Batroxobin

Batroxobin, also known as reptilase, is a snake venom enzyme with Venombin A activity produced by Bothrops atrox and Bothrops moojeni, venomous species of pit viper found east of the Andes in South America. It is a hemotoxin which acts as a serine protease similarly to thrombin, and has been the subject of many medical studies as a replacement of thrombin. Different enzymes, isolated from different species of Bothrops, have been called batroxobin, but unless stated otherwise, this article covers the batroxobin produced by B. moojeni, as this is the most studied variety. Batroxobin, also known as reptilase, is a snake venom enzyme with Venombin A activity produced by Bothrops atrox and Bothrops moojeni, venomous species of pit viper found east of the Andes in South America. It is a hemotoxin which acts as a serine protease similarly to thrombin, and has been the subject of many medical studies as a replacement of thrombin. Different enzymes, isolated from different species of Bothrops, have been called batroxobin, but unless stated otherwise, this article covers the batroxobin produced by B. moojeni, as this is the most studied variety. Bothrops atrox was described by Carl Linnaeus as early as 1758, but batroxobin, the active compound in its venom, was first described only in 1954 by H. Bruck and G. Salem. In the years following, this first description of batroxobin was shown to have several uses in surgery. Because of the increasing interest in the properties of batroxobin, several studies on its hemostatic effect and coagulation have been published. More recently, in 1979, a German study showed the uses of batroxobin (reptilase clot retraction test) as a replacement test for the more commonly used thrombin time. Because the enzyme is unaffected by heparin, it is mostly used when heparin is present in blood. Recent studies emphasize more on improving its uses in surgery, mostly spinal surgery, and the uses as serine protease. Batroxobin is a protein of the serine protease family. Batroxobin is closely related in physiological function and molecular size to thrombin. Five subspecies for the Brazilian lancehead snake (Bothrops atrox) are found. Batroxobin obtained from certain subspecies exhibits the hemostatic efficacy, whereas the protein obtained from other subspecies exhibits the cleavage of fibrinogen. Some of the forms have hemostatic efficacy as main effect, where the other forms have degradation of fibrinogen as main effect. Batroxobin that is naturally extracted from the snake venom is mainly obtained from the snake Bothrops moojeni. But the concentration is low and it is difficult to purify the protein. Often the product remains polluted, this makes it harder to use for clinical purposes. Theoretically, the molecular weight of batroxobin should be around 25.5 kDa. Often, isolated batroxobin is heavier, around 33 kDa. The higher molecular weight is caused by a glycosylation modification during the secretion. The differences in weight result from different possible purification procedures, which can remove different sugar(chains) from the enzyme. Because the batroxobin isolated from venom is highly irregular in quality, it is now more often synthesized in organisms using Bothrops moojeni cDNA. The structure and working mechanism of batroxobin extracted from the Bothrops moojeni have been thoroughly studied. Various subspecies exist and the working mechanisms of each batroxobin differ. As such, the structure of Bothrops moojeni batroxobin is further elucidated. The structure of batroxobin has been studied by various research groups throughout the years. These studies have mostly been performed by biologically synthesizing batroxobin from Bothrops moojeni cDNA, and analyzing this product and using homology models based on other proteases, such as thrombin and trypsin, among others. One of the earlier studies from 1986 showed that the molecular weight is 25.503 kDa, 32.312 kDa with the carbohydrate, and it consists of 231 amino acids. The amino acid sequence exhibited significant homology with other known mammalian serine proteases, such as trypsin, thrombin, and most notably pancreatic kallikrein. It was therefore concluded that it is indeed a member of the serine protease family. Based on the homology, the disulfide bridges were identified and the structure was elucidated further. A later molecular modelling study from 1998 used the homology between glandular kallikrein from the mouse and batroxobin, which is about 40%, to propose a 3D structure for biologically active batroxobin. To date no definite 3D structure has been proposed. After the cDNA nucleotide sequence of batroxobin from Bothrops moojeni was determined back in 1986, a research group from the Kyoto Sangyo university successfully expressed the cDNA for batroxobin in E. Coli in 1990. The recognition sequence for thrombin was used to obtain mature batroxobin. The fusion protein which was obtained was insoluble and was easily purified. After cleaving the fusion protein, the recombinant batroxobin could be isolated by electrophoresis and it was then successfully refolded to produce biologically active batroxobin. This study showed that it was possible to produce batroxobin using micro-organisms, a method which was more promising than isolating the enzyme from extracted snake venom. In 2004, a research group from Korea produced batroxobin by expressing it in the yeast species Pichia pastoris. This recombinant enzyme had a molecular weight of 33 kDa and included the carbohydrate structure. This method of expressing it in Pichia pastoris turned out to be more effective, as the produced enzyme showed cleaving activity which was more specific than thrombin in some cases and was more specific than non-recombinant batroxobin. Therefore, synthesis using Pichia pastoris seems promising for producing high quality recombinant batroxobin.