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 http://br.expasy.org/ 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.
Bibliographical noteFunding 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.
- Bothrops andianus
- Primary sequence
- Serine protease
- Snake venom
- Thrombin-like enzyme