Characterization of One Novel Venom Protease with β-Fibrinogenase Activity from the Taiwan Habu ( Trimeresurus mucrosquamatus): Purification and cDNA Sequence Analysis

Abstract

Several fibrinogenolytic proteases were isolated from the venom of Taiwan habu, Trimeresurus mucrosquamatus, a snake species belonging to the Crotalidae family. One protease with strong fibrinogenolytic activity was further purified to homogeneity through multiple-step chromatographies including ion-exchange chromatography, gel permeation and reversed-phase HPLC. In vitro, the purified enzyme cleaved β-chain of fibrinogen molecules efficiently and showed relatively lower activity on α-chain, with almost no activity on γ-chain even after a long period of incubation. Further characterization indicated that it is a single-chain polypeptide with molecular weight of about 28,000. Its stability at high temperatures (> 90°C) distinguished it from the previously reported venom fibrinogenases. N-Terminal sequence analysis revealed that it is similar to batroxobin and ancrod, which were shown to possess either fibrinogen-clotting or antithrombotic effect. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A) +RNA of fresh venom glands of the same snake species to facilitate the cloning and sequencing of this important fibrinogenase. Sequencing several positive clones corresponding to the coding sequence of the enzyme revealed the existence of a family of novel thrombin-like fibrinogenases in the Taiwan habu, which are heat-stable and may be useful as strong antithrombotic agents.