Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta).
Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.
Sánchez
E F
EF
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Brazil.
Magalhës
A
A
Mandelbaum
F R
FR
Diniz
C R
CR
D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000032:analysis; D002413:Cations, Divalent; D002853:Chromatography, Liquid; D003435:Crotalid Venoms / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D005779:Immunodiffusion; D007526:Isoelectric Point; D008274:Magnesium / Q000032:analysis; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D008970:Molecular Weight; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D002413:Cations, Divalent; D003435:Crotalid Venoms; D005340:Fibrinogen; D008666:Metalloendopeptidases; C068795:hemorrhagic factor LHFII; D008274:Magnesium; D015032:Zinc; D002118:Calcium