Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes

Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera amm odytes ammodytes venom. They are monomeric glycoproteins of an apparent mol ecular mass of 70 kDa and multiple isoelectric points around pH 5.5. Both m olecules are proteolytically active against azocasein as substrate. VaH1, w hich was characterised in detail, showed maximum activity at pH 7.5. Ethyle nediaminetetraacetic acid eliminated the proteolytic as well as the hemorrh agic activity of VaH1 while iodoacetamide, phenylmethylsulfunyl fluoride an d pepstatin A, inhibitors of cysteine, serine and aspartic proteinases resp ectively, had no effect. VaH1 is therefore a nictalloproteinase whose hemor rhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the A alpha -chain of fibrinog en. In the B-chain of insulin it cleaved with a high preference the bond be tween Ala(14) and Leu(15). Based on its molecular mass, VaH1 (as well as Va H2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin s ubfamily of zinc-metalloproteinases. (C) 2001 Elsevier Science Ltd. All rig hts reserved.