PURIFICATION AND CHARACTERIZATION OF ECAMULIN - A NEW PROTHROMBIN ACTIVATOR FROM THE ECHIS-MULTISQUAMATUS SNAKE-VENOM

Enzyme ecamulin, a novel prothrombin activating enzyme, has been isola ted and purified 63-fold with a 57% yield from the venom of the Middle -Asian sand viper Echis multisquamatus using three-step ion exchange c hromatography. The enzyme was shown to activate prothrombin similarly to ecarin, a prothrombin-converting enzyme from Echis carinatus venom, however, differing from the latter by structural and chemical propert ies. The enzyme is a Zn-proteinase: it contains 1 mole of Zn per 1 mol e of protein. The molecular mass of the enzyme as determined by gel fi ltration on Sephacryl S-400 SF in the presence of 8 M urea is 93 +/- 2 kD. Upon SDS-PAGE ecamulin produces two bands with M(r) of 67 and 27 kD under non-reducing conditions, and three bands with M(r) of 67, 14 and 13 kD in the presence of DTT. During native PAGE without SDS, the activator yields one slow mobility band; two bands are observed after addition of DTT or EDTA. Carbohydrates containing N-acetyl-alpha D-glu cosamine residues are localized in the 67 kD chain. Ecamulin has two i soforms, S2 and S3, that are distinguished by the charge and specific coagulation activities: form S2 has 250 NIH units/mg, while the form S 3 has 524 NIH units/mg. The amino acid sequences of the both isoforms are similar but the more active S3 form has a 4 times higher content o f Gin and 4 times less of Gly than the S2 form. The isoelectric point is 4.3-4.5; A(280) Of 1% solution is 10.2. Forms S2 and S3 of ecamulin hydrolyze chromogenic substrate of plasma kallikrein S2302 and glandu lar kallikrein S2266. Ecamulin does not hydrolyze BAEE, TAME, TLME, su bstrates of thrombin (Chromozym TH and S2160), factor Xa (S2222), prot ein C (Chromozym PCa), and plasmin (S2251). The amidase activity is in hibited by EDTA (irreversibly), o-phenanthroline (the activity is reco vered by addition of Zn2+), Cys, or DTT. EGTA, DFP, PMSF, or pCMB do n ot inhibit the enzyme activity. Ecamulin converts prothrombin to alpha -thrombin passing by a shunt via the mesothrombin stage. The reaction of prothrombin activation by ecamulin does not require Ca2+, phospholi pids or factor Va.