High yield purification AND characterization of serine proteinases from the venoms of Bothropsjararaca, Bothrops jararacussu, Bothrops moojeni and Crotalus durissus terrificus 

Murakami, M.T.(1), Laure, H.J.(2), Greene L.J.(2), Arni, R.K.(1)

(1)Department of Physics, IBILCE, UNESP, São José do Rio Preto, SP, (2)Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP.

The serine proteinases encountered in snake venoms, especially in the venoms of Viperidae e Crotalidae families, have been the focus of attention since they have many medical applications, for example, Calloselasma rodhostoma Ancrod, Bothrops atrox moojeni Batroxobin, Bothrops jararaca Bothrombin, Crotalus atrox Calobin, Crotalus adamanteus Crotalase and Trimeresurus flavoviridis Flavoxobin. These enzymes are very important because that are involved in many biological process, such as plasminogen activation, factor II_a, V, X activation in the blood coagulation cascade and platelet aggregation. The aim of this work was to develop a rapid procedure for purifying large quantities of the serine proteases from the venom of Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Crotalus durissus terrificus in high purity for crystallographic and biochemical studies. The serine proteinases from the venoms were purified using affinity chromatography on benzamidine-sepharose 6B as an important step. The purity and molecular masses were estimated by SDS-PAGE. The crystallizations were carried out by the hanging-drop vapor-diffusion method using 24-well tissue-culture plates. The activities of these enzymes were tested using bovine fibrinogen as substrates. These enzymes were present as single bands in silver stained SDS-PAGE gels and had molecular masses of approximately 28kDa. The sequence of the first fifteen amino acids of the enzyme from the venom of Bothrops jararacussu was determined and compared to the cDNA sequences of venom proteases available in the data bank that demonstrated high homology. The single crystals obtained prove the purity of these enzymes. X-ray diffraction data of the enzymes from Bothrops e Crotalus sp. have been collected to high resolution and the structure determination is in progress. We are currently trying to improve the quality of the crystals of the other enzymes by including substrates and inhibitors. This work will contribute to improve our understanding of the stereochemical requirements of these enzymes.

Financial support: FAPESP and CNPq.

CORRESPONDENCE TO:

Mário Tyago Murakami, Rua Belmonte, nº1184, São José do Rio Preto, SP, CEP: 15054-120, Brasil, Email: qualitus@uol.com.br