Purification and partial characterization of a haemorrhagin (VRH-1) from Vipera russelli russelli venom

A haemorrhagic toxin (VRH-1) has been purified to homogeneity from Vipera russelli russelli venom by subjecting it to chromatography twice successively on CM-Sephadex C-50. It is a protein of mol. wt 22,000 and contains one mole of Mg2+. Intradermal administration of this haemorrhagin in mice resulted in severe lung haemorrhage but produced little haemorrhage in skin. This apparent organ preference led us to develop a new haemorrhage assay method utilizing dye diffusion from lung in vitro. Proteolytic activity of VRH-1 was demonstrated using dimethylcasein as substrate following quantitation by reaction with trinitrobenzoyl sulfonic acid. Both haemorrhagic and proteolytic activities of VRH-1 were inhibited by serine protease inhibitors like phenylmethyl sulfonyl fluoride and chymostatin, but metal chelators had no effect. Lung haemorrhage is unlikely to be a direct reflection of a high local concentration of VRH-1. The administration of supernatant generated by incubation of chopped liver from untreated mouse and VRH-1 (in subhaemorrhagic dose) results in severe lung haemorrhage. This raises the possibility that VRH-1 leads to the formation of intermediate(s) which causes the haemorrhage.