title: Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom.
abstract: This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the Bβ chain and BpirSP41 on both Aα and Bβ chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of ∼3.5 μg versus 20 μg for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu(2+) ion and specific serine protease inhibitors. In addition, BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure-function relationship of SVSPs.
authors: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Av. do Café, s/n°, CEP 14040-903, Ribeirão Preto, SP, Brazil. danmenaldo@yahoo.com.br
authors: Danilo Luccas
authors: DL
authors: Menaldo
authors: Carolina Petri
authors: CP
authors: Bernardes
authors: Norival Alves
authors: NA
authors: Santos-Filho
authors: Laura de Andrade
authors: Lde A
authors: Moura
authors: André Lopes
authors: AL
authors: Fuly
authors: Eliane Candiani
authors: EC
authors: Arantes
authors: Suely Vilela
authors: SV
authors: Sampaio
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D001777:Blood Coagulation / Q000187:drug effects; D017837:Bothrops / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005260:Female; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012910:Snake Venoms / Q000201:enzymology* / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D055815:Young Adult
chemical_list: D004151:Dipeptides; D007527:Isoenzymes; D012910:Snake Venoms; C021129:S 2238; D057057:Serine Proteases