title: Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca (Jararaca).
abstract: Bothropasin, one of the proteases from the venom of Bothrops jararaca active on casein, was isolated by ammonium sulfate precipitation, DEAE-cellulose and DEAE-Sephadex A-50 chromatographies and Sephadex G-100 column filtration. The preparation possessed no other detectable activities which are present in the crude venom. Addition of Ca2+ during purification stabilized the enzyme. The endopeptidase was inhibited by EDTA and EGTA; Ca2+ did not restore the activity of the inhibited enzyme. The material was homogeneous by polyacrylamide gel electrophoreses at different pH values, immunoprecipitation and crossed immunoelectrophoresis. By SDS-polyacrylamide gel electrophoresis the denatured and reduced enzyme had only a 48,000 molecular weight band. In the presence of 6 M guanidine-HCl and 0.1 M beta-mercaptoethanol the preparation showed a value of 49,870 by sedimentation equilibrium. The native tertiary structure of the protein is dependent on S-S and metal bonds. The denatured and reduced enzyme, in the presence of EDTA, showed a molecular weight of 37,300 by sedimentation equilibrium, a value which was also confirmed in SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed five peptide bonds: His-Leu (5-6), His-Leu(10-11), Ala-Leu(14-15), Tyr-Leu(16-17) and Phe-Phe(24-25) in the B-chain of oxidized insulin.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000032:analysis*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007328:Insulin / Q000032:analysis; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000494:pharmacology; D012910:Snake Venoms / Q000302:isolation & purification* / Q000494:pharmacology; D014461:Ultracentrifugation
chemical_list: D000596:Amino Acids; D003435:Crotalid Venoms; D007328:Insulin; D012910:Snake Venoms; C030961:botrocetin; D004492:Edetic Acid; D010447:Peptide Hydrolases