BjSP, a novel serine protease from Bothrops jararaca snake venom that degrades fibrinogen without forming fibrin clots (original) (raw)
Related papers
2013
A thrombin-like enzyme named TLBbar was isolated from Bothrops barnetti snake venom and its biochemical and pharmacological characteristics were determined. TLBbar was purified using size exclusion chromatography and reverse phase HPLC, showing molecular mass of 28750.7 Da determined by mass spectrometry. TLBbar serine protease is basic (pI 7.4) and its structure shows similarity with other serine proteases of snake venom. Optimal proteolytic activity was at 37°C and pH 8; this activity was strongly inhibited by PMSF and Leupeptin, however; heparin, and soybean trypsin inhibitor (SBT-I) were ineffective. Kinetic studies on BApNA chromogenic substrate have revealed that TLBbar presents a Michaelis-Menten kinetics, with values of and of 0.433 mM and 0.42 nmol/min, respectively. TLBbar showed high clotting activity upon bovine and human plasma, presenting IC of 125 and minimum dose coagulant (MDC) of 2.23 μg/μL. TLBbar cleavages the Aα chain of bovine fibrinogen, with maximal efficienc...
Acta biochimica et …, 2004
A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bβ chain of fibrinogen while the Aα chain and γ chain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bβ chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mm×0.2 mm×0.2 mm) and used for X-ray diffraction experiments.
2012
This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the Bb chain and BpirSP41 on both Aa and Bb chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of w3.5 mg versus 20 mg for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu 2þ ion and specific serine protease inhibitors. In addition, BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structureefunction relationship of SVSPs. Ó 2012 Elsevier Masson SAS.
Biochimie, 2012
This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry 121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the Bb chain and BpirSP41 on both Aa and Bb chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of w3.5 mg versus 20 mg for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu 2þ ion and specific serine protease inhibitors. In addition, BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structureefunction relationship of SVSPs.
Acta Biochimica et Biophysica Sinica, 2004
A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bβ chain of fibrinogen while the Aα chain and γ chain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bβ chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mm×0.2 mm×0.2 mm) and used for X-ray diffraction experiments.
New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom
Toxins, 2018
Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80–6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC anal...
Proteases from Vipera lebetina Venom Affecting Coagulation and Fibrinolysis
Pathophysiology of Haemostasis and Thrombosis, 2001
Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants in the same venom. We showed that V. lebetina venom contains: (1) proteases that degrade fibrinogen, but not fibrin; (2) fibrinolytic enzyme (lebetase); (3) factor X activator (VLFXA); (4) factor V activator (VLFVA). Fibrinolytic enzyme and VLFXA are metalloproteases; the other studied enzymes are serine proteases. α-Fibrinogenase has no homolog among known serine proteases. β-Fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the β-chain of fibrinogen. Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins. We used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique for the recovery and identification of peptides released by protease hydrolysis and for the detection of human factor X cleav...
Toxins, 2020
A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell’s viper venom in lupus anticoagulant testing, thus achieving greater reliabi...