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

Abstract

Two isoenzymes of phospholipase A (fraction DE-I and DE-11) were isolated from the venom of Hemachatus haemachatus (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. and Porath [ 5 ] , with gel filtration, succeeded in obtaining four protein peaks from Ringhals venom. Phosphodiesterase, acetylcholine esterase, and 5'-nucleotidase appeared together in the first peak while lecithinase A was eluted in the second peak.In

The Primary Structure of Bovine Pancreatic Phospholipase A2

“…The active-center peptide T3 could be easily sequenced and no indication was found for a drop in the yield passing the glutamine residue. Working with active-center peptides from snake venom phospholipases, Joubert [9,28,29] reported this glutamine to be very sensitive to cyclization. Parallel to this study we also isolated the active-center peptide from porcine 1271.…”

“…Working with active-center peptides from snake venom phospholipases, Joubert [9,28,29] reported this glutamine to be very sensitive to cyclization. Parallel to this study we also isolated the active-center peptide from porcine 1271.…”

Application of electrospray mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for molecular weight assignment of peptides in complex mixtures

“…Several proteins have been separated and sequenced from the venoms of Dendroaspis jamesoni kaimoeae (Jameson's mamba, D. jameson i), Dendroaspis polylepis polylepis (black mamba, D. polylepis), Dendroaspis augusticeps (eastern green mamba, D. augusticeps), and Hemachatus haemachatus . (Ringhals cobra, H. haemachatus), but the venom of Micrurus fulvius (eastern coral snake, M. fulvius) is totally uncharacterized [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40].…”

Snake Venom Toxins

“…The source of the Hemachatus haernachatus venom, trypsin, a-chymotrypsin, thermolysin and chemical reagents have been described previously [3,10]. The physicochemical methods, the toxicity determinations by intravenous injection, the immunochemical examination .by gel-diffusion, the reduction and S-carboxymethylation, the digestion with trypsin, a-chymotrypsin and thermolysin, the fractionation of enzyme digests by chromatography on DEAE-cellulose and ' After 12-h hydrolysis.…”

“…paper chromatography and/or high-voltage paper electrophoresis, the amino acid analyses of the toxins and of the peptides, the sequence determination of reduced and S-carboxymethylated toxins and peptides by Edrnan degradation with a Beckman sequencer or manually and the nomenclature of the peptide have all been detailed in previous communications [3,10].…”