Purification and Properties of Phospholipase A from Venom of Trimeresurus flavoviridis (Habu Snake)

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Abstract
Phospholipase A was purified from venom of Trimeresurus flavoviridis (Habu snake) via three steps consisting of Sephadex G-100, CM-cellulose, and DEAE-cellulose column chromatographies. The apparent molecular weights determined by gel filtration on Sephadex G-75 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 28,000 and 14,000, respectively, suggesting that the enzyme is composed of two identical subunits. The isoelectric point was 7.9. The enzyme is characterized by high contents of aspartic acid, glycine, tyrosine, and lysine and contains one residue each of histidine and methionine, three or four tryptophans, and eight disulfide bonds per subunit. The enzyme was inactivated by reaction with p-bromophenacyl bromide following pseudo first order kinetics. The loss of activity was accompanied by loss of histidine, indicating that a single histidine residue is essential for activity. Oxidation with N-bromosuccinimide decreased the enzymic activity. Tryptophan residues appear to play some role in catalysis.