RESUMO
The high content and quality of protein in Andean legumes make them valuable for producing protein hydrolysates using proteases from bacteria isolated from extreme environments. This study aimed to carry out a single-step purification of a haloprotease from Micrococcus sp. PC7 isolated from Peru salterns. In addition, characterize and apply the enzyme for the production of bioactive protein hydrolysates from underutilized Andean legumes. The PC7 protease was fully purified using only tangential flow filtration (TFF) and exhibited maximum activity at pH 7.5 and 40 °C. It was characterized as a serine protease with an estimated molecular weight of 130 kDa. PC7 activity was enhanced by Cu2+ (1.7-fold) and remained active in the presence of most surfactants and acetonitrile. Furthermore, it stayed completely active up to 6% NaCl and kept Ì´ 60% of its activity up to 8%. The protease maintained over 50% of its activity at 25 °C and 40 °C and over 70% at pH from 6 to 10 for up to 24 h. The determined Km and Vmax were 0.1098 mg mL-1 and 273.7 U mL-1, respectively. PC7 protease hydrolyzed 43%, 22% and 11% of the Lupinus mutabilis, Phaseolus lunatus and Erythrina edulis protein concentrates, respectively. Likewise, the hydrolysates from Lupinus mutabilis and Erythrina edulis presented the maximum antioxidant and antihypertensive activities, respectively. Our results demonstrated the feasibility of a simple purification step for the PC7 protease and its potential to be applied in industrial and biotechnological processes. Bioactive protein hydrolysates produced from Andean legumes may lead to the development of nutraceuticals and functional foods contributing to address some United Nations Sustainable Development Goals (SDGs).
Assuntos
Fabaceae , Micrococcus , Hidrolisados de Proteína , Micrococcus/metabolismo , Micrococcus/enzimologia , Concentração de Íons de Hidrogênio , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Peso Molecular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Peru , Temperatura , Serina Proteases/metabolismo , Serina Proteases/isolamento & purificação , Serina Proteases/química , Estabilidade Enzimática , Cloreto de Sódio/metabolismo , Cloreto de Sódio/farmacologia , Hidrólise , CinéticaRESUMO
Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.
Assuntos
Fibrinogênio , Mannheimia haemolytica , Serina Proteases , Animais , Mannheimia haemolytica/enzimologia , Ovinos , Bovinos , Fibrinogênio/metabolismo , Concentração de Íons de Hidrogênio , Serina Proteases/metabolismo , Serina Proteases/isolamento & purificação , Temperatura , Proteólise , Peso Molecular , Gelatina/metabolismo , Estabilidade Enzimática , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Espectrometria de Massas , Cromatografia por Troca Iônica , Suínos , Fatores de Virulência/metabolismo , Fatores de Virulência/isolamento & purificaçãoRESUMO
A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.
Assuntos
Cajanus , Folhas de Planta , Humanos , Cajanus/química , Folhas de Planta/química , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Proteínas de Plantas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismoRESUMO
Proteases can be used in several biotechnological processes including detergent, food and leather industries. In the leather industry, dehairing is carried out by chemicals, which pollute the environment. Therefore, to make the hair removal process environmentally friendly, a protease produced by Aspergillus terreus has been purified, biochemically characterized and had an efficient ability to remove hair from bovine leather. The protease was produced using 1% wheat bran and was purified 2.3-fold using two chromatographic steps showing a molecular weight of 90 kDa. Optimal temperature and pH were 50 °C and 6.5, respectively. Thermal stability was up to 1 h at 50 °C. Protease was stable to detergents like Tween 80 and to organic solvents. The activity was activated by Ca2+ and inhibited by Hg2+ and Cu2+. The enzyme was classified as serine protease, by the inhibition by PMSF and was stable to reducing agents. It hydrolyzed casein, azocasein, BSA, egg albumin and BTpNA. The Km and Vmax values were 0.65 ± 0.03 mg/mL and 3.66 ± 0.18 µmol/min, respectively. Remarkable properties about temperature, pH, stability to detergents and reducing agents ensure that the protease from A. terreus can be an excellent candidate for industrial applications, particularly in the leather industry.
Assuntos
Aspergillus/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Química Verde/métodos , Remoção de Cabelo/métodos , Serina Proteases/química , Serina Proteases/metabolismo , Animais , Biotecnologia/métodos , Cálcio/metabolismo , Bovinos , Cobre/metabolismo , Detergentes/química , Ativação Enzimática , Estabilidade Enzimática , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Mercúrio/metabolismo , Peso Molecular , Substâncias Redutoras/química , Serina Proteases/isolamento & purificação , Solventes/química , TemperaturaRESUMO
The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (MPEG), concentrations of PEG (CPEG) and citrate (CCIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°m (-10.89 kJ mol-1), ΔHm (-5.0 kJ mol-1) and partition ΔSm (19.74 J mol-1 K-1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40-50 °C and 9.0-11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.
Assuntos
Aspergillus/enzimologia , Polietilenoglicóis/química , Serina Proteases/biossíntese , Serina Proteases/isolamento & purificação , Citrato de Sódio/química , Termodinâmica , Água/química , Concentração de Íons de Hidrogênio , Íons/farmacologia , Metais/farmacologia , Peso Molecular , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacos , TemperaturaRESUMO
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 5.80â»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 Bß chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.
Assuntos
Proteínas de Répteis , Serina Proteases , Venenos de Serpentes/enzimologia , Adulto , Animais , Coagulação Sanguínea/efeitos dos fármacos , Bothrops , Estabilidade Enzimática , Feminino , Fibrinogênio/metabolismo , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Proteínas de Répteis/química , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/farmacologia , Adulto JovemRESUMO
An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0-11.0, the optimum being at pHâ¯9.0. The enzyme was stable at 40⯰C for 180â¯min, enhanced by Mg++ and Ca++, but inhibited by Zn++, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Kmâ¯=â¯0.434â¯mg/mL and Vmaxâ¯=â¯7.739â¯mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8â¯U/mg) had a molecular mass of 49.3â¯kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.
Assuntos
Aspergillus/enzimologia , Colagenases/química , Colagenases/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Cromatografia por Troca Iônica , Colagenases/metabolismo , Detergentes/farmacologia , Ativação Enzimática , Estabilidade Enzimática/efeitos dos fármacos , Espaço Extracelular/enzimologia , Fermentação , Concentração de Íons de Hidrogênio , Íons , Cinética , Metais , Peso Molecular , Proteólise , Serina Proteases/metabolismo , TemperaturaRESUMO
A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1â¯M NaCl solution) and eluted 3 times (1â¯M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40â¯kDa on SDS-PAGE and optimum activity at 45⯰C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55⯰C, did not lose any activity over 48â¯hâ¯at 25⯰C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Nanopartículas de Magnetita/química , Penicillium/enzimologia , Serina Proteases/isolamento & purificação , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Caseínas/química , Domínio Catalítico , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular , Serina Proteases/química , Serina Proteases/metabolismoRESUMO
Plant proteases are capable of performing several functions in biological systems, and their use is attractive for biotechnological process due to their interesting catalytic properties. Bromelia pinguin (aguama) is a wild abundant natural resource in several regions of Central America and the Caribbean Islands but is underutilized. Their fruits are rich in proteases with properties that are still unknown, but they represent an attractive source of enzymes for biotechnological applications. Thus, the proteolytic activity in enzymatic crude extracts (CEs) from wild B. pinguin fruits was partially characterized. Enzymes in CEs showed high proteolytic activity at acid (pH 2.0-4.0) and neutral alkaline (pH 7.0-9.0) conditions, indicating that different types of active proteases are present. Proteolytic activity inhibition by the use of specific protease inhibitors indicated that aspartic, cysteine, and serine proteases are the main types of proteases present in CEs. Activity at pH 3.0 was stable in a broad range of temperatures (25-50 °C) and retained its activity in the presence of surfactants (SDS, Tween-80), reducing agents (DTT, 2-mercapoethanol), and organic solvents (methanol, ethanol, acetone, 2-propanol), which suggests that B. pinguin proteases are potential candidates for their application in brewing, detergent, and pharmaceutical industries.
Assuntos
Ácido Aspártico Proteases/química , Bromelia/enzimologia , Cisteína Proteases/química , Frutas/enzimologia , Proteínas de Plantas/química , Serina Proteases/química , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/isolamento & purificação , Bromelia/química , Cisteína Proteases/isolamento & purificação , Ditiotreitol/química , Ensaios Enzimáticos , Frutas/química , Concentração de Íons de Hidrogênio , Cinética , Mercaptoetanol/química , Extratos Vegetais/química , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Polissorbatos/química , Inibidores de Proteases/química , Proteólise , Serina Proteases/isolamento & purificação , Dodecilsulfato de Sódio/química , Solventes/químicaRESUMO
Filamentous fungi secrete diverse peptidases with different biochemical properties, which is of considerable importance for application in various commercial sectors. In this study, we describe the isolation of two fungal species collected from the soil of decayed organic matter: Aspergillus fischeri and Penicillium citrinum. In a submerged bioprocess, we observed better peptidase production with the fungus P. citrinum, which reached a peak production at 168 h with 760 U/mL, in comparison with the fungus A. fischeri, which reached a peak production at 72 h with 460 U/mL. In both situations, the fermentative medium contained 0.5% crushed feathers as a source of nitrogen. On performing biochemical characterization, we detected two alkaline serine peptidases: The one secreted by P. citrinum had optimal activity at pH 7.0 and at 45°C, while the one secreted by A. fischeri had optimal activity in pH 6.5-8 and at 55-60°C. Metallic ions were effective in modulating these peptidases; in particular, Cu2+ promoted negative modulation of both peptidases. The peptidases were stable and functional under conditions of nonionic surfactants, temperatures up to 45°C for 1 h, and incubation over a wide pH range. In addition, it was observed that both peptidases had the capacity to hydrolyze collagen and performed well in removing an egg protein stain when supplemented into a commercial powder detergent; this was especially true for the peptidase from P. citrinum.
Assuntos
Aspergillus/enzimologia , Colagenases/isolamento & purificação , Penicillium/enzimologia , Serina Proteases/isolamento & purificação , Aspergillus/química , Aspergillus/metabolismo , Colagenases/química , Colagenases/metabolismo , Detergentes/metabolismo , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Metais/metabolismo , Penicillium/química , Penicillium/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , TemperaturaRESUMO
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.
Assuntos
Venenos de Crotalídeos/química , Serina Proteases/isolamento & purificação , Sequência de Aminoácidos , Animais , Bothrops , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Serina Proteases/químicaRESUMO
Peptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45°C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S2 did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S1 showed higher catalytic efficiency than the S2 and S3 subsites.
Assuntos
Aspergillus/enzimologia , Serina Proteases/isolamento & purificação , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Serina Proteases/química , Serina Proteases/metabolismo , TemperaturaRESUMO
Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.
Assuntos
Venenos de Crotalídeos/enzimologia , Crotalus , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , Animais , Coagulação Sanguínea , Bovinos , Clonagem Molecular , Cobre/metabolismo , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Fibrinogênio/metabolismo , Fibrinopeptídeo A/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Proteases/química , Serina Proteases/genética , Temperatura , Zinco/metabolismoRESUMO
BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Mariposas/enzimologia , Serina Proteases/farmacologia , Anafilatoxinas/química , Anafilatoxinas/isolamento & purificação , Animais , Complexo de Ataque à Membrana do Sistema Complemento/química , Eritrócitos/efeitos dos fármacos , Humanos , Proteínas de Insetos/isolamento & purificação , Proteólise , Serina Proteases/isolamento & purificaçãoRESUMO
This paper presents a novel serine protease (SP) isolated from Bothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu(2+) significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated from Bothrops pirajai snake venom.
Assuntos
Venenos de Crotalídeos , Fator VIII/química , Fibrinólise , Serina Proteases/química , Serina Proteases/isolamento & purificação , Animais , Bothrops , Humanos , Peso Molecular , Fluoreto de Fenilmetilsulfonil/química , Inibidores de Serina Proteinase/químicaRESUMO
The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.
Assuntos
Venenos de Aranha/toxicidade , Aranhas/química , Animais , Antivenenos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/isolamento & purificação , Proteínas de Artrópodes/toxicidade , Biomarcadores Tumorais/química , Biomarcadores Tumorais/isolamento & purificação , Feminino , Humanos , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/isolamento & purificação , Hialuronoglucosaminidase/toxicidade , Masculino , Modelos Moleculares , Fosfolipase D/química , Fosfolipase D/isolamento & purificação , Fosfolipase D/toxicidade , Proteômica , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/toxicidade , Picada de Aranha/patologia , Venenos de Aranha/química , Venenos de Aranha/imunologia , Aranhas/anatomia & histologia , Aranhas/fisiologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Envenomation by Bothrops species results, among other symptoms, in hemostatic disturbances. These changes can be ascribed to the presence of enzymes, primarily serine proteinases some of which are structurally similar to thrombin and specifically cleave fibrinogen releasing fibrinopeptides. A rapid, three-step, chromatographic procedure was developed to routinely purify serine proteinases from the venoms of Bothrops alternatus and Bothrops moojeni. The serine proteinase from B. alternatus displays an apparent molecular mass of ~32 kDa whereas the two closely related serine proteinases from B. moojeni display apparent molecular masses of ~32 kDa and ~35 kDa in SDS-PAGE gels. The partial sequences indicated that these enzymes share high identity with serine proteinases from the venoms of other Bothrops species. These proteins coagulate plasma and possess fibrinogenolytic activity but lack fibrinolytic activity.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Serina Proteases/isolamento & purificação , Sequência de Aminoácidos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Plasma/efeitos dos fármacos , Alinhamento de Sequência , Análise de Sequência de Proteína , Serina Proteases/farmacologiaRESUMO
We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BA ρ NA, with a broad optimum pH (7-10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, "ex vivo", a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Serina Proteases/isolamento & purificação , Serina Proteases/farmacologia , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/química , Fibrinogênio/metabolismo , Cinética , Masculino , Camundongos , Dados de Sequência Molecular , Peso Molecular , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Serina Proteases/química , Espectrometria de Massas por Ionização por Electrospray , Viperidae/metabolismoRESUMO
Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca(2+) ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators.
Assuntos
Bothrops/metabolismo , Protrombina/metabolismo , Serina Proteases/metabolismo , Venenos de Serpentes/enzimologia , Animais , Ativação Enzimática/fisiologia , Serina Proteases/isolamento & purificaçãoRESUMO
The present study aimed to evaluate the effects of two serine proteases from Bothrops pirajai snake venom, named BpirSP27 and BpirSP41, on the complement system and the inflammatory response. The effects of these enzymes on the human complement system were assessed by kinetic hemolytic assays, evaluating the hemolysis promoted by the classical/lectin (CP/LP) and alternative (AP) pathways after incubation of normal human serum with the serine proteases. The results suggested that these enzymes were able to induce modulation of CP/LP and AP at different levels: BpirSP41 showed higher inhibitory effects on the hemolytic activity of CP/LP than BpirSP27, with inhibition values close to 40% and 20%, respectively, for the highest concentration assayed. Regarding AP, both enzymes showed percentages of inhibition of the hemolytic activity around 20% for the highest concentrations tested, indicating similar effects on this complement pathway. The proinflammatory effects of B. pirajai serine proteases were evaluated regarding their ability to induce paw edema, variations in the pain threshold and leukocyte recruitment at the site of injection. Both showed mild effects on these inflammatory processes, leading to low levels of increase of paw volumes and decrease in pain thresholds in rats up to 6 h after injection, and inducing neutrophil recruitment without significant increases in the total number of leukocytes in the inflammatory exudates after 6 and 24 h of administration into mice peritoneal cavity. These results suggest that serine proteases must present a minor role in the inflammation caused by B. pirajai snake venom.