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BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF THROMBINLIKE FROM Bothrops andianus SNAKE VENOM
1Valeriano-Zapana
J.A., 1Vilca-Quispe, A., 1Romero-Vargas F.F., 1HuancahuireVega, S., 1Ponce-Soto, L.A., 1Marangoni, S.1
1
Department of Biochemistry / Institute of Biology, UNICAMP-SP, Brazil
Aim:
Characterization and study of the properties biological of a new serine protease
with activity thrombin-like from B. andianus snake venom.
Methods:
A new serine protease with thrombin-like activity from <i>B. andianus</i> snake
venom (TLBan) was obtained through two chromatographic steps, molecular
exclusion chromatography on G-75 column (1 cm x 60 cm) and RP-HPLC on μBondapak C-5 column. The molecular mass was analyzed by MALDI-TOF
spectrometry, its kinetic activity was evaluated in the presence of a synthetic
substrate N-benzoyl-L-arginine-ρ-nitroanilide (BAρNA) as your inhibitory activity
whit ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride
(PMSF) and soybean trypsin inhibitor (SBTI). Fibrinogenolytic activity was
determined mixing TLBan with bovine fibrinogen solution at various time
intervals (0 to 24h) and then applied to SDS-PAGE. The effect of ions (Zn2+,
Ca2+, Mg2+ and Mn2+) and pH (5 to 10) on the fibrigenolytic activity was
determined mixing TLBan with bovine fibrinogen against solution of different
ions and pHs. Platelet aggregation assays were performed on platelets-rich
plasma (PRP) isolated by centrifugation and the hemorrhagic activity was
determinated by the area of hemorrhagic spot. On the other hand the studies of
the ability of TLBan to induce inflammation were tested using the mouse
footpad oedema model.
Isolated thrombin like (TLBan) showed proteolytic and fibrinogenolytic activities,
presented a molecular mass of 25835,65Da. Kinetics parameters were Km =
2.9x10-1 M and Vmax = 5.4 x10-1 nmoles ρ-NA/lt/min and was strongly
inhibited by phenylmethanesulfonyl fluoride (PMSF). TLBan evidenced a
fibrinogenolitic activity above the bovine fibrinogen, and hydrolyzed α and β
chains, such fibrigenolitic activity was elevated in presence of Ca2+ and pH 8.0.
Biological activities such as hemorrhage and inflammatory effect reveal that the
TLBan is completely deprived which is characteristic of the family of proteins,
and has the property of inducing platelet aggregation on platelets-rich plasma
(PRP), as well as was inhibited on the presence of phenylmethanesulfonyl
fluoride (PMSF).
Results:
Isolated thrombin like (TLBan) showed proteolytic and fibrinogenolytic activities,
presented a molecular mass of 25835,65Da. Kinetics parameters were Km =
2.9x10-1 M and Vmax = 5.4 x10-1 nmoles ρ-NA/lt/min and was strongly inhibited
by phenylmethanesulfonyl fluoride (PMSF). TLBan evidenced a fibrinogenolitic
activity above the bovine fibrinogen, and hydrolyzed α and β chains, such
fibrigenolitic activity was elevated in presence of Ca2+ and pH 8.0. Biological
activities such as hemorrhage and inflammatory effect reveal that the TLBan is
completely deprived which is characteristic of the family of proteins, and has the
property of inducing platelet aggregation on platelets-rich plasma (PRP), as well
as was inhibited on the presence of phenylmethanesulfonyl fluoride (PMSF).
Conclusion:
TLBan exhibited a high specificity for BAρNA and was inhibited by PMSF. This
enzyme has the ability to degrade fibrinogen α and β chains and the platelet
aggregation was inhibited strongly when was incubated with PMSF.
key-word: serine protease, Bothrops andianus, platelet aggregation, MALDITOF mass spectrometry, fibrinogenolitic activity.
Supported by: CAPES