Purification and characterization of a proteinase in the venom of Trimeresurus flavoviridis: Complete separation of the enzyme from hemorrhagic activity

Abstract

1. 1. A proteinase was purified from the venom of Trimeresurus flavoviridis, by gel filtration on Sephadex G-100 and column chromatography on Amberlite CG-50 (Type 2), with a 27% over-all yield and a 9-fold increase in specific activity. This enzyme, named H 2-proteinase, is the major proteinase in the venom. 2. 2. The purified enzyme was completely free from hemorrhagic activity. It was proven that large amounts of this enzyme had contaminated all preparations of Hemorrhagic Principle 2 so far obtained. 3. 3. The purified enzyme was proved to be homogeneous by ultracentrifugation, sucrose-density-gradient centrifugation, electrophoresis on cellulose acetate membrane and from the absence of any of the other enzyme activities detected in the original venom. 4. 4. H 2-proteinase had an s 20,w of 2.43 S and a molecular weight of 24 000, as shown by the sedimentation velocity and the approach-to-equilibrium methods. The enzyme was most active at pH 9.5 with casein as substrate and the enzyme activity was abolished completely by addition of Cd 2+, Ni 2+, EDTA or cysteine. 5. 5. H 2-proteinase split the oxidized B chain of insulin at the three peptide bonds, Asn 3Gln 4, His 10Leu 11 and Ala 14Leu 15. Of the synthetic leucine-containing peptides of varying sizes tested, the longest one, ZGlyProLeuGlyPro·OH, alone was split by the enzyme at the peptide bond ProLeu. A certain dependence of the enzymatic hydrolysis on the chain length of the substrate was suggested.