Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis.
The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BArhoNA, Michaelis-Menten behavior with K(m) 2.3x10(-1)M and the V(max) 0.52x10(-1) nmoles rho-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 alpha- and beta-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.
Vilca-Quispe
Augusto
A
Department of Biochemistry, Institute of Biology, Campinas, SP, Brazil.
Ponce-Soto
Luis Alberto
LA
Winck
Flavia Vischi
FV
Marangoni
Sergio
S
D000818:Animals; D017837:Bothrops; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003435:Crotalid Venoms; D057057:Serine Proteases