BbrzSP-32, the first serine protease isolated from Bothrops brazili venom: Purification and characterization

Abstract

Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated.

Venomics and antivenomics of the poorly studied Brazil’s lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

“…In the murine model, Peruvian B. brazili exhibited minimum hemorrhagic dose (MHD) of 7.40 μg/ mouse), minimum dermonecrotic dose (MND) of 152.15 μg/ mouse, minimum coagulant dose against plasma (MCD-P) and fibrinogen (MCD-F) of 19.20 and 1020.0 μg/mL, respectively, and minimum defibrinogenating dose (MDD) of 7.0 μg/mouse [11]. Although described as a new Bothrops from Brazil 65 years ago [15], very few studies have been reported on the toxin arsenal of the Brazil's lancehead venom, and these were mainly focused on the pharmacological effects and possible biotechnological applications of isolated toxins [21][22][23][24][25][26][27][28][29][30][31], including acidic and basic phospholipase A 2 (PLA 2 ) molecules (myotoxic Braziliase I and II, MTX I and II, brazilitoxins II and III) [23][24][25][26]; a PI-snake venom metaloproteinase (SVMP), with in vitro antiplasmodial properties [27]; coagulant thrombin-like and pro-angiogenic snake venom serine proteinase (SVSP) [28,29]; and a hyaluronidase [30].…”

Agkihpin, a Distinct SVTLE from the Venom of Gloydius halys Pallas: Purification, Characterization and Structure–Activity Determination

“…To investigate the impacts of VSCAs, VSCGHs or VSCGs on the fibrinogenolytic activity of SVTLEs, we compared the surface‐charge potentials and size of active sites pocket (ASP) where those variable sites located. Homology modelling is usually the method of choice when a clear relationship of homology between the sequence of the target protein and at least one known structure is found . By using Swiss‐Model, thirty 3D models for agkihpin and some other SVTLEs proteins from Viperidae snakes and O. hannah , made with crystal structure of a thrombin‐like enzyme isolated from the venom of the snake G .…”

“…Homology modelling is usually the method of choice when a clear relationship of homology between the sequence of the target protein and at least one known structure is found. [9] By using Swiss-Model, thirty 3D models for agkihpin and some other SVTLEs proteins from Viperidae snakes and O. hannah, made with crystal structure of a thrombin-like enzyme isolated from the venom of the snake G. halys, Ahv_TL-I (PDB IDs: 4e7n.1.A), as the best templates, were refined with suitable stereochemical parameters and had highest structure quality. [10] Templates with percent identities above 30% are sufficient to predict the three-dimensional structure between templateprotein and target-protein, [11] and AhV_TL-I has 62.93 -96.20% identity ( Table 3) with the sequence of agkihpin and some similar SVTLEs.…”

PURIFICACIÓN Y CARACTERIZACIÓN BIOQUÍMICA DE LA ENZIMA SIMILAR A TROMBINA DEL VENENO DE LA SERPIENTE Bothrops brazili

“…Los resultados obtenidos se corroboran con los ya reportados por Limán 7 , donde la enzima de B. brazili presenta actividad máxima hasta los 40 ºC, pero pierde rápidamente su actividad llegando a tener solamente el 17 % de actividad a los 70 ºC. Estos resultados indican que la enzima de esta especie no es muy estable al tratamiento térmico (igualmente señalado por Zaqueo et al 8 ), a diferencia de las enzimas que poseen otras especies peruanas, como es el caso de B. pictus, cuya actividad amidolítica se mantiene por encima del 50 % hasta los 90 ºC 4 . La TLE de B. barnetti mostró óptima actividad amidolítica a 40 ºC y mantuvo más del 50 % de la misma hasta los 60 ºC 19 .…”

“…como "jergón shushupe", solo se cuenta con un estudio preliminar de la TLE en su ponzoña, a partir de una purificación parcial y cuya actividad solo ha sido ensayada sobre sustratos cromogénicos 7 , sin embargo, la referencia más reciente con respecto a serinoproteasas de esta especie fue reportada por Zaqueo et al 8 , quienes lograron purificar una de estas enzimas de un espécimen oriundo de Brasil. B. brazili es una especie particularmente abundante en la selva norte del Perú abarcando los departamentos de Loreto y Amazonas, frontera con Ecuador, y es una serpiente capaz de inocular hasta 4mL de veneno por mordedura.…”