Uns Purification of an α-Fibrinogenase with thrombin-like and kallikrein-like activity from Bothrops leucurus snake venom

Bothrops leucurus snake venom

Resumen

A thrombin-like enzyme from Bothrops leucurus snake venom, named TL-Leuc, was purified in two chromatography steps on CM-Sepharose fast flow and HiPrep Sephacryl S-300. This protease was homogeneous on SDS-PAGE and when analyzed in MS/MS (MALDI TOF\TOF) showed mass of 30710.25 and 32109.69Da after alkylation and reduction. The peptides with significant score were compared with those of other proteins used NCBI-BLAST2 alignment display, showing similarity with other serine proteases. TL-Leuc showed proteolytic activity on fibrinogen, being able to degrade only the Aα-chain. This enzyme caused defibrinogenation when administered intraperitoneally in mice, making the plasma incoagulable. Tl-Leuc showed high ability to coagulate bovine plasma and showing a high clotting activity upon bovine plasma and fibrinogen solution. The hydrolytic activity of Tl-Leuc upon S-2238, S-2266 and S-2302 substrates suggests that this enzyme has thrombin and kallikrein-like activities, respectively. TL-Leuc not show hemorrhagic, myotoxic and  fibrinolytic activities. Taken together, our data showed that TL-Leuc is in fact, a thrombin-like enzyme isoform isolated from Bothrops leucurus snake venom.