Partial Purification and Characterization of Bothrops jararaca (Bj) fibrinogen
Vieira, C.O.¹, Tanaka-Azevedo, A.M.¹
1Laboratório de Fisiopatologia, Instituto Butantan, São Paulo, Brasil
Blood coagulation is a complex process composed by sequential chain reactions, which participates many blood proteins resulting in prevention of blood loss. Fibrinogen is a plasma glycoprotein that acts in the end of coagulation cascade. This protein consists in two halves with two pairs of three no identical polypeptide chains Aa, Bb and g, interlinked by disulfide bonds. Thrombin releases fibrinopeptides A and B from Aa and Bb fibrinogen chains, respectively, which results in the fibrinogen conversion to insoluble fibrin.
This work presents the partial purification and characterization of Bj plasma fibrinogen. Briefly, the purification of Bj fibrinogen was done by ammonium sulphate precipitation, followed by gel filtration chromatography (Sephacryl S300) and ionic exchange chromatography (HiTrap DEAE “fast flow”). The protein concentration was determined by absorbance at 280 nm. Fibrinogen measurement was made according to Ratnoff & Menzie method (1951). The fibrinogen purification and recovery were 9.3 fold .and 22.3%, respectively. The molecular masses of fibrinogen chains were 115, 53 and 44 kDa, for Aa, Bb and g, respectively, by SDS-PAGE. The molecular mass of Bj fibrinogen in non reduced condition was about 428 kDa, this protein is bigger than bovine and human fibrinogen (340 kDa). The perspective of this work is to determine the N-terminal sequence of the Bj fibrinogen chains to compare them with other protein chains, previously described.
Ratnoff O. D. & Menzie C. J. Lab. Clin. Med. 37: 316, 1951.
Supported by CNPq and FAPESP (04/02224-4)
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