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A method for faster purification of serine proteinases from Bothrops alternatus and Bothrops moojeni snake venoms

Snake venoms are important sources of substances with a variety of pharmacological activities. Among the different proteins present in these venoms, snake venom serine proteinases (SVSPs) have important effects on the hemostatic system that influence the hemodynamic properties of blood. Bothrops genus snakes presented their venom richly composed of SVSPs thrombin-like, and the isolation of these enzymes is of great interest. In 1994, the Center for the Study of Venoms and Venomous Animals (CEVAP) - UNESP standardized the fibrin sealant derived from snake venom, replacing the bovine thrombin by gyroxin thrombin-like enzyme from Crotalus durissus terrificus (Rattlesnake) and human plasma fibrinogen by buffaloes cryoprecipitate. Despite chromatographic techniques for the purification of gyroxin be well grounded in the literature, that income is considered low. Thus, in addition to gyroxin, other thrombin-like enzymes could be employed in the composition of the new fibrin sealant after being standardized to the purifying and chromatographic performance and widely evaluated for biological activities. Therefore, it is extremely important that in our lab is deployed, standardized and validated a method for the chromatographic purification of other thrombin-like enzymes such as found in Bothrops snake venoms. Thus a two-step chromatographic procedure was developed to routinely purify serine proteinases from Bothrops alternatus and B. moojeni snakes venoms to provide new enzymes for improving the CEVAP’s heterologous fibrin sealant.


INTRODUCTION
The Bothrops snake venoms contain a large variety of proteins and peptides affecting the hemostatic system. These proteins are classified as coagulant, anticoagulant or fibrinolytic factors , Marsh (1994). The high selectivity of these snake venom proteins for especific blood coagulation factors makes these componentes potentially helpful tools to study the mechanisms of action, the regulation and the structure-function relationships of coagulation factors. Among these proteins, serine proteinases serve as tools to study molecular details in the activation of specific factors involved in coagulation and fibrinolytic cascades and are useful in treating various thrombotic and hemostatic conditions. Serine proteinases are found in microorganisms (Siezen, 1999), plants (Rudenskaya, 1995) and numerous animals (Neurath, 1984(Neurath, e 1985. Snake venom proteases, in addition to their contribution to the digestion of the prey, affect various physiological functions. They affect platelet aggregation, blood coagulation, fi brinolysis, complement system, blood pressure and nervous system (Kini. 2005).
These enzymes share many biochemical and structural properties such as a conserved catalytic triad (Ser195, His57, Asp102), and its three-dimensional structure is highly conserved (Greer, 1990;Perona, 1995). Their structure, taken as a whole, is made of two β-barrels constituted of six-strands and separated by the catalytic residues, and of a Cterminal helical segment. Snake venom serine proteinases (SVSPs) found in Bothrops snake venoms are functionally similar to endogenous blood clotting enzymes. They interfere with maintenance and regulation of the blood coagulation cascade by cleaving specific bonds and activating proteins involved in blood coagulation, platelet aggregation, fibrinolysis and in the proteolytic degradation of cells resulting in an imbalance of the hemostatic system (Kini, 2005;Serrano and Maroun, 2005). Many other SVSPs convert fibrinogen into fibrin by cleaving fibrinopeptides A and/or B. As this activity resembles the activity of thrombin, these venom components are commonly named "thrombin-like" enzymes. Snake Venom Serine Proteinases are encountered in the venoms of several Bothrops species. The amino acid sequence homology shared between the SVSPs mentioned above is approximately 65%, however, the homology exhibited by these enzymes with mammalian serine proteinases such as thrombin and trypsin, ranges from 30% to 40%. (Costa et al., 2013) The mechanism of action of MSP1 (Bothrops moojeni), cerastobin (Cerastes vipera), cerastocytin and cerastotin (Cerastes cerastes) on platelet activation is unknown (Marrakchi et al., 1995;Serrano et al.,1995). Bothropase and Reptilase are thrombin-like serine proteinase from Bothrops snake venoms and their administration in vivo results in a transient hypercoagulable state. They have been used to treat patients with urinary or gastrointestinal bleedings. Ancrod and batroxobin, two other serine proteinases from Bothrops atrox venom, have a fibrinogenolytic action and have been used in the treatment of deep vein thrombosis and peripheral vascular occlusive diseases.
In 1994, the Center for the Study of Venoms and Venomous Animals (CEVAP) -UNESP standardized the fibrin sealant derived from snake venom, replacing the bovine thrombin by thrombin-like enzyme from Crotalus durissus terrificus gyroxin, and human plasma fibrinogen by buffaloes cryoprecipitate. Despite chromatographic techniques for the purification of gyroxin be well grounded in the literature, that income is considered low.
Thus, in addition to gyroxin, other thrombin-like enzymes could be employed in the composition of the new fibrin sealant after being standardized to the purifying and chromatographic performance and widely evaluated for biological activities. Therefore, it is extremely important that in our lab be deployed, standardized and validated a method for the chromatographic purification of other thrombin-like enzymes such as found in Bothrops snake venoms.

PURPOSE
The purpose of this work was to certify a faster methodology than usual to isolate TLEs from Bothrops alternatus and B. moojeni snake venoms, carachterize them and compare with other SVSP TLE deposited in databases, aiming to obtain new enzymes for improving CEVAP's heterologous fibrin sealant.

METHODS
The venoms were obtained from the "pool" of venoms extracted from adult Bothrops

1-Isolation of TLE serine proteinases from Bothrops snake venoms.
A B Figure 1. 500 mg of crude venom of each Bothrops snake venom were applied to a Benzamidine-Seharose 6B affinity column. The first column figures represents the chromatograms obtained in this experimental phase, where the serine proteinases were located in peak III. Subsequently, the fraction III was subjected to RP-HPLC (second column) in a Shimadzu C18 column, revealing new fractions. The fractions marked with * showed coagulant activity. Insert: Electrophoretic profiles on SDS-PAGE (13%) under denaturing and reducing conditions. A-B. alternatus; B-B. moojeni.    Several serine proteinases from Bothrops snakes venom have been purified and characterized, such as Bhalternin and Balterobin, wich have been isolated from Bothrops alternatus venom (Costa et al., 2010;Smolka et al., 1998), MSP 1, MSP 2, MMO3 and Batroxobin, isolated from Bothrops moojeni venom (Oliveira et al., 1999;Serrano et al., 1993;Stocker and Barlow, 1976), serine proteinases also have been identified in the venoms of Bothrops jararacussu (Bortoleto et al., 2002;Hill-Eubanks et al., 1989),

4-Serine Proteinases N-terminal amino acids sequencing
Bothrops atrox (Itoh et al., 1987;Kirby et al., 1979;Petretski et al., 2000), Bothrops jararaca (Mandelbaum and Henriques, 1964;Nishida et al., 1994;Serrano et al., 1995), BpSP-I from B. pauloensis (Costa et al.,2009)  According to their specificity for cleaving fibrinogen chains, the serine proteinases have been classified as α, β and γ-fibrinogenases. SVSPs preferentially cleave the Bb-chain The knowledge about the components constituents of snake venoms has a great importance in the understanding of the venom as a whole. To understand the extreme complexity of these venoms, it is necessary to isolate these constituents and identify their individual properties. From this identification, we can search how to combat or explore the pharmacological effects caused by specific components of the venom.

CONCLUSION
The purification process developed in this study is faster and more economical compared to the other existing methods, since it uses only two chromatographic steps for obtaining the serine proteinases of B. alternatus and B. moojeni. All isolated TLEs showed molecular mass of 30-40 kDa, and their partial sequences sharing high identity with other SVSPs, such as venom plasminogen activator, platelet-aggregating enzyme and lectins, according