Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom Nget Hong Tan ⁎, Shin Yee Fung, Yeannie Hui Yeng Yap Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia abstract article info Article history: Received 3 August 2011 Received in revised form 21 September 2011 Accepted 21 September 2011 Available online 29 September 2011 Keywords: Cryptelytrops albolabris venom Thrombin-like enzyme ELISA A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatogra- phy. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3 mg/mL bovine fibrinogen, and showed distinct species differ- ences in the following decreasing order: bovine fibrinogen N dog fibrinogen ≈human fibrinogen N goat fibrino- gen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sand- wich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Snake venom thrombin-like enzymes (SV-TLEs) are enzymes that are functionally related to thrombin, being capable of clotting fibrino- gen. They belong to the group of snake venom serine proteinases (SVSPs). According to Rawlings and Barrett (2004), SVSPs belong to the clan PA subclain S and family S1. In snake envenomation, the enzyme may cause coagulopathies in the victim, via the depletion of plasma fibrinogen and thus contributes to the hemorrhagic effects of the venom. The defibrinogenating action of the SV-TLEs is usually be- nign and they may lower blood viscosity (Castro et al., 2004). These properties make SV-TLEs useful as anti-thrombotic agents. As such, many SV-TLEs have been isolated from various snake venoms and char- acterized (Mackessy, 2010; Phillips et al., 2010). As these TLEs differ in their thrombin-like action, comparative study, in particular the sub- strate specificity of the enzyme will contribute to the understanding of the structural basis of the thrombin-like action. Cryptelytrops albolabris (white-lipped tree viper, formerly known as Trimeresurus albolabris) is a medically important snake in South- east Asia (Hutton et al., 1990). The venom exhibits moderate throm- bin-like enzyme activity (Tan et al., 1989). In severe C. albolabris envenomation, hemorrhage, defibrination, thrombocytopenia and increased fibrinolytic activity were observed (Mahasandana et al., 1980). Rojnuckarin et al. (2006) reported the molecular cloning of five novel serine proteases from the venom gland of Thai C. albolabris, two of which were predicted to be SV-TLEs (termed GPV-TL1 and GPV-TL2), each with 260 amino acids and are highly homologous to stejnobin, the SV-TLE isolated from Trimeresurus (now renamed Viridovipera) stejnegeri venom (Zhang et al., 1998). Earlier, Deng et al. (2008) reported the isolation and partial characterization of chitribrisin, a AB-class SV-TLE from the Chinese green pit viper (T. albolabris, now renamed C. albolabris). Subsequently, they reported the functional char- acterization of the recombinant enzyme (Lin et al., 2009). In this paper, we present the results of purification of a thrombin- like enzyme from C. albolabris venom of Thai origin. This enzyme differs from chitribrisin which reflects the geographic differences between venoms (and venom proteins) of Thai C. albolabris and the Chinese green pit viper. We also investigated the substrate specificity and immunological cross reactivity of the purified SV-TLE, which have not been investigated previously for chitribrisin. 2. Materials and methods 2.1. Materials Cryptelytrops albolabris venom was obtained from Liverpool Tropical School of Medicine (U.K.) and is of Thailand origin. All other venoms were obtained from Latoxan (Valence, France). Sephadex G25, Resource Comparative Biochemistry and Physiology, Part B 161 (2012) 79–85 ⁎ Corresponding author. Tel.: + 60 3 79674912; fax: + 60 3 79674957. E-mail address: tanngethong@yahoo.com.sg (N.H. Tan). 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.09.009 Contents lists available at SciVerse ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb