High-yield Purification Of Serine Proteases From Snake Venoms. 

Zela, S.P., Murakami, M.T., Michelan-Duarte, S., Gava, L.M. and Arni, R.K.

Departamento de Física – IBILCE/UNESP – São José do Rio Preto, São Paulo, Brazil.

Venom thrombin-like and trypsin-like serine proteases have been the focus of recent research since they possess the potential to be used clinically in the treatment of cardiovascular diseases and to control and regulate steps in the blood coagulation cascade. These proteases interfere in the coagulation and fibrinolytic pathways by hydrolyzing fibrinogen releasing fibrinopeptide (FPA), FPB or both. Since they share significant sequence homology with serine proteases such as trypsin and thrombin, they serve as ideal model systems to improve our understanding of the structural and stereo-specific constraints.

A rapid, two-step high-yield process was developed to purify serine proteases from the venoms of B. jararaca, B. jararcussu, Lachesismuta muta and Crotalusdurissus terrificus. Initial purification was achieved by applying the samples of crude venom on an affinity column (benzamidine sepharose 6B) at basic pH. After washing with a buffer containing a high salt concentration to remove unspecifically bound proteins, the serine protease were eluted by reducing the pH or by using a buffer containing benzamidine. Further purification was achieved by ion exchange chromatography (mono-Q). These steps were modified to optimize yield and purity. The samples were characterized for their purity and activity using a variety of spectroscopic and biochemical techniques.

This research was supported by grants from FAPESP, CNPq and CAPES.

Correspondence to: sandropz@bol.com.br