Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake (Agkistrodon contortrix contortrix).
A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom has been purified by combination of CM-cellulose chromatography, molecular sieve chromatography on Sephadex G-100, p-aminobenzamidine-agarose affinity chromatography, and DEAE-cellulose chromatography. The enzyme, fibrolase, has a molecular weight of 23,000-24,000 and an isoelectric point of pH 6.8. It is composed of approximately 200 amino acids, possesses a blocked NH2-terminus and contains little or no carbohydrate. The enzyme shows no activity against a series of chromogenic p-nitroanilide substrates and is not inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, Trasylol, or p-chloromercuribenzoate. However, the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator). Metal analysis revealed 1 mol of zinc/mol of protein. Study of cleavage site preference of the fibrinolytic enzyme using the oxidized B chain of insulin revealed that specificity is similar to other snake venom metalloproteinases with cleavage primarily directed to an X-Leu bond. Interestingly, unlike some other venom fibrinolytic metalloproteinases, fibrolase exhibits little if any hemorrhagic activity. The enzyme exhibits direct fibrinolytic activity and does not activate plasminogen. In vitro studies revealed that fibrolase dissolves clots made either from purified fibrinogen or from whole blood.
Guan
A L
AL
Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
Retzios
A D
AD
Henderson
G N
GN
Markland
F S
FS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography; D003435:Crotalid Venoms / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
D000596:Amino Acids; D003435:Crotalid Venoms; D008666:Metalloendopeptidases; C058789:fibrolase