Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca).
Moojeni protease A was purified from the venom of Bothrops moojeni by chromatography on Sephadex G-100, DEAE Sephadex A-50 and rechromatography on Sephadex G-100. The enzyme shows one protein band in polyacrylamide gel electrophoresis at pH 8.5 or at pH 4.3. The pI of moojeni protease A was approximately 7.7. In immunoelectrophoresis it migrates to the cathode. The enzyme was homogeneous by polyacrylamide gel electrophoresis, immunoelectrophoresis and analyses in the ultracentrifuge. The S20,w and D20,w are 2.68 S and 10.34 X 10(-7) cm2/sec, respectively. The molecular weight calculated by s/D ratio was 22,500 and a value of 22,800 was obtained by sedimentation equilibrium. In SDS-polyacrylamide gel electrophoresis the enzyme exhibits a single polypeptide chain of approximately 20,400 mol. wt under denaturating conditions. In water or low salt solution it undergoes denaturation and autolysis. The enzyme is also unstable at acidic pH and to heat treatment and precipitates in the presence of metal chelating compounds such as EDTA or 1,10 phenanthroline. Leucine, the NH2-terminal amino acid of moojeni protease A is blocked after EDTA treatment. The proteolytic activity of this enzyme increases about 20% in the presence of Ca2+; Mg2+ has no effect and other divalent cations cause inhibition. The removal of Ca2+ ions by oxalate causes about 20% inhibition; the activity was restored by addition of Ca2+.
D000818:Animals; D003435:Crotalid Venoms / Q000032:analysis*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis / Q000302:isolation & purification*; D006470:Hemorrhage / Q000139:chemically induced; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases; D008667:Metalloproteins / Q000032:analysis; D008670:Metals / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000032:analysis / Q000302:isolation & purification*; D010741:Phospholipases A / Q000032:analysis; D011817:Rabbits; D014461:Ultracentrifugation
D003435:Crotalid Venoms; D008667:Metalloproteins; D008670:Metals; D004492:Edetic Acid; D010741:Phospholipases A; D010450:Endopeptidases; D010447:Peptide Hydrolases; C046860:Bothrops moojeni protease A; D008666:Metalloendopeptidases