Hemachatus haemachatus (Ringhals) Venom. Purification, Some Properties and Amino‐Acid Sequence of Phospholipase A (Fraction DE‐I)

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Abstract
Two isoenzymes of phospholipase A (fraction DE‐I and DE‐II) were isolated from the venom of Hemachatus haemuchatus (Ringhals) by gel filtration on Sephadex G‐50 and by ion‐exchange chromatography on DEAE‐cellulose. Fraction DE‐I was homogeneous by various physicochemical criteria. The enzyme comprises 119 amino acid residues and is cross‐linked by seven disulphide bridges. The values of the molecular weight of fraction DE‐I from gel filtration in the presence of calcium ions, also by the dodecylsulphate gel method and calculated from the amino acid composition were close to 13 500. The complete amino acid sequence of phospholipase A (fraction DE‐I) was elucidated. The reduced and S‐carboxymethylated enzyme was digested with trypsin, chymotrypsin, thermolysin and subtilisin and the peptides purified by DEAE‐cellulose chromatography, gel filtration and chromatography or electrophoresis on paper. The amino acid sequences of the intact enzyme and the pure peptides were determined by the Edman procedure, either through the use of the automatic sequencer or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The amino acid sequence of Hemachatus haemachatus phospholipase A (fraction DE‐I) shows a high degree of homology with the three isoenzymes of phospholipase A from Naja melanoleuca and is also homologous with the phospholipase A from Bitis gabonica and porcine pancreas.