Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom

José Antonio Valeriano-Zapana, Fernando Steve Segovia-Cruz, José Miguel Rojas Hualpa, Daniel Martins-de-Souza, Luis Alberto Ponce-Soto, Sergio Marangoni

Producción científica: Contribución a una revistaArtículorevisión exhaustiva

17 Citas (Scopus)


A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr ∼29kDa under reducing conditions and non-reducing ∼25kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4×10-1 M and the Vmax 7.9×10-1 nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60°C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF).The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom. TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF).In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical-scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources.

Idioma originalInglés
Páginas (desde-hasta)231-240
Número de páginas10
EstadoPublicada - feb. 2012

Nota bibliográfica

Funding Information:
The authors thank Paulo A. Baldasso for technical assistance. This work was supported by CAPES and is part of MSc thesis of José Antonio Valeriano-Zapana.


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