A phospholipase A2 with anticoagulant activity. I. Isolation from Vipera berus venom and properties.
An anticoagulant protein has been isolated by DEAE cellulose chromatography and gel filtration from the venom of the Vipera berus orientale (Eastern Europe). Purification has been completed by elution on carboxymethyl cellulose with continuous gradient at constant pH. The inhibitor of coagulation was separated from the other venom enzymes, e.g. procoagulant, fibrinogenolytic, aminoesterase and amino acid oxidase activities. It was also separated from other phospholipase components which were not related to the anticoagulant property. The inhibitor appeared as a simgle polypeptidic chain protein, formed by 119 amino acid residues, with a molecular weight of 13400 and an isoelectric point of 9.2. At low saline molarity, a monomer-trimer transition of this protein was observed. Both forms had the same amino acid composition. There were six disulfide bridges without free SH groups per phospholipase molecule. Deprived of any proteolytic activity, the clotting inhibitor displayed a high phospholipase activity in the presence of calcium. Activity did no appear with EDTA buffer deprived of cation. Finely dispersed micellar suspensions were found suitable for obtaining the highest phospholipase activity. High sodium cholate concentration or methanol/chloroform/ether solvent were effective without loss of enzymatic activity. As characteristis of phospholipase A2 (EC 3.1.1.4), the degradation products identified on thin-layer chromatography induced hemolysis of human erythrocytes. The apparent Km value 1.25 - 10(-3) M was determined on phosphatidylcholine isolated from ovolecithin. This purified berus inhibitor would be of value for investigating the involvement of phospholipids in the clotting mechanism.
D000596:Amino Acids / Q000032:analysis; D000925:Anticoagulants / Q000302:isolation & purification*; D001777:Blood Coagulation; D002118:Calcium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D012910:Snake Venoms
D000596:Amino Acids; D000925:Anticoagulants; D012910:Snake Venoms; D004492:Edetic Acid; D010740:Phospholipases; D002118:Calcium