Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom.
A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.
Costa
Júnia de O
Jde O
Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, 38400-902 Uberlândia-MG, Brazil. juniacosta@iftriangulo.edu.br
Fonseca
Kelly C
KC
Mamede
Carla C Neves
CC
Beletti
Marcelo E
ME
Santos-Filho
Norival A
NA
Soares
Andreimar M
AM
Arantes
Eliane C
EC
Hirayama
Silvia N S
SN
Selistre-de-Araújo
Heloísa S
HS
Fonseca
Fernando
F
Henrique-Silva
Flávio
F
Penha-Silva
Nilson
N
de Oliveira
Fábio
F
D000418:Albumins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D017837:Bothrops; D002364:Caseins / Q000737:chemistry; D003435:Crotalid Venoms / Q000201:enzymology* / Q000494:pharmacology; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005088:Exocrine Glands / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D005343:Fibrinolytic Agents / Q000494:pharmacology; D015723:Gene Library; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000473:pathology; D009336:Necrosis / Q000139:chemically induced / Q000473:pathology; D010447:Peptide Hydrolases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D057057:Serine Proteases / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry*; D014757:Viper Venoms / Q000201:enzymology* / Q000494:pharmacology
D000418:Albumins; D002364:Caseins; D003435:Crotalid Venoms; D018076:DNA, Complementary; D005343:Fibrinolytic Agents; D011506:Proteins; D014757:Viper Venoms; C026018:azocasein; D005340:Fibrinogen; D010447:Peptide Hydrolases; D057057:Serine Proteases; C550861:bhalternin protein, Bothrops alternatus; D013917:Thrombin