ISOLATION OF A NEW SERINE PROTEASE (URUPROT) FROM Bothrops alternatus SNAKE VENOM

RIBEIRO, D. A.(1), SILVA, J. A.(1), MARANGONI, S.(1),NOVELLO, J. C.(1)

(1)Departaments of Biochemistry, Institute of Biology State University of Campinas (UNICAMP) Campinas, SP, Brazil.

Objective: Isolation and partial biochemical and biological characterization of a Uruprot serine protease from the venom of Bothrops alternatus.

Methods and Results: The serine protease named Uruprot was isolated using a combination of molecular exclusion and ion-exchange HPLC. 

The approximate molecular mass of the reduced (DTT-treated) protein was approximately 32 kDa as determined by Tricine SDS-PAGE 12.5%. Two dimensional (2D) electrophoresis of uruprot showed an isoelectric point of around 4.6 and confirming the molecular mass in SDS-PAGE.

Amino acid analysis showed Asx/12, Glx/22, Ser/30, Gly/54, His/1, Arg/2, Thr/45, Ala/13, Pro/14, Tyr/6, Val/10, Met/0, half Cys/6, Ile/31, Leu/10, Phe/33 and Lys/9. Uruprot showed a high content of acidic residues (Asp and Glu), and a high content of hydrophobic amino acids (Ser, Thr, Pro, Ile, Leu and Phe). These data corroborate the acidic nature of the protein as well as its hydrophobicity. The N-terminal sequence of Uruprot was VVGGDECNINEHRSLVAIFDS. N-terminal sequence of uruprot showed high homology sequence with the N-terminal sequences of other serine proteases as catroxobin, crotalase and bilineobin. Uruprot had hemolytic activity and inhibited ristocetin-induced aggregation of washed platelets at a minimum concentration of 10 mg/ml. 

Conclusion: In this work, we purified and characterized a serine protease from the venom of a South American pit viper, the “urutu” (Bothrops alternatus).

Thus, uruprot is a serine protease acid with a only band and showed hemolytic activity and inhibit the platelet aggregation induced for ristocetin.

Financial support: CAPES, CNPq and FAPESP.

CORRESPONDENCE TO:

DulcinéiaAparecida Ribeiro, Av. Santa Isabel, 1338, Campinas, SP, CEP: 13084471, Brasil, Email: dribeiro@unicamp.br