Purification and partial characterization of an a-fibrinogenase (patagonfibrase) from venom of the colubrid snake Philodryas patagoniensis

MARÍA ELISA PEICHOTO; LAURA LEIVA; OFELIA ACOSTA; FLÁVIO TAVARES; LUIS ROBERTO CAMARGO GONÇALVES; ANITA MITIKO TANAKA-AZEVEDO; MARCELO LARAMI SANTORO

San Pablo (Brasil)

Otro; VII Annual Scientific Meeting of Instituto Butantan; 2005

Instituto Butantan

We describe the first enzyme, an α-fibrinogenase, to be isolated from Philodryas patagoniensis venom, named patagonfibrase. Isolation of patagonfibrase was undertaken by chromatography on Mono-Q (FPLC) and HiTrap Blue HP, and it was homogenous by SDS-PAGE and C8 reversed-phase Äkta FPLC. Patagonfibrase is a single-chain protein, with a molecular mass of 57.8 kDa under non-reducing conditions, and has an acidic isoelectric point (5.8). It hydrolyzed selectively the Aα-chain of fibrinogen, leaving the Bβ- and γ-chains unaffected. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase, and its minimum hemorrhagic dose was 0.27 μg. Furthermore, it also possessed proteolytic activity toward azocasein, with a specific activity of 6.08 units/mg of protein. The optimal temperature and pH for patagonfibrase activity on azocasein were 37ºC and 7.5, respectively. Azocaseinolytic activity was enhanced by Ca2+ and inhibited by Zn2+, cysteine, dithiothreitol and Na2EDTA, but PMSF had no effect on it. Patagonfibrase showed neither pro-aggregating nor anti-aggregating activity on platelet-aggregation induced by collagen. This is the first report on the isolation and characterization of a metalloproteinase from P. patagoniensis venom, which may be homologous to other metalloproteinases isolated from the venom of other snake families.