RESUMO
Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO2 to conventional liquid mobile phases. The addition of liquefied CO2 increases diffusivity and decreases viscosity, which inherently leads to a more efficient separation. Herein, EFLC is applied to hydrophobic interaction chromatography (HIC) stationary phases for the first time to study the impact of liquefied CO2 to the chromatographic behavior of proteins. The effects of liquefied CO2 on chromatographic properties, charge state distributions (CSDs), and ionization efficiencies were evaluated. EFLC offered improved chromatographic performance compared to conventional liquid chromatography (LC) methods including a shorter analysis time, better peak shapes, and higher plate numbers. The addition of liquefied CO2 to the mobile phase provided an electrospray ionization (ESI)-friendly and "supercharging" reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in large-scale application.
Assuntos
Quimotripsina/isolamento & purificação , Quimotripsinogênio/isolamento & purificação , Muramidase/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Ribonuclease Pancreático/isolamento & purificação , Animais , Bovinos , Galinhas , Cromatografia Líquida , Quimotripsina/química , Quimotripsina/metabolismo , Quimotripsinogênio/química , Quimotripsinogênio/metabolismo , Espectrometria de Massas , Muramidase/química , Muramidase/metabolismo , Proteínas de Plantas/química , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismoRESUMO
A novel magnetic solid-phase extraction (MSPE) method based on 1-hexyl-3-methyl imidazolium chloride ionic liquid (IL) modified magnetic Fe3O4 nanoparticles, hydroxylated multiwall carbon nanotubes (MWCNTs-OH) and zeolitic imidazolate frameworks (ZIFs) nanocomposites (Fe3O4-MWCNTs-OH@ZIF-67@IL) were proposed and applied to extract α-chymotrypsin. The magnetic materials were synthesized successfully and characterized by X-ray diffraction (XRD), transmission electron microscope (TEM), thermal gravimetric analysis (TGA), fourier transform infrared spectrometry (FT-IR), vibrating sample magnetometer (VSM) and zeta potentials. Subsequently, the UV-vis spectrophotometer at about 280â¯nm was utilized to quantitatively analyze the α-chymotrypsin concentration in the supernatant. Furthermore, single factor experiments revealed that the extraction capacity was influenced by initial α-chymotrypsin concentration, ionic strength, extraction time, extraction temperature and pH value. The extraction capacity could reach up to about 635â¯mgâ¯g-1 under the optimized conditions, absolutely higher than that of extraction for Ovalbumin (OVA), Bovine serum albumin (BSA) and Bovine hemoglobin (BHb). In addition, the regeneration studies showed Fe3O4-MWCNTs-OH@ZIF-67@IL particles could be reused several times and kept a high extraction capacity. Besides, the study of enzymatic activity also indicated that the activity of the extracted α-chymotrypsin was well maintained 93% of initial activity. What's more, the proposed method was successfully applied to extract α-chymotrypsin in porcine pancreas crude extract with satisfactory results. All of above conclusions highlight the great potential of the proposed Fe3O4-MWCNTs-OH@ZIF-67@IL-MSPE method in the analysis of biomolecules.
Assuntos
Quimotripsina/isolamento & purificação , Líquidos Iônicos/química , Nanopartículas de Magnetita/química , Estruturas Metalorgânicas/química , Extração em Fase Sólida/métodos , Animais , Bovinos , Quimotripsina/química , Misturas Complexas/química , Ensaios Enzimáticos , Reutilização de Equipamento , Óxido Ferroso-Férrico/química , Hemoglobinas/isolamento & purificação , Nanopartículas de Magnetita/ultraestrutura , Nanotubos de Carbono/química , Concentração Osmolar , Ovalbumina/isolamento & purificação , Pâncreas/química , Sensibilidade e Especificidade , Soroalbumina Bovina/isolamento & purificação , Suínos , Zeolitas/químicaRESUMO
In this work, quartz crystal microbalance with dissipation (QCM-D) was employed to study the kinetic processes involved in the interaction of proteins with self-assembled monolayers (SAMs) of multimodal (MM) ligands. SAMs were fabricated to mimic two chromatographic multimodal resins with varying accessibility of the aromatic moiety to provide a well-defined model system. Kinetic parameters were determined for two different proteins in the presence of the arginine and guanidine and a comparison was made with chromatographic retention data. The results indicated that the accessibility of the ligand's aromatic moiety can have an important impact on the kinetics and chromatographic retention behavior. Interestingly, arginine and guanidine had very different effects on the protein adsorption and desorption kinetics in these MM systems. For cytochrome C, arginine resulted in a significant decrease and increase in the adsorption and desorption rates, respectively, while guanidine produced a dramatic increase in the desorption rate, with minimal effect on the adsorption rate. In addition, at different concentrations of arginine, two distinct kinetic scenarios were observed. For α-chymotrypsin, the presence of 0.1 M guanidine in the aromatic exposed ligand system produced an increase in the adsorption rate and only a moderate increase in the desorption rate, which helped to explain the surprising increase in the chromatographic salt elution concentration. These results demonstrate that protein adsorption kinetics in the presence of different mobile phase modifiers and MM ligand chemistries can play an important role in contributing to selectivity in MM chromatography.
Assuntos
Quimotripsina/isolamento & purificação , Citocromos c/isolamento & purificação , Técnicas de Microbalança de Cristal de Quartzo , Adsorção , Quimotripsina/química , Quimotripsina/metabolismo , Citocromos c/química , Cinética , Ligantes , Modelos Moleculares , Estrutura Molecular , Espectroscopia Fotoeletrônica , Ligação Proteica , Propriedades de SuperfícieRESUMO
In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10-5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by ß chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.
Assuntos
Quimotripsina/química , Quimotripsina/metabolismo , Neurospora/enzimologia , Plasminogênio/química , Plasminogênio/metabolismo , Quimotripsina/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Peso Molecular , Plasminogênio/isolamento & purificação , Inibidores de Proteases/farmacologia , TemperaturaRESUMO
Herein, we present a water dispersable, magnetic nanoparticle supported "click and release" system. The cleavable linker has been synthesized by using a strain-promoted copper-free "click" reagent to establish the specific link and a fluoride cleavable silane moiety for mild cleavage. Small organic molecules, azide-bearing dyes and functionalized enzymes have been bound to the magnetic particle and released in a bioorthogonal way.
Assuntos
Azidas/isolamento & purificação , Quimotripsina/isolamento & purificação , Química Click , Corantes Fluorescentes/isolamento & purificação , Fluoretos/química , Animais , Azidas/química , Bovinos , Quimotripsina/química , Quimotripsina/metabolismo , Corantes Fluorescentes/química , Nanopartículas de Magnetita/química , Silanos/química , Água/químicaRESUMO
A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3.0, 5.0, and 12.0), showing higher stability at pH 8.0, and was inactivated after 20 min at 55 °C. Lobster chymotrypsin was able to hydrolyze protein substrates at as low as pH 3.0. These results are consistent with the findings of enzyme stability. Activity was assessed after incubation of enzyme with different organic solvents (in the range of 10-50 %); when tested in the presence of acetone, ethanol, propanol, and butanol, lobster chymotrypsin residual activity was >80 %; whereas in the presence of dimethyl sulfoxide (DMSO) and toluene, lobster chymotrypsin residual activity was <80 %. Deduced amino acid sequence, corroborated by mass spectrometry, was determined.
Assuntos
Quimotripsina/análise , Quimotripsina/isolamento & purificação , Suco Gástrico/química , Palinuridae/química , Sequência de Aminoácidos , Análise de Variância , Animais , Sequência de Bases , Cromatografia de Afinidade , Quimotripsina/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Dimetil Sulfóxido , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Oligopeptídeos , Fluoreto de Fenilmetilsulfonil , Proteólise , Análise de Sequência de DNA , Temperatura , ToluenoRESUMO
A novel fibrinolytic enzyme was purified from Lyophyllum shimeji, a popular edible mushroom in Asia. The enzyme was purified using combination of anion exchange chromatography on a Mono Q 5/5 column and size exclusion gel filtration chromatography on Superdex 200 100/300 column. This purification protocol resulted 80.9-fold purification of the enzyme and a final yield of 5.7%. The molecular weight of the purified enzyme was estimated to be 21 kDa by SDS-PAGE and size exclusion gel filtration. The N-terminal amino acid sequence was found to be ITFQSASP, which is dissimilar from that of known fibrinolytic enzymes. The purified enzyme was a neutral protease with an optimal reaction pH and temperature of 8.0 and 37°C, respectively. Enzymatic activity was inhibited by Cu(2+) and Co(2+). It was also significantly inhibited by PMSF and TPCK. Furthermore, it was found to exhibit a higher specificity for S-7388, a well-known chymotrypsin chromogenic substrate, indicating chymotrypsin like serine metalloprotease. The relative fibrinolytic activity of 5 µg purified enzyme have two fold more activity than 1 unit/ml of plasmin on fibrin plate. Furthermore, purified enzyme preferentially hydrolyzed the Aα-chain followed by the Bß- and γ-chain of fibrinogen, which is precursor of fibrin. Therefore, these data suggests that the fibrinolytic enzyme derived from edible mushroom, L. shimeji, might be useful for thrombolytic therapy and preventing thrombotic disease.
Assuntos
Agaricales/enzimologia , Quimotripsina/isolamento & purificação , Quimotripsina/metabolismo , Fibrinólise , Sequência de Aminoácidos , Cromatografia em Gel , Quimotripsina/química , Eletroforese em Gel de Poliacrilamida , Fibrina/metabolismo , Fibrinogênio/metabolismo , Concentração de Íons de Hidrogênio , Metaloproteases/química , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Peso Molecular , Serina/metabolismo , Temperatura , TromboseRESUMO
In this paper, we present a comparative analysis of different methods of purification of proteasomes from the culture medium in which proerithroleukemia human K562 cells were grown. The results obtained allowed us to purify proteasomes from samples of conditioned cell culture medium and control the quality of the proteasome preparations at all stages of their separation. Extracellular proteasomes purified via different approaches possess all the three types of peptidase activity described for intracellular counterparts.
Assuntos
Caspases/isolamento & purificação , Quimotripsina/isolamento & purificação , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Tripsina/isolamento & purificação , Caspases/química , Quimotripsina/química , Meios de Cultivo Condicionados/química , Humanos , Células K562 , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tripsina/química , UbiquitinaçãoRESUMO
Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30-70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg(2+), Fe(2+), Cu(2+), Li(1+), Mg(2+), K(1+), Mn(2+), while Ca(2+) had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K m and V max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 µmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.
Assuntos
Peixes-Gato/metabolismo , Quimotripsina/isolamento & purificação , Vísceras/química , Animais , Fracionamento Químico , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida/veterinária , Concentração de Íons de Hidrogênio , Cinética , Metais Pesados/metabolismo , México , Cloreto de Sódio/metabolismo , TemperaturaRESUMO
Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules. Precipitation with increasing amounts of ammonium sulphate led to increasing size of aggregates. While light scattering estimated the hydrodynamic diameter of these aggregates in the range of 242-1124 nm; Nanoparticle tracking analysis (NTA) gave the value as 130-462 nm. Scanning electron microscopy and gel filtration on Sephadex G-200 showed extensive aggregation in these preparations. Transmission electron microscopy showed that the aggregates had irregular shapes. All the aggregates had about 3× higher catalytic activity than the native enzyme. These aggregates did not differ in λ(max) of fluorescence emission which was around 340 nm. However, all the aggregates showed higher fluorescence emission intensity. Far-UV and near-UV circular dichroism also showed no significant structural changes as compared to the native molecule. Interestingly, HPLC gel filtration (on a hydroxylated silica column) gave 14 nm as the diameter for all preparations. Light scattering of preparations in the presence of 10% ethylene glycol also dissociated the aggregates to monomers of 14 nm. Both these results indicated that hydrophobic interactions were the driving force behind this aggregation. These results indicate: (1) Even without any major structural change, three phase partitioning led to protein molecules becoming highly prone to aggregation. (2) Different methods gave widely different estimates of sizes of aggregates. It was however possible to reconcile the data obtained with various approaches. (3) The nature of the gel filtration column is crucial and use of this technique for refolding and studying aggregation needs a rethink.
Assuntos
Sulfato de Amônio/química , Quimotripsina , Substâncias Macromoleculares , Quimotripsina/química , Quimotripsina/isolamento & purificação , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Tamanho da Partícula , Estrutura Secundária de Proteína , terc-Butil Álcool/químicaRESUMO
We report here a label-free method for ultrasensitive and selective assay of protease activity based on the peptide-induced fluorescence quenching of conjugated polyelectrolyte (PPESO(3)). It is very interesting to find that there is a critical length of oligo-polyarginine (i.e., Arg(5)) below which 1) the quenching efficiency of PPESO(3) is sharply decreased, and more importantly, 2) the trypsin-catalyzed hydrolysis is greatly slowed down. This opens good opportunities for not only the ultrasensitive assay of trypsin, but the specific detection of other proteases if carefully designing an appropriate peptide length and the cleavage site. Herein, the enzyme selected as a proof of concept is chymotrypsin. Due to the essence that any cleavage of the designed peptide probes will result in a notable decrease or even a complete loss of their capability to quench the emission of PPESO(3), the limits of detection for trypsin and chymotrypsin have been found as low as 0.25 ng/mL (11 pM) and 0.15 ng/mL (6 pM), respectively. Both are superior to those of most previous methods by 1-2 orders or higher.
Assuntos
Técnicas Biossensoriais/métodos , Quimotripsina/isolamento & purificação , Oligopeptídeos/química , Peptídeo Hidrolases/isolamento & purificação , Peptídeos/química , Quimotripsina/química , Fluorescência , Hidrólise , Peptídeo Hidrolases/química , Sensibilidade e EspecificidadeRESUMO
Affinity partitioning combines the partitioning behavior of biological macromolecules in aqueous two-phase systems with the principle of biorecognition. Among the numerous substances that have been evaluated as ligands, the reactive dyes constitute a group of low cost textile dyes which have proved to act as biomimetic ligands for many enzymes. The ability of reactive yellow 2 (RY2) to interact with trypsin (TRP) and chymotrypsin (ChTRP) and its behavior in aqueous two-phase systems formed by polyethylene glycol (PEG) and sodium citrate (NaCit) - were investigated. Different variables such as PEG molecular weight, tie line length and dye concentration were analyzed. RY2 showed to bind specifically to both TRP and ChTRP with affinity constants near to 10(3)M(-1). Its partition equilibrium is practically displaced to the top phase in systems formed by PEG of different molecular weight. Addition of this dye to PEG 8000/NaCit systems until a final concentration of 0.196% (w/w) induced an increase in TRP and ChTRP partition coefficients of at least 2 times over that in the absence of the ligand. These findings demonstrate that RY2 fulfils all the requirements to be considered as an affinity ligand in aqueous two-phase partitioning of TRP and ChTRP.
Assuntos
Compostos Azo/química , Extração Líquido-Líquido/métodos , Pâncreas/enzimologia , Serina Endopeptidases/isolamento & purificação , Triazinas/química , Animais , Compostos Azo/farmacologia , Soluções Tampão , Bovinos , Quimotripsina/antagonistas & inibidores , Quimotripsina/isolamento & purificação , Citratos/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Ligantes , Polietilenoglicóis/química , Serina Endopeptidases/química , Citrato de Sódio , Triazinas/farmacologia , Tripsina/isolamento & purificaçãoRESUMO
In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated L-phenylalanine and L-tyrosine methyl ester hydrolysis.
Assuntos
Aminoácidos/metabolismo , Quimotripsina/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Sequência de Bases , Quimotripsina/genética , Quimotripsina/isolamento & purificação , DNA Fúngico/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Esterificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Hidrólise , Cinética , Metarhizium/enzimologia , Metarhizium/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMO
A fibrinolytic enzyme from Bacillus subtilis strain Al was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain Al was a chymotrypsin-like serine protease. In addition, the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0-10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N, which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain Al.
Assuntos
Bacillus subtilis/enzimologia , Fibrina/metabolismo , Fibrinolíticos/metabolismo , Serina Proteases , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Quimotripsina/química , Quimotripsina/isolamento & purificação , Quimotripsina/metabolismo , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Concentração de Íons de Hidrogênio , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , TemperaturaRESUMO
Proteins (bovine serum albumin (BSA), α-chymotrypsin, cytochrome c, and lysozyme) were extracted from 0.5 to 2.0 g L(-1) aqueous solution by adding an equal volume of isooctane solution that contained a surfactant mixture (Aerosol-OT, or AOT, and a 1,3-dioxolane (or cyclic ketal) alkyl ethoxylate, CK-2,13-E5.6), producing a three-phase (Winsor-III) microemulsion with a middle, bicontinuous microemulsion, phase highly concentrated in protein (5-13 g L(-1)) and small in volume (12-20% of entire volume). Greater than 90% forward extraction was achieved within a few minutes. Robust W-III microemulsion systems were formulated at 40°C, or at 25°C by including a surfactant with shorter ethoxylate length, CK-2,13-E3 , or 1.5% NaCl (aq). Successful forward extraction correlated with high partitioning of AOT in the middle phase (>95%). The driving force for forward extraction was mainly electrostatic attractions imposed by the anionic surfactant AOT, with the exception of BSA at high ionic strength, which interacted via hydrophobic interactions. Through use of aqueous stripping solutions of high ionic strength (5.0 wt %) and/or pH 12.0 (to negate the electrostatic attractive driving force), cytochrome c and α-chymotrypsin were back extracted from the middle phase at >75% by mass, with the specific activity of recovered α-chymotrypsin being >90% of its original value.
Assuntos
Ácido Dioctil Sulfossuccínico/química , Emulsões , Proteínas/isolamento & purificação , Quimotripsina/química , Quimotripsina/isolamento & purificação , Citocromos c/química , Citocromos c/isolamento & purificação , Modelos Teóricos , Muramidase/química , Muramidase/isolamento & purificação , Proteínas/química , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificaçãoRESUMO
Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum. In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structure-function studies.
Assuntos
Quimotripsina/genética , Quimotripsina/isolamento & purificação , Sequência de Aminoácidos , Animais , Quimotripsinogênio/química , Quimotripsinogênio/isolamento & purificação , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Corpos de Inclusão/genética , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Pâncreas/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Serina Endopeptidases/química , Serina Endopeptidases/isolamento & purificação , SuínosRESUMO
The condition of chymotrypsin (ChTRP)-Eudragit® (Eu) insoluble complex formation was studied with the aim of applying this information to the separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate. The optimal pH of the complex precipitation was 4.60 for ChTRP-Eudragit® L100 and 5.40 for ChTRP-Eudragit® S100. The polyelectrolyte concentration necessary for the commercial enzyme precipitation was lower than 0.1% (w/v). The complex formation was inhibited by NaCl for both polyelectrolytes. The method was applied to recover the enzyme from bovine homogenate; ChTRP was precipitated by Eudragit® addition. The non-soluble complexes were separated by simple centrifugation and re-dissolved by a pH change to 8.20. The best conditions to recover ChTRP brought about a purification factor of around 4 and 90% yield.
Assuntos
Quimotripsina/isolamento & purificação , Pâncreas/química , Ácidos Polimetacrílicos/química , Animais , Bovinos , Centrifugação/métodos , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Nefelometria e Turbidimetria , Cloreto de Sódio/química , SolubilidadeRESUMO
An alkaline chymotrypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified by precipitation with ammonium sulfate, Sephadex G-100 gel filtration, Mono Q-Sepharose anion-exchange chromatography, ultrafiltration, second Sephadex G-100 gel filtration, and a second Mono Q-Sepharose anion-exchange chromatography with a 80-fold increase in specific activity. The molecular weight of the purified alkaline chymotrypsin was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 10.0-11.0 using succinyl-L-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPNA) as a substrate. The relative activities at pH 7.0 and 12.0 were about 66% and 45.5%, respectively. Further, the enzyme was extremely stable over a broad pH range (6.0-12.0). The optimum temperature for enzyme activity was 50 degrees C, and the enzyme displayed higher enzyme activity at low temperatures when compared to other enzymes. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulfonyl-fluoride (PMSF), a serine protein inhibitor, and N-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), a chymotrypsin specific inhibitor. The N-terminal amino acid sequence of the first nine amino acids was IVNGEEAVP. The chymotrypsin kinetic constants, Km and kcat on SAAPNA as a substrate, were 30.7 microM and 14.35 s(-1), respectively, while the catalytic efficiency kcat/Km was 0.465 microM(-1) s(-1). The high activity at high alkaline pH and low temperatures make this protease a potential candidate for future use in detergent processing industries.
Assuntos
Quimotripsina/isolamento & purificação , Dourada , Sequência de Aminoácidos , Animais , Catálise , Cromatografia em Gel , Cromatografia por Troca Iônica , Quimotripsina/química , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Cinética , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Two chymotrypsins (chymotrypsins A and B) have been purified to homogeneity from the hepatopancreas of Japanese sea bass ( Lateolabrax japonicus ) by ammonium sulfate fractionation and chromatographies on DEAE-Sepharose and Phenyl-Sepharose. Two-dimensional electrophoresis (2-DE) analysis revealed that the molecular masses of chymotrypsins A and B were approximately 27.0 and 27.5 kDa, respectively. Their respective isoelectric points were 8.0 and 7.0. Purified chymotrypsins also revealed a single band on native-PAGE, whereas their mobilities were quite different. Optimum temperature and pH of chymotrypsins A and B were 45 degrees C and 8.0, respectively. Both enzymes were strongly inhibited by chymostatin, phenylmethanesulfonyl fluoride (PMSF), and Pefabloc SC, but slightly inhibited by metalloproteinase inhibitor of 1,10-phenanthroline and EDTA. Using Suc-Leu-Leu-Val-Tyr-MCA as substrate, apparent K(m) values of chymotrypsins A and B were 0.8 and 1.1 microM and k(cat) values were 2.7 and 2.0 s(-1), respectively. The N-terminal amino acid sequences of chymotrypsins A and B were determined to the 21st and 18th residues, respectively, and were identical. These sequences exhibited high identities to chymotrypsins from other animals. The digestive effect of the two chymotrypsins on myofibrillar proteins indicated their effectiveness in the degradation of food proteins.
Assuntos
Bass , Quimotripsina/química , Proteínas de Peixes/química , Hepatopâncreas/enzimologia , Animais , Quimotripsina/isolamento & purificação , Estabilidade Enzimática , Proteínas de Peixes/isolamento & purificação , Hepatopâncreas/química , Ponto Isoelétrico , Peso MolecularRESUMO
Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Four insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera, and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization using different colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe p-nitroanilide). Chloromethyl ketones (TPCK, N- alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N- carbobenzoxy-Gly-Gly-Phe-CK) inactivated all chymotrypsins tested. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1) s(-1); Z-GGF-CK, 150 to 450 M(-1) s(-1)) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1) s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa value. This is proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed to be an adaptation to the presence of dietary ketones.