TY - JOUR
T1 - Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis
AU - Vilca-Quispe, Augusto
AU - Ponce-Soto, Luis Alberto
AU - Winck, Flavia Vischi
AU - Marangoni, Sergio
N1 - Funding Information:
The authors thank Paulo A. Baldasso for technical assistance. This work was supported by the São Paulo State Research Foundation, (FAPESP) and is part of Ms Sc thesis of Augusto Vilca Quispe (Process 06/54275-7).
PY - 2010/4/1
Y1 - 2010/4/1
N2 - The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BAρNA, Michaelis-Menten behavior with K m 2.3×10 -1M and the V max 0.52×10 -1 nmoles ρ-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity.
AB - The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BAρNA, Michaelis-Menten behavior with K m 2.3×10 -1M and the V max 0.52×10 -1 nmoles ρ-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity.
UR - http://www.scopus.com/inward/record.url?scp=77953443800&partnerID=8YFLogxK
U2 - 10.1016/j.toxicon.2009.11.006
DO - 10.1016/j.toxicon.2009.11.006
M3 - Article
C2 - 19931298
AN - SCOPUS:77953443800
SN - 0041-0101
VL - 55
SP - 745
EP - 753
JO - Toxicon
JF - Toxicon
IS - 4
ER -