Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca (Jararaca).
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.
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
D000596:Amino Acids; D003435:Crotalid Venoms; D007328:Insulin; D012910:Snake Venoms; C030961:botrocetin; D004492:Edetic Acid; D010447:Peptide Hydrolases