title: Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom.
abstract: 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 ∼29 kDa under reducing conditions and non-reducing ∼25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4 × 10(-1) M and the V(max) 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.
authors.affiliation: Department of Biochemistry, Institute of Biology (IB), State University of Campinas (UNICAMP), P.O. Box 6109, Zip code 13083-970, Campinas, SP, Brazil.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Valeriano-Zapana
authors.forename: Fernando Steve
authors.initials: FS
authors.lastname: Segovia-Cruz
authors.forename: José Miguel
authors.initials: JM
authors.lastname: Rojas-Hualpa
authors.forename: Daniel
authors.initials: D
authors.lastname: Martins-de-Souza
authors.forename: Luis Alberto
authors.initials: LA
authors.lastname: Ponce-Soto
authors.forename: Sergio
authors.initials: S
authors.lastname: Marangoni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D017837:Bothrops; D003435:Crotalid Venoms / Q000201:enzymology*; D005340:Fibrinogen / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010974:Platelet Aggregation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000302:isolation & purification* / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
chemical_list: D003435:Crotalid Venoms; D005340:Fibrinogen; D057057:Serine Proteases; D013917:Thrombin