. Fingerprint maps of the basic enzymes showed only slight differences. 7. NH2 - and C-terminal sequence analyses indicated a striking homology between the three Crotalus phospholipase A isoenzymes and several phospholipases from other sources.