TY - JOUR
T1 - Molecular identification and activity upon chromogenic substrates of a venombin A from Bothrops atrox Peruvian snake venom
AU - Sandoval Peña, Gustavo Adolfo
AU - Lazo Manrique, Fanny Elizabeth
AU - Rodriguez Quispe, Edith Fanincia
AU - Yarleque Chocas, Armando
AU - Zingali, Russolina B.
N1 - Publisher Copyright:
© Facultad de Ciencias Biológicas UNMSM
Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 2010/12
Y1 - 2010/12
N2 - In this work, the thrombin-like enzyme (TLE) from Bothrops atrox has been identified by mass spectrometry and its enzymatic activity evaluated upon several synthetic substrates. The enzyme was purified to homogeneity using three chromatography steps on Sephadex G-75, CM-Sephadex C-50 and Agarose-PAB. Also, molecular weight by PAGE-SDS was determined. For molecular identification of this enzyme, mass spectrometry-based peptide mass fingerprinting was used and later in silico analysis. Enzymatic activities were determined using bovine fibrinogen, BApNA and also upon specific chromogenic substrates such as S-2238, S-2251 y S-2266. As a result of these biochemical and structural procedures, we obtained a TLE from B. atrox venom with a molecular weight of 29,6 kDa. Mass spectrometry analysis of obtained peptides, allow us to identify this enzyme as a venombin A, showing a 75% sequence homology. After recording enzymatic activity, this TLE showed coagulant activity on bovine fibrinogen and upon BApNA, S-2238 y S-2266, being unable to hydrolyze S-2251 substrate. Using this combination of structural and functional approaches, we have identified the main component of B. atrox venom related to its coagulant activity, as well as a detailed evaluation of its enzymatic activity upon several substrates.
AB - In this work, the thrombin-like enzyme (TLE) from Bothrops atrox has been identified by mass spectrometry and its enzymatic activity evaluated upon several synthetic substrates. The enzyme was purified to homogeneity using three chromatography steps on Sephadex G-75, CM-Sephadex C-50 and Agarose-PAB. Also, molecular weight by PAGE-SDS was determined. For molecular identification of this enzyme, mass spectrometry-based peptide mass fingerprinting was used and later in silico analysis. Enzymatic activities were determined using bovine fibrinogen, BApNA and also upon specific chromogenic substrates such as S-2238, S-2251 y S-2266. As a result of these biochemical and structural procedures, we obtained a TLE from B. atrox venom with a molecular weight of 29,6 kDa. Mass spectrometry analysis of obtained peptides, allow us to identify this enzyme as a venombin A, showing a 75% sequence homology. After recording enzymatic activity, this TLE showed coagulant activity on bovine fibrinogen and upon BApNA, S-2238 y S-2266, being unable to hydrolyze S-2251 substrate. Using this combination of structural and functional approaches, we have identified the main component of B. atrox venom related to its coagulant activity, as well as a detailed evaluation of its enzymatic activity upon several substrates.
KW - Bothrops atrox
KW - Chromogenic substrates
KW - Thrombin-like enzyme MALDI-TOF
UR - http://www.scopus.com/inward/record.url?scp=84969314369&partnerID=8YFLogxK
M3 - Artículo
AN - SCOPUS:84969314369
SN - 1561-0837
VL - 17
SP - 365
EP - 370
JO - Revista Peruana de Biologia
JF - Revista Peruana de Biologia
IS - 3
ER -