title: Purification and characterisation of a haemorrhagic fraction from the venom of the Uracoan rattlesnake Crotalus vegrandis.
abstract: Uracoan rattlesnake (Crotalus vegrandis) venom was subjected to chromatographic, electrophoretic, biochemical and in vivo haemorrhagic analysis. A haemorrhagic toxin (Uracoina-1) active on skin at the site of inoculation in mice was purified by Mono Q2 anion-exchange chromatography and size exclusion (SE) high-performance liquid chromatography. The purified preparation was a protein of M(r) 58,000 as revealed by sodium dodecyl sulphate--polyacrylamide gel electrophoresis under denatured conditions and with silver staining. The use of EDTA, EGTA and 1,10-phenanthroline inhibited haemorrhagic and proteolytic activities. Inhibitors of serine proteinases such as PMSF and TCLK had no effect on the haemorrhagic fraction. Uracoina-1 hydrolyses casein, hide powder azure and fibrinogen have an optimal pH of 8.2. It rapidly digests the A alpha-chain of fibrinogen. Thermal denaturation of Uracoina-1 after exposure at 60 degrees C for 15 min led to inactivation of the haemorrhagic activity. In addition, Uracoina-1 is myotoxic, lacking haemolytic, defibrinating and lethal effects. The N-terminal amino acid sequence (20 residues) was determined.
authors.affiliation: Tropical Medicine Institute, Immunochemistry Section, Universidad Central de Venezuela, Caracas, Venezuela.
authors.forename: I
authors.initials: I
authors.lastname: Aguilar
authors.forename: M E
authors.initials: ME
authors.lastname: Girón
authors.forename: A
authors.initials: A
authors.lastname: Rodríguez-Acosta
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D003402:Creatine Kinase / Q000097:blood; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D017839:Crotalus; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006470:Hemorrhage / Q000139:chemically induced; D006863:Hydrogen-Ion Concentration; D007271:Injections, Intradermal; D007273:Injections, Intramuscular; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009135:Muscular Diseases / Q000097:blood / Q000139:chemically induced; D011506:Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D012867:Skin / Q000187:drug effects; D017819:Viperidae
chemical_list: D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D011506:Proteins; C433203:uracoina-1; D003402:Creatine Kinase