Enzyme ecamulin, a novel prothrombin activating enzyme, has been isolated and purified 63-fold with a 57% yield from the venom of the Middle-Asian sand viper Echis multisquamatus using three-step ion exchange chromatography. 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 properties. The enzyme is a Zn-proteinase: it contains 1 mole of Zn per 1 mole of protein. The molecular mass of the enzyme as determined by gel filtration on Sephacryl S-400 SF in the presence of 8 M urea is 93 ± 2 kD. Upon SDS-PAGE ecamulin produces two bands with Mr of 67 and 27 kD under non-reducing conditions, and three bands with Mr 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-αD-glucosamine residues are localized in the 67 kD chain. Ecamulin has two isoforms, S2 and S3, that are distinguished by the charge and specific coagulation activities: form S2 has 250 NIH units/mg, while the form S3 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 of Gln and 4 times less of Gly than the S2 form. The isoelectric point is 4.3-4.5; A280 of 1% solution is 10.2. Forms S2 and S3 of ecamulin hydrolyze chromogenic substrate of plasma kallikrein S2302 and glandular kallikrein S2266. Ecamulin does not hydrolyze BAEE, TAME, TLME, substrates of thrombin (Chromozym TH and S2160), factor Xa (S2222), protein C (Chromozym PCa), and plasmin (S2251). The amidase activity is inhibited by EDTA (irreversibly), o-phenanthroline (the activity is recovered by addition of Zn2+), Cys, or DTT. EGTA, DFP, PMSF, or pCMB do not inhibit the enzyme activity. Ecamulin converts prothrombin to α-thrombin passing by a shunt via the mesothrombin stage. The reaction of prothrombin activation by ecamulin does not require Ca2+, phospholipids or factor Va.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Dec 1 1996|
- Echis multisquamatus
- Prothrombin activator
- Snake venom
ASJC Scopus subject areas