RESUMO
In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.
Assuntos
Antioxidantes , Caseínas , Enzimas Imobilizadas , Glutaral , Cabras , Iridoides , Pepsina A , Peptídeos , Antioxidantes/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Caseínas/química , Animais , Pepsina A/metabolismo , Pepsina A/química , Glutaral/química , Peptídeos/química , Iridoides/química , Hidrólise , Carvão Vegetal/químicaRESUMO
OBJECTIVE: The aim of this study was to compare two in vitro erosion protocols, in which one simulates in vivo conditions experienced by patients with gastroesophageal disorders or bulimia (HCl-pepsin protocol), and the other simulates the diet of an individual who consumes a high volume of erosive beverages (citric acid protocol). In addition, the mechanical properties and surface gloss of eroded human dentin were compared with those of sound human dentin. MATERIALS AND METHODS: Blocks of cervical dentin were used: sound human dentin (n=10), human dentin with erosive lesions (n=10), and bovine dentin (n=30). Twenty bovine blocks were subjected to either of two erosion protocols (n=10/protocol). In the first protocol, samples were demineralized using HCl-pepsin solution, then treated with trypsin solution. In the second protocol, samples were demineralized with 2% citric acid. Toothbrushing was performed in both protocols using a toothbrushing machine (15 s with a 150 g load). Ten bovine dentin blocks were not subjected to any erosive treatment. All samples of bovine and human dentin were analyzed to obtain Martens hardness values (MH), elastic modulus (Eit*) and surface gloss. One-way ANOVA and Tukey's test were performed to analyze the data (α=0.05). RESULTS: Sound human and eroded human dentin groups showed similar MH and Eit* values (p>0.05); however, sound human dentin showed a higher surface gloss value when compared to eroded human dentin (p<0.05). Sound bovine dentin and HCl-pepsin-treated bovine dentin treatments resulted in similar values for both MH and Eit* (p>0.05), but HCl-pepsin-treated bovine dentin and citric acid-treated bovine dentin resulted in lower surface gloss than sound bovine dentin (p<0.05). CONCLUSIONS: The HCl-pepsin protocol modified bovine dentin properties that could be similar to those that occur on human dentin surfaces with erosive lesions.
Assuntos
Dentina/efeitos dos fármacos , Técnicas In Vitro/métodos , Erosão Dentária/etiologia , Análise de Variância , Animais , Bovinos , Ácido Cítrico/química , Dentina/química , Módulo de Elasticidade , Testes de Dureza , Humanos , Pepsina A/química , Valores de Referência , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Propriedades de Superfície/efeitos dos fármacosRESUMO
Abstract Objective The aim of this study was to compare two in vitro erosion protocols, in which one simulates in vivo conditions experienced by patients with gastroesophageal disorders or bulimia (HCl-pepsin protocol), and the other simulates the diet of an individual who consumes a high volume of erosive beverages (citric acid protocol). In addition, the mechanical properties and surface gloss of eroded human dentin were compared with those of sound human dentin. Materials and Methods Blocks of cervical dentin were used: sound human dentin (n=10), human dentin with erosive lesions (n=10), and bovine dentin (n=30). Twenty bovine blocks were subjected to either of two erosion protocols (n=10/protocol). In the first protocol, samples were demineralized using HCl-pepsin solution, then treated with trypsin solution. In the second protocol, samples were demineralized with 2% citric acid. Toothbrushing was performed in both protocols using a toothbrushing machine (15 s with a 150 g load). Ten bovine dentin blocks were not subjected to any erosive treatment. All samples of bovine and human dentin were analyzed to obtain Martens hardness values (MH), elastic modulus (Eit*) and surface gloss. One-way ANOVA and Tukey's test were performed to analyze the data (α=0.05). Results Sound human and eroded human dentin groups showed similar MH and Eit* values (p>0.05); however, sound human dentin showed a higher surface gloss value when compared to eroded human dentin (p<0.05). Sound bovine dentin and HCl-pepsin-treated bovine dentin treatments resulted in similar values for both MH and Eit* (p>0.05), but HCl-pepsin-treated bovine dentin and citric acid-treated bovine dentin resulted in lower surface gloss than sound bovine dentin (p<0.05). Conclusions The HCl-pepsin protocol modified bovine dentin properties that could be similar to those that occur on human dentin surfaces with erosive lesions.
Assuntos
Humanos , Animais , Bovinos , Técnicas In Vitro/métodos , Dentina/efeitos dos fármacos , Valores de Referência , Propriedades de Superfície/efeitos dos fármacos , Erosão Dentária/etiologia , Reprodutibilidade dos Testes , Análise de Variância , Pepsina A/química , Estatísticas não Paramétricas , Ácido Cítrico/química , Dentina/química , Módulo de Elasticidade , Testes de DurezaRESUMO
Hen eggs are a source of bioactive compounds, of which the hen egg white lysozyme (HEWL) protein. HEWL has a demonstrated antibacterial activity. The aim of this study was to evaluate the antimicrobial activity of native and heated HEWL hydrolysates obtained through hydrolysis with pepsin and to identify their peptides using the reversed phase high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (RP-HPLC-ESI-MS-MS) analysis. Native and heat-treated HEWL was hydrolyzed with pepsin at pH 1.2, and their antibacterial activity was tested against Escherichia coli and Staphylococcus carnosus. Two of the hydrolysates obtained presented high antibacterial activity against Gram-positive and Gram-negative bacteria. Native HEWL hydrolysate was a bactericide at 2.0 mg/mL against E. coli. Fifty-one peptide sequences were identified on the two hydrolysates. Peptides identified are cationic peptides. These peptides are rich in Lys and Arg cationic amino acids and have Trp in their sequences.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Clara de Ovo/química , Muramidase/química , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Galinhas , Cromatografia Líquida de Alta Pressão , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Hidrólise , Muramidase/farmacologia , Pepsina A/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/crescimento & desenvolvimento , Espectrometria de Massas em TandemRESUMO
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab')2 antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab')2 fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab')2 fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab')2 affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab')2 presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab')2 fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.
Assuntos
Anticorpos Antibacterianos , Anticorpos Monoclonais Murinos , Proteínas de Bactérias/imunologia , Fragmentos Fab das Imunoglobulinas , Staphylococcus aureus Resistente à Meticilina/imunologia , Proteínas de Ligação às Penicilinas/imunologia , Pepsina A/química , Animais , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , CamundongosRESUMO
The aim was to investigate the potential of germinated soybean proteins asa source of peptides with anticancer and anti-inflammatory activities produced after simulated gastrointestinal digestion. Protein concentrate from germinated soybean was hydrolysed with pepsin/pancreatin and fractionated by ultrafiltration. Whole digest and fractions>10, 5-10, and<5kDa caused cytotoxicity to Caco-2, HT-29, HCT-116 human colon cancer cells, and reduced inflammatory response caused by lipopolysaccharide in macrophages RAW 264.7. Antiproliferative and anti-inflammatory effects were generally higher in 5-10kDa fractions. This fraction was further purified by semi-preparative chromatography and characterised by HPLC-MS/MS. The most potent fraction was mainly composed of ß-conglycinin and glycinin fragments rich in glutamine. This is the first report on the anti-cancer and anti-inflammatory effects of newly isolated and identified peptides from germinated soybean released during gastrointestinal digestion. These findings highlight the potential of germination as a process to obtain functional foods or nutraceuticals for colon cancer prevention.
Assuntos
Neoplasias do Colo/metabolismo , Glycine max/metabolismo , Peptídeos/metabolismo , Proteínas de Soja/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/fisiopatologia , Digestão , Germinação/efeitos dos fármacos , Humanos , Hidrólise , Camundongos , Pancreatina/química , Pancreatina/metabolismo , Pepsina A/química , Pepsina A/metabolismo , Peptídeos/química , Células RAW 264.7 , Proteínas de Soja/química , Glycine max/química , Glycine max/crescimento & desenvolvimento , Espectrometria de Massas em TandemRESUMO
High pressure processing (HPP) is able to promote changes in enzymes structure. This study evaluated the effect of HP on the structural changes in milk-clotting enzymes processed under activation conditions for recombinant camel chymosin (212MPa/5min/10°C), calf rennet (280MPa/20min/25°C), bovine rennet (222MPa/5min/23°C), and porcine pepsin (50MPa/5min/20°C) and under inactivation conditions for all enzymes (600MPa/10min/25°C) including the protease from Rhizomucor miehei. In general, it was found that the HPP at activation conditions was able to increase the intrinsic fluorescence of samples with high pepsin concentration (porcine pepsin and bovine rennet), increase significantly the surface hydrophobicity and induce changes in secondary structure of all enzymes. Under inactivation conditions, increases in surface hydrophobicity and a reduction of intrinsic fluorescence were observed, suggesting a higher exposure of hydrophobic sites followed by water quenching of Trp residues. Moreover, changes in secondary structure were observed (with minor changes seen in Rhizomucor miehei protease). In conclusion, HPP was able to unfold milk-clotting enzymes even under activation conditions, and the porcine pepsin and bovine rennet were more sensitive to HPP.
Assuntos
Quimosina , Manipulação de Alimentos/métodos , Pressão , Animais , Camelus , Bovinos , Quimosina/química , Quimosina/metabolismo , Quimosina/efeitos da radiação , Estabilidade Enzimática , Interações Hidrofóbicas e Hidrofílicas , Pepsina A/química , Pepsina A/metabolismo , Pepsina A/efeitos da radiação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Suínos , TemperaturaRESUMO
Germination is an inexpensive process to improve the nutritional properties of legumes. The effect of germinating black bean seeds on the production of cotyledon protein hydrolysates (CPH) with antioxidant and antiinflammatory activities was analyzed in this research. After simulated enzymatic digestion, the oxygen radical absorbance capacity (ORAC) of CPH obtained from germinated black beans was lower than that observed for raw cotyledons. There were no significant differences among CPH cellular antioxidant activities (CAA), except for the high CAA of the 120 min hydrolysate obtained from one day germinated black bean cotyledons. The most significant changes due to germination and enzymatic hydrolysis were observed for the inhibition of nitric oxide (NO) production in macrophages. The NO synthesis inhibition observed for raw CPH was reduced after simulated gastrointestinal digestion but for germinated samples the inhibition was doubled. Peptides derived from cell wall proteins produced during germination could be responsible of antiinflammatory activity.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Germinação , Phaseolus/química , Proteínas de Plantas/química , Hidrolisados de Proteína/farmacologia , Animais , Anti-Inflamatórios/química , Antioxidantes/química , Células CACO-2 , Cotilédone/química , Digestão/fisiologia , Humanos , Hidrólise , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Modelos Biológicos , Óxido Nítrico/antagonistas & inibidores , Pancreatina/química , Pepsina A/química , Phaseolus/crescimento & desenvolvimento , Hidrolisados de Proteína/química , Sementes/química , Sementes/crescimento & desenvolvimentoRESUMO
The effects of different thermal (raw, autoclaving or boiling for 5 and 20min) and soaking (with or without) treatments on the degree of hydrolysis (DH) of protein were investigated for selected legumes (Canavalia brasiliensis; Lablab purpureus; pink, red and white colour hulls Vigna unguiculata). Each legume preparation underwent in vitro simulated gastrointestinal tract digestion comprising either pepsin (120min) or pepsin/pancreatin (120/240min) digestion. The DH was determined based on the amount of free amino groups released. Autoclaving for 5min increased the pepsin/pancreatin DH for all the unsoaked and soaked legumes (+20% to 46% units) except Canavalia, while boiling for 5min only increased DH for two soaked legumes (+12% to 28% units). Extending boiling from 5 to 20min increased the DH for three soaked legumes (+5% to 29% units). In conclusion, autoclaving, in general, extensively increased the sequential pepsin/pancreatin DH, while boiling only increased it for selected legumes.
Assuntos
Fabaceae/química , Nitrogênio/química , Pancreatina/química , Pepsina A/química , Proteínas de Plantas/química , Amônia/química , Animais , Canavalia/química , Digestão , Enzimas/química , Temperatura Alta , Hidrólise , Suínos , Verduras/químicaRESUMO
This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G' value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.
Assuntos
Pepsina A/química , Pepsina A/metabolismo , Pressão , Animais , Ativação Enzimática , Estabilidade Enzimática , Géis , Leite , Proteólise , Reologia , SuínosRESUMO
The objectives of this study were to characterize peptides found in unprocessed amaranth hydrolysates (UAH) and extruded amaranth hydrolysates (EAH) and to determine the effect of the hydrolysis time on the profile of peptides produced. Amaranth grain was extruded in a single screw extruder at 125 °C of extrusion temperature and 130 rpm of screw speed. Unprocessed and extruded amaranth flour were hydrolyzed with pepsin/pancreatin enzymes following a kinetic at 10, 25, 60, 90, 120 and 180 min for each enzyme. After 180 min of pepsin hydrolysis, aliquots were taken at each time during pancreatin hydrolysis to characterize the hydrolysates by MALDI-TOF/MS-MS. Molecular masses (MM) (527, 567, 802, 984, 1295, 1545, 2034 and 2064 Da) of peptides appeared consistently during hydrolysis, showing high intensity at 10 min (2064 Da), 120 min (802 Da) and 180 min (567 Da) in UAH. EAH showed high intensity at 10 min (2034 Da) and 120 min (984, 1295 and 1545 Da). Extrusion produced more peptides with MM lower than 1000 Da immediately after 10 min of hydrolysis. Hydrolysis time impacted on the peptide profile, as longer the time lower the MM in both amaranth hydrolysates. Sequences obtained were analyzed for their biological activity at BIOPEP, showing important inhibitory activities related to chronic diseases. These peptides could be used as a food ingredient/supplement in a healthy diet to prevent the risk to develop chronic diseases.
Assuntos
Amaranthus/química , Proteínas de Plantas/química , Hidrolisados de Proteína/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Pancreatina/química , Pepsina A/química , Fragmentos de Peptídeos/química , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
The aqueous solution behavior of polyethyleneimine (a synthetic cationic polymer) in the presence of anions with two or more electrical charges (citrate, phosphate, sulphate, malate, malonate and succinate) was studied by means of turbidimetry and light scattering. Polyethyleneimine forms non-soluble complexes with these anions, which behave as a pseudo-polyampholyte with an isoelectrical pH value dependent on the type of anion. The effect of pH, polymer concentration and ionic strength on the non-soluble complexes formation was examined. The complex precipitation pH range was between 3.5 and 8.0 and also depended on the type of anion. The complex formation was inhibited by the ionic strength in agreement with the electrostatic mechanism of the non-soluble complex formation. Model proteins with isoelectric pH from 1 to 10 were assayed in orden to be precipitated by these complexes. It was found that the non-soluble polyethyleneimine-anion complexes have the property to precipitate macromolecules charged with an opposite electrical charge.
Assuntos
Ânions/química , Polietilenoimina/química , Animais , Bovinos , Quimotripsina/química , Concentração de Íons de Hidrogênio , Íons , Substâncias Macromoleculares/química , Tamanho da Partícula , Pepsina A/química , Polímeros/química , Proteínas/química , Sais/química , Soroalbumina Bovina/química , SolubilidadeRESUMO
The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H(+)-treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 - EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (K(I) (*)) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection.
Assuntos
Echinococcus granulosus/metabolismo , Serina Proteases/química , Sequência de Aminoácidos , Animais , Cães , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Genes de Helmintos , Interações Hospedeiro-Parasita , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pepsina A/química , Inibidores de Proteases/química , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina Proteases/metabolismoRESUMO
The interaction between the acidic protein, pepsin, and the non-charged polyethyleneglycol polymer was studied by dynamic light scattering, fluorescence spectroscopy and measurements of the protein thermal stability at neutral pH. Polyethyleneglycol of average molecular mass 1450 showed a higher interaction capacity with the protein than polyethyleneglycol of average molecular mass 8000. Polyethyleneglycol of average molecular mass 1450 showed a molecular mechanism where the interpolymer interaction led to the complex formation. This fact can be explained taking into account that the extended form on this polymer molecule favours the interaction with the protein, which is highly dependent of the polymer total concentration. Polyethyleneglycol of average molecular mass 8000 showed a cooperative interaction between the polymer and protein molecules which was independent of the PEG concentration.
Assuntos
Pepsina A/química , Polietilenoglicóis/química , Fracionamento Químico , Concentração de Íons de Hidrogênio , Espalhamento de Radiação , Espectrometria de FluorescênciaRESUMO
The influence of the phase volume ratio and polymer pausidispersity on chymosin and pepsin partition in polyethylenglycol-phosphate aqueous two-phase systems was studied. Both proteins showed a high affinity for the polyethylenglycol rich phase with a partition coefficient from 20 to 100 for chymosin and from 20 to 180 for pepsin, when the polyethyleneglycol molecular mass in the system varied between 1450 and 8000. The partition coefficient of chymosin was not affected by the volume phase ratio, while the pepsin coefficient showed a significant decrease in its partition coefficient with the increase in the top/bottom phase volume ratio.
Assuntos
Fracionamento Químico/métodos , Quimosina/química , Pepsina A/química , Polietilenoglicóis/química , Água/química , Peso Molecular , Transição de Fase , Fosfatos/química , Polímeros/químicaRESUMO
Oral administration of an antigen can result in local and systemic priming or tolerance and the basis of this dichotomy is poorly understood. The intestinal microenvironment, and factors such as nature of the antigen, dose, genetic background, uptake and concentration of the antigen that gain access to the internal milieu via the mucosa influence these active immunologic processes. Chitosan is a biocompatible natural polysaccharide able to promote the transmucosal absorption of peptides and proteins. The aim of our work was to study the effect of the co-administration of type II collagen (CII) and chitosan during the initial contact of the antigen with the immune system. Sixteen hours after feeding we evaluated several molecular events in mucosal and in systemic lymphoid tissues. We determined in Peyer's patches (PP) and spleen cells the number and activation of T cells, the arrival of the antigens, and the cytokine profile. In PP we found a reduction in the cell number without changes in CD3(+) cells. In spleen, instead, we observed an increase in CD3(+) cells as well as the internalization of the CD3 complex. CII:chitosan-fed animals exhibited a reduced secretion of IL-2 with an increase of IL-10 in PP and spleen respectively. In addition, in PP, CII:chitosan-fed rats showed increased levels of mRNA for transforming growth factor-beta, IL-4 and IL-10. Together, our data suggest that the co-administration with chitosan modifies the uptake and/or the distribution of the relevant antigen, and promotes an anti-inflammatory environment early after feeding.
Assuntos
Quitina/análogos & derivados , Quitina/farmacologia , Colágeno Tipo II/farmacologia , Citocinas/biossíntese , Imunidade nas Mucosas , Administração Oral , Animais , Complexo CD3/imunologia , Complexo CD3/metabolismo , Morte Celular , Quitina/administração & dosagem , Quitina/metabolismo , Quitosana , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/química , Citocinas/genética , Feminino , Expressão Gênica , Interleucina-10/análise , Interleucina-2/análise , Pepsina A/antagonistas & inibidores , Pepsina A/química , Nódulos Linfáticos Agregados/imunologia , RNA Mensageiro , Ratos , Ratos Wistar , Baço/citologia , Baço/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Thermal denaturation of porcine pepsin in 10% ethanol was studied by circular dichroism (CD) spectroscopy. It was observed that the process is markedly irreversible. The denaturation unfolding process was strongly dependent on the heating rate, as is expected for an unfolding process kinetically controlled due to the presence of an irreversible reaction. Experimentally, we demonstrate the existence of an unfolded (U) state in equilibrium with the native (N) state. The U state is observed to exist at temperatures lower than 45 degrees C. The van't Hoff enthalpy, DeltaH(vH), was determined from direct estimation of the equilibrium constant at several temperatures (DeltaH(vH)=304.3 kJ/mol). To explain the observed behavior, we have considered a Lumry-Eyring model, which takes into account the presence of the U state in addition to N and denatured (D) states (i.e. N<-->U-->D).
Assuntos
Pepsina A/química , Animais , Dicroísmo Circular , Etanol/química , Temperatura Alta , Desnaturação Proteica , Dobramento de Proteína , Suínos , Temperatura , TermodinâmicaRESUMO
Pepsin-catalyzed synthesis of protected peptides was studied in two-phase systems containing up to 5% (by volume) of aqueous phase. A methodological study was carried out to determine the optimum conditions for the synthesis of the model protected peptide Z-Phe-Phe-OMe. Several parameters such as concentrations of carboxylic and amino components, pH of the aqueous phase, ratio of organic to aqueous phase volumes and nature of the organic solvent were investigated. It was observed that the most hydrophobic solvents produced the best yields, despite the low solubility of substrates in these media. The log P of the solvent could be used to predict the solvent effect over the reaction yields. Pepsin immobilized by adsorption onto the solid supports Celite and Chromosorb was employed to perform a study of secondary specificity of the enzyme in organic media through the coupling between Z-X-Phe-OH (X = Ala, Asp, Glu, Gly, Phe, Ile, Val, Trp and Tyr) and Phe-OMe. This investigation was performed in two solvent systems: (A) ethyl acetate:citrate buffer pH 4.5 (98:02, v:v) and (B) acetonitrile:citrate buffer pH 4.5 (96:04, v:v). Reaction rate data showed that pepsin had a preference for more hydrophilic substituents in the P2 position. These data are in contrast to the literature for a similar reaction performed in predominantly aqueous media. Thus, for mainly organic media, partition phenomena are very important and may cause an apparent modification of enzyme specificity.
Assuntos
Enzimas Imobilizadas/química , Pepsina A/química , Peptídeos/síntese química , Sequência de Aminoácidos , Catálise , Técnicas de Química Analítica , Cromatografia Líquida de Alta Pressão , Dipeptídeos/síntese química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Sensibilidade e EspecificidadeRESUMO
Pepsin catalyzed peptide synthesis in biphasic systems containing more than 95% (v/v) of organic phase was studied. Good yields were only obtained with more hydrophobic solvents, in spite of the low solubility of the substrates. The effects of the following parameters were also investigated: concentration of amino and carboxylic components, pH and buffer concentration, ratio between the aqueous and organic phases. The influence of different amino acid residues in the P2 position was investigated through the coupling between Z-Xyz-Phe-OH (where Xyz = Ala, Phe, Trp and Tyr) and Phe-OMe.