Abstract

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 s 20,w and D 20,w are 2.68 S and 10.34 × 10 −7 cm 2/ 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 ~ 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 NH 2-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 Ca 2+; mg 2+ has no effect and other divalent cations cause inhibition. The removal of Ca 2+ ions by oxalate causes about 20% inhibition; the activity was restored by addition of Ca 2+.

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