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A novel method of antithrombin (AT) purification from Bothrops jararaca snake plasma was developed to obtain this protein using a waste supernatant from B. jararaca fibrinogen purification. The AT purification was achieved by affinity chromatography on HiTrap Heparin HP. The results showed an efficient purification process yielding pure AT (purity 65-fold and specific activity 368.91). In conclusion, we showed a feasible purification method of AT from B. jararaca plasma using a discarded material. This feature is important, considering the limitation of material, such as snake plasma, and could also be useful to obtain pure plasma proteins from other animals, including human plasma.
Antithrombin (AT), a serine protease inhibitor (serpin), is the most important anticoagulant molecule in mammalian blood. It is the most effective and versatile inhibitor of coagulation proteases, controlling enzymes such as thrombin, factors Xa, IXa, XIa, XIIa, and plasma kallikrein. It is believed that AT plays a major role in controlling blood coagulation, thereby preventing widespread thrombosis.
Although some hemostatic diseases, such as myocardial infarction and thrombotic disorders, have stimulated research about human blood coagulation to search selective antithrombotics, little information is available about hemostasis in other vertebrates.1 At present, few studies about blood coagulation have been carried out in reptiles, and so far, the reptilian blood coagulation mechanisms differ from mammals as a result of the absence or low concentrations of intrinsic clotting factors, e.g., factors VIII and IX.2–4 Furthermore, anticoagulant activities have been reported in the blood of the lizard Trachydosaurus rugosus and of snake Bothrops jararaca and noted that blood coagulation is slower in these reptiles than in mammals.5,6 In addition, an anticoagulant protein named BjI was purified from B. jararaca plasma. This protein is a specific thrombin inhibitor that prolongs the blood coagulation of these animals.7 Recently, our group has purified B. jararaca AT, which has similar features to human AT.8
Although the traditional plasma fractionation methods are based on ethanol fractionation, more and more plasma fractionation technologies include chromatographic steps. The use of those methods has improved dramatically the production of pure plasma proteins, such as coagulation factors.9 In addition, protein purification with column chromatographies allows for the discovery of new proteins or new functions of proteins.10 Many years ago, affinity chromatography had been introduced in the large-scale extraction of plasma AT and other proteins, such as factor VIII, von Willebrand-factor VIII complex, factor IX, and Protein C.11
The aim of this paper was to develop a new strategy for purification of B. jararaca AT using the discarded material from fibrinogen purification to conserve valuable material and contribute to evolutionary studies. In addition, this new methodology will allow for the purification of more plasma proteins from a unique protein source.
The Laboratory of Herpetology of Butantan Institute (São Paulo, Brazil) supplied specimens of B. jararaca. The Committee for the Ethical Use of Animals of Butantan Institute approved these experimental protocols (Number 156/04).
Bovine thrombin was purchased from Roche (USA) and chromogenic substrate S-2238 (D-Phe-Pip-Arg-pNA) from Chromogenix (Italy). HiTrap Heparin HP column (1 mL) and precast polyacrylamide gels (PhastGel IEF 3-10) were purchased from GE Healthcare (Sweden). Microplates were acquired from Nalgene Nunc International (USA). All other reagents were of analytical grade or better.
Adult snakes (n=14) were anesthetized with pentobarbital (30 mg kg–1) before being bled by puncturing the aorta. Samples of snake blood were collected in the proportion of 9 vol blood to 1 vol 3.8% sodium citrate solution. Plasma was obtained by centrifugation at 1200 g for 15 min at room temperature and stored at –20°C.
This step has been done following Vieira et al.12 Briefly, 80 mM BaCl2, 50 mM [epsion]-amino caproic acid, 1 mM PMSF, and 5 mM benzamidine were added to plasma (90 mL), which was homogenized for 30 min at 4°C. The plasma was centrifuged at 4500 g for 20 min at 4°C, and the pellet was discarded. Ammonium sulfate was added to the supernatant to achieve 25% of saturation. The solution was stirred for 1 h at 4°C and centrifuged at 7000 g for 20 min at 4°C. The supernatant was kept frozen at –20°C.
The supernatant from fibrinogen purification was centrifuged at 5000 g for 15 min at 4°C, and the pellet was discarded. This supernatant was filtered through paper filter and cellulose membrane Millex (0.45 μm) to remove salt precipitates or to clarify the sample. This solution (7 mL) was diluted in 3.5 mL 0.1 M Tris and 0.01 M sodium citrate buffer, pH 7.4, containing 0.25 M NaCl. This solution was applied to a HiTrap Heparin HP column (1 mL), equilibrated previously with the same buffer, connected to an ÄKTA chromatography system (GE Healthcare) at a flow rate of 1 mL/min. The elution of proteins was performed by a step-wise gradient with 2 M NaCl in the same buffer. Protein concentration was monitored by measuring the absorbance at λ = 280 nm.13 Fractions (1 mL) were collected, and the AT activity was measured by thrombin inhibition using the chromogenic substrate S-2238. Fractions containing AT activity were pooled and submitted to SDS-PAGE.
AT inhibitory activity was measured using the chromogenic substrate S-2238 after addition of an excess of heparin and thrombin, according to the manufacturer's recommendations (Chromogenix). Briefly, 100 μL sample (prediluted 60-fold in 50 mM Tris buffer, pH 8.4, containing 7.5 mM EDTA, 3 U heparin, and 175 mM NaCl) was incubated at 37°C for 5 min, followed by the addition of 25 μL bovine thrombin (2 U mL–1) and incubation at 37°C for 30 s. Afterwards, 15 μL chromogenic substrate S-2238 (4 mM) were added. The thrombin residual activity was measured by absorbance at λ = 405 nm after 5 min incubation at 37°C.
Plasma from different species is a valuable source for biomarker discovery in clinical and animal samples16 and contributes to evolutionary studies or for therapeutic purposes. The novelty of this work is the development of a new strategy for purification of B. jararaca AT using the discarding material from fibrinogen isolation to conserve material (Figure 1,a).
The first step of AT purification was an adsorption using BaCl2, which was essential to remove vitamin K-dependent proteins (factors II, VII, IX, X)17–19 to avoid fibrinogen and AT inactivation during the purification process. The ammonium sulfate precipitation step results in fibrinogen precipitation,12 allowing the separation of AT and fibrinogen.
B. jararaca AT activity was purified by a unique chromatography step using an affinity chromatography on a HiTrap Heparin HP column. AT inhibitory activity was eluted by step-wise increasing NaCl to 2 M (Figure 1, b). This procedure was used to isolate bovine AT, and our group had also used the same method for human (results not shown) and B. jararaca AT,8 in which a high degree of purification was obtained (results not shown). The fractions were pooled and dialyzed against distilled water during 16 h at 4°C. AT purification was about 65-fold, and specific activity was 368.91 (Table 1). These results are similar to those obtained to AT, purified straight from plasma,8 but it is interesting to note that the yield and the specific activity of AT purified in this work are lower than AT purified directly from the plasma (AT purification was approximately 86-fold, and specific activity was 500).8 This could be explained by the use of ammonium sulfate precipitation, as ammonium sulfate fractionation usually causes a considerable loss of activity, which may be a result of protein denaturation by sulfate ion.20 However, Frost et al.21 had purified AT from ostrich plasma by heparin-Sepharose and Super Q-650S chromatography, without the use of ammonium sulfate precipitation, and AT purification and specific activity were approximately 7.98-fold and 231.5, respectively, which are lower than our results, which showed the advantageous use of this method to obtain high yield and specific activity to AT, combined with improving the purification process and decreasing the costs.
This novel process is fast and reproducible, yielding a homogenous product, as determined by SDS-PAGE (Figure 2).Human AT was the first example of a plasma protein industrially isolated by affinity chromatography. This was achieved by exploiting the ability of AT to bind to heparin, its physiological cofactor.22 This strategy was also used to purify salmon AT.23
We have shown a novel method to purify AT from supernatant discarded from snake plasma fibrinogen purification, based on a classical purification method using affinity chromatography for blood coagulation proteins. Our results were achieved using commercially available columns, which allowed the attainment of a high pure AT from snake B. jararaca using material discarded previously from fibrinogen purification. In conclusion, we showed the possibility to purify two plasma proteins from the same source; this feature is very important considering the limitation of material such as snake plasma. Our present finding could also be useful to obtain high amounts of pure plasma proteins from other animals.
We gratefully acknowledge Fundão de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 04/02224-4, 08/08140-8, and 05/03514-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenão de Aperfeio¸amento de Pessoal de Nível Superior (CAPES) for the financial support and Carolina O. Vieira for supplying the supernatant from fibrinogen purification.