Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2

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Abstract
The water-soluble venom of B. asper (San Juan Evangelista, Veracruz, Mexico) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-L-arginine ethyl hydrolase, N-benzoyl-L-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded 7 protein fractions, which were apparently less toxic than the whole venom (LD50 = 4.3 .mu.g/g mouse wt). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH 7.55) gave several fractions, 2 being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH 8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding 2 more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes showed that all 3 phospholipases belong to type A2. Amino acid analysis showed that phospholipase A2 (type 1) has 97 residues with a calcualted MW 10,978 .+-. 11. Phospholipase A2 (type 2) has 96 residues with a MW 10,959 .+-. 11. Phospholipase A2 (type 3) has 266 residues with 16 half-cystine residues and a calculated MW 29,042 .+-. 31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val-Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A2 (type 2).