β-Fibrinogenase from the venom of Vipera lebetina

Abstract

An arginine esterase was purified from the venom of Vipera lebetina by gel filtration on Sephadex G-100 and by affinity and DEAE-cellulose chromatography. The enzyme has a mol. wt of 52,500 and p I ∼ 3. It is a glycoprotein containing 23% of neutral sugars, and has extremely high thermostability. The esterase activity is inhibited by diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF). The K m and k cat values are for α-N- benzoyl- l-arginine ethyl ester (BAEE) 7.7 × 10 −5M and 43.8 sec −1, for p- tosyl- l-arginine methyl ester (TAME) 3.6×10 −4M and 39.8 sec −1 (pH 8.5, 25°C, and for α-N- benzoyl- dl-arginine -4- nitroanilide (BAPNA) 1.8×10 −4M and 0.94 sec −1 (pH 8.3, 25°C), respectively. Lysine esters are not hydrolyzed. The enzyme has weak caseinolytic activity and hydrolyzes glucagon at the sites Lys 12-Tyr 13, Arg 17-Arg 18 and Arg 18-Ala 19. In fibrinogen it cleaves Bβ-chain first and later also the Aα-chain.