Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom

A factor V activator (VLFVA) was separated from Vipera lebetina venom by ge l filtration on Sephadex G-100 superfine, followed by chromatography on CM- cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russell i factor V activator RVV-V gamma (90% identity). Aside from factor V, no ot her protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes s everal synthetic arginine ester substrates, such as benzoylarginine ethyl e ster (BAEE), tosylarginine methyl ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine ester hydrolase activity of the enzyme is ma rkedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine pr oteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable prote in, heating for 20 min at 70 degrees C does not alter the arginine esterase activity of the enzyme. (C) 1998 Elsevier Science B.V. All rights reserved .