Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities.
Crotoxin, the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus is a mixture of several isoforms that differ slightly in their molecular structure. The toxin consists of two nonidentical subunits: a basic and weakly toxic phospholipase A2, component B, and an acidic and nontoxic subunit, component A. In the present investigation, we have used fast-performance liquid chromatography (FPLC) on anionic and cationic exchange columns to purify isoforms of both crotoxin subunits. Two component A isoforms and four component B isoforms were obtained in a homogeneous state, and their purity was verified by isoelectric focusing in polyacrylamide gels. The amino acid composition of the purified component A and component B isoforms was in good agreement with the protein sequences determined previously with mixtures of isoforms. The amino acid compositions indicated that for both crotoxin components the isoforms differed only by the replacement of few amino acid residues. Eight crotoxin complexes have been prepared in a homogeneous state by reassociation of pure component A and component B isoforms. The quantitative comparison of enzymatic and pharmacological properties of the reconstituted crotoxins indicated that the two component A isoforms had identical properties, whereas the four component B isoforms fell in two classes: crotoxin complexes formed with component B isoforms of the first class were enzymatically less active and pharmacologically more potent than those obtained with component B isoforms of the second class.
Faure
G
G
Unité associée Pasteur/INSERM 285, Institut Pasteur, Paris, France.
Bon
C
C
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000302:isolation & purification*; D003439:Crotoxin / Q000302:isolation & purification* / Q000633:toxicity; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D009498:Neurotoxins; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D013329:Structure-Activity Relationship
D000596:Amino Acids; D003435:Crotalid Venoms; D007527:Isoenzymes; D046911:Macromolecular Substances; D009498:Neurotoxins; D003439:Crotoxin; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2