Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24.132
Filtrar
1.
Toxicol Lett ; 321: 138-145, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31891759

RESUMO

Organophosphorus (OP)1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE),2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.


Assuntos
Antídotos/farmacologia , Proteínas de Bactérias/farmacologia , Caulobacteraceae/enzimologia , Agentes Neurotóxicos/envenenamento , Intoxicação por Organofosfatos/tratamento farmacológico , Compostos Organofosforados/toxicidade , Hidrolases de Triester Fosfórico/farmacologia , Antídotos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacteraceae/genética , Estabilidade de Medicamentos , Estabilidade Enzimática , Temperatura Alta , Mutação , Intoxicação por Organofosfatos/enzimologia , Compostos Organotiofosforados/envenenamento , Oxirredução , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Desnaturação Proteica , Proteínas Recombinantes/farmacologia , Sarina/envenenamento , Soman/envenenamento
2.
Food Chem ; 302: 125296, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31400698

RESUMO

The process of manufacturing infant milk formulas (IMFs) involves heat treatments that can lead to whey protein denaturation. The objective of the study was to determine how protein composition affects the denaturation kinetics of the whey proteins within IMFs. Three model IMFs (1.3% of cow's milk protein) were produced with a caseins: whey proteins ratio of 40:60, differing only by the whey protein composition. The kinetics of heat-induced denaturation of α-lactalbumin, ß-lactoglobulin and lactoferrin were investigated between 67.5 °C and 80 °C by chromatographic quantification of the residual native proteins. Results showed that the heat-denaturation of α-lactalbumin was reduced when ß-lactoglobulin was absent. The heat-denaturation of lactoferrin was not affected by the composition of the IMFs but its presence enhanced the heat-denaturation of ß-lactoglobulin. This study revealed that, for higher heat treatments (90 °C/15 s, 75 °C/15 min), IMF containing α-lactalbumin and lactoferrin preserved a higher proportion of native whey proteins than IMFs containing ß-lactoglobulin.


Assuntos
Temperatura Alta , Fórmulas Infantis/química , Desnaturação Proteica , Proteínas do Soro do Leite/química , Animais , Bovinos , Humanos , Lactente , Cinética
3.
Food Chem ; 305: 125454, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505413

RESUMO

The effects of the (-)-Epigallocatechin gallate (EGCG)-gelatin biofilm treatment (EGT) on microbial composition and quality of tilapia fillets stored at low temperatures were evaluated. The changes in mechanical properties, microbial reproduction, as well as lipid and protein oxidation during fillets storage were determined. The results showed that EGT reduced the microbial count and the relative abundance of the fillets. And EGT delayed the rate of lipid oxidation and protein denaturation in fillets. Compared with the control group, EGT samples had lower K values (74% on 18 d) and biogenic amines (39 mg/kg for putrescine and 50 mg/kg for cadaverine on 21 d). According to sensory evaluation, the shelf life of tilapia fillets was extended by 6 d in the EGT group. Therefore, EGT improved the quality of cryopreserved tilapia fillets and could be considered as a potential method for fish fillet preservation.


Assuntos
Materiais Biomiméticos/química , Catequina/análogos & derivados , Armazenamento de Alimentos/métodos , Gelatina/química , Alimentos Marinhos/análise , Aeromonas/efeitos dos fármacos , Animais , Aminas Biogênicas/análise , Materiais Biomiméticos/farmacologia , Catequina/química , Peroxidação de Lipídeos/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Proteínas/química , Pseudomonas/efeitos dos fármacos , Alimentos Marinhos/microbiologia , Paladar/efeitos dos fármacos , Temperatura Ambiente
4.
J Phys Chem Lett ; 11(1): 292-296, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31841337

RESUMO

Urea is a strong denaturing osmolyte that disrupts noncovalent bonds in proteins. Here, we present a small-angle neutron scattering (SANS) and neutron spin-echo spectroscopy (NSE) study on the structure and dynamics of the intrinsically disordered myelin basic protein (MBP) denatured by urea. SANS results show that urea-denatured MBP is more compact than ideal polymers, while its secondary structure content is entirely lost. NSE experiments reveal concomitantly an increase of the relaxation time and of the amplitude of internal motions in urea-denatured MBP as compared to native MBP. If interpreted in terms of the Zimm model including internal friction (ZIF), the internal friction parameter decreased by a factor of 6.5. Urea seems to not only smooth local energy barriers, reducing internal friction on a local scale, but also significantly reduces the overall depth of the global energy landscape. This leads to a nearly complete loss of restoring forces beyond entropic forces and in turn allows for larger motional amplitudes. Obviously, the noncovalent H-bonds are largely eliminated, driving the unfolded protein to be more similar to a synthetic polymer.


Assuntos
Proteína Básica da Mielina/química , Ureia/química , Fricção , Modelos Moleculares , Difração de Nêutrons , Conformação Proteica , Desnaturação Proteica , Espalhamento a Baixo Ângulo , Software
5.
Phys Chem Chem Phys ; 22(1): 354-367, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31815262

RESUMO

Surface-specific spectroscopic data has shown that urea undergoes a shift in orientation at protein surfaces in acidic media. Since urea denatures proteins at a wide range of pHs, the variable chemical nature of protein-urea interactions has been used to support an indirect mechanism of urea-induced denaturation. Here, we use molecular dynamics simulations, minimum-distance distribution functions (MDDFs), and hydrogen-bond analysis, to characterize the interactions of urea with proteins at neutral and low pH, as defined by the protonation state of acidic residues. We obtain the expected preferential solvation by urea and dehydration, consistently with urea-induced denaturation, while the MDDFs allow for a solvent-shell perspective of protein-urea interactions. The distribution functions are decomposed into atomic contributions to show that there is indeed a shift in the orientation of urea molecules in the vicinity of acidic side-chains, as shown by the experimental spectroscopic data. However, this effect is local, and the interactions of urea with the other side chains and with the protein backbone are essentially unaffected at low pH. Therefore, hydrophobic solvation and urea-backbone hydrogen bonds can play a role in a direct mechanism of urea-induced protein denaturation without contradicting the observed variations in the chemical nature of protein-urea interactions as a function of the acidity of the solution.


Assuntos
Lipase/química , Ureia/química , Burkholderia cepacia/enzimologia , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Desnaturação Proteica
6.
Phys Rev Lett ; 123(15): 158002, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31702313

RESUMO

We study the interplay between a crack tip slowly propagating through a hydrogel and nanoparticles suspended in its liquid environment. Using a proteinic gel enables us to tune the electrostatic interaction between the network and silica colloids. Thereby, we unveil two distinct, local toughening mechanisms. The primary one is charge independent and involves the convective building of a thin particulate clog, hindering polymer hydration in the crack process zone. When particles and network bear opposite charges, transient adhesive bonding superimposes, permitting the remarkable pinning of a crack by a liquid drop.


Assuntos
Biopolímeros/química , Hidrogéis/química , Nanopartículas/química , Colágeno/química , Modelos Químicos , Desnaturação Proteica , Sílica Gel/química , Eletricidade Estática
7.
J Phys Chem Lett ; 10(23): 7406-7413, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31721587

RESUMO

Using enhanced-sampling replica exchange fully atomistic molecular dynamics simulations, we show that, individually, urea and guanidinium chloride (GdmCl) denature the Trpcage protein, but remarkably, the helical segment 1NLYIQWL7 of the protein is stabilized in mixed denaturant solutions. GdmCl induces protein denaturation via a combination of direct and indirect effects involving dehydration of the protein and destabilization of stabilizing salt bridges. In contrast, urea denatures the protein through favorable protein-urea preferential interactions, with peptide-specific indirect effects of urea on the water structure around the protein. In the case of the helical segment of Trpcage, urea "oversolvates" the peptide backbone by reorganizing water molecules from the peptide side chains to the peptide backbone. An intricate nonadditive thermodynamic balance between GdmCl-induced dehydration of the peptide and the urea-induced changes in solvation structure triggers partial counteraction to urea denaturation and stabilization of the helix.


Assuntos
Guanidina/química , Peptídeos/química , Ureia/química , Sequência de Aminoácidos , Ligações de Hidrogênio , Conformação Proteica em alfa-Hélice , Desnaturação Proteica
8.
Chemistry ; 25(55): 12820-12829, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411775

RESUMO

An inorganic sandwich molecule, Na[Co(C2 B9 H11 )2 ], able to produce vesicles through self-assembly and known to produce strong dihydrogen-bond interactions with amine groups is capable of interacting with proteins. This dual non-bonding ability of Na[Co(C2 B9 H11 )2 ] is what makes this molecule unique: it can be firmly anchored to a protein surface and is capable of extending over it. To prove this, the widely available bovine serum albumin (BSA), which has many pendant amino groups in its structure, has been taken as the model protein. It has been found that around 100 molecules of Na[Co(C2 B9 H11 )2 ] preserve the native structure of BSA, while endorsing it with a significantly increased stability with respect to chemical- and thermal-induced denaturation due to efficient encapsulation. The advantages of this encapsulation technique are two-fold; the first is its simplicity as it relies on the anchoring capacity of Na[Co(C2 B9 H11 )2 ] to the surface of the protein through the amine-containing residues and the second is its self-assembling capacity allowing it to spread across the surface. The dense shield of protection offered by Na[Co(C2 B9 H11 )2 ] has been demonstrated by the inhibition of BSA pseudo-esterase activity, which indicates that the inorganic corset around BSA protects its reactive surface residues, thereby preventing their acetylation.


Assuntos
Boranos/química , Compostos Organometálicos/química , Soroalbumina Bovina/metabolismo , Animais , Desnaturação Proteica , Soroalbumina Bovina/química
9.
Pak J Pharm Sci ; 32(3 (Supplementary)): 1167-1173, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31303586

RESUMO

Medicinal plants are playing an imperative role in the therapy for treating various chronic ailments including arthritis. The present study was focused on finding in-vitro and in-vivo anti-arthritic potential of P. braunii roots. In vitro protein denaturation, membrane stabilization and anti-trypsinase assays were carried out to demonstrate anti-arthritic activity of the extracts. Furthermore, the extracts exerting promising in vitro anti-arthritic potential were tested orally at 150, 300 and 600mg/kg/day against formaldehyde induced arthritis in Wistar rats. The methanolic, aqueous and ethyl acetate extracts of the plant revealed noteworthy in vitro anti-arthritic activities while mitigating formaldehyde induced paw edema in dose dependent manner. Methanolic and aqueous extracts showed the highest inhibition (p<0.05) of paw edema, arthritic indices, reduced elevated level of platelets and leukocytes while increasing hemoglobin and body weight of arthritic rats. Anti-arthritic activity of the plant extracts may be due to inhibition of protein denaturation and lysosomal membrane stabilization. The plant exhibited good anti-arthritic potential.


Assuntos
Artrite Experimental/tratamento farmacológico , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Polystichum/química , Albuminas/química , Albuminas/efeitos dos fármacos , Animais , Artrite Experimental/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos , Membrana Eritrocítica/efeitos dos fármacos , Feminino , Formaldeído/toxicidade , Humanos , Masculino , Medicina Tradicional do Leste Asiático , Paquistão , Extratos Vegetais/química , Raízes de Plantas/química , Desnaturação Proteica/efeitos dos fármacos , Ratos Wistar , Soroalbumina Bovina/efeitos dos fármacos
10.
J Chromatogr A ; 1603: 190-198, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31277950

RESUMO

The heat-induced (80 ±â€¯1 ℃, 1 h) aggregates of bovine serum albumin and ovalbumin at neutral pH and low ionic strength (10 mM sodium phosphate buffer, pH 6.9) were characterized using size-exclusion chromatography combined with small-angle X-ray scattering measurement. The values calculated for the radius of gyration and molecular weight of the eluted aggregates of the bovine serum albumin were 9-11 nm and 540,000-820,000, respectively. Those of ovalbumin were 11-16 nm and 500,000-1,820,000, respectively. The overall linear conformation of the bovine serum albumin aggregates slightly differed from that of the ovalbumin aggregates since the mass fractal dimensions were found from calculation to be 1.63-1.7 for the bovine serum albumin and 1.36-1.51 for the ovalbumin. The surface property of the aggregates of both proteins was suggested to be similar to that of their native monomer since all the surface fractal dimensions were almost equivalent. The dimensionless Kratky plots of the eluted aggregates indicated that the non-globular conformation of the bovine serum albumin aggregates differs from that of the ovalbumin aggregates. These analyses using size-exclusion chromatography combined with the solution X-ray scattering measurement will be helpful for characterization of the components of the denatured protein aggregation in solution.


Assuntos
Cromatografia em Gel/métodos , Temperatura Alta , Concentração Osmolar , Agregados Proteicos , Proteínas/química , Espalhamento de Radiação , Água/química , Animais , Bovinos , Galinhas , Fractais , Peso Molecular , Ovalbumina/química , Conformação Proteica , Desnaturação Proteica , Soroalbumina Bovina/química , Soluções , Raios X
11.
Food Chem ; 299: 125104, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31279125

RESUMO

The role of protein denaturation in formation of thaw loss is currently not well understood. This study investigated denaturation of myofibrillar and sarcoplasmic proteins of pork loins caused by freezing-thawing in relation to freezing rate. Compared to fast freezing, slow freezing caused 28% larger thaw loss, decreased water-holding capacity of myofibrils and increased surface hydrophobicity, indicating more pronounced denaturation of myofibrillar proteins. We here propose a model: In slow freezing protons are concentrated in the unfrozen water resulting in reduced pH in proximity of structural proteins causing protein denaturation. In parallel, large ice crystals are formed outside of muscle fibers resulting in transversal shrinkage. In fast freezing small ice crystals trap protons and cause less severe protein denaturation and reduced thaw loss. Differential scanning calorimetry and tryptophan fluorescence spectra indicated sarcoplasmic protein denaturation in drip due to freezing-thawing. However, sarcoplasmic protein denaturation was independent of freezing rate.


Assuntos
Congelamento , Proteínas Musculares/química , Miofibrilas/química , Desnaturação Proteica , Carne Vermelha , Animais , Varredura Diferencial de Calorimetria , Conservação de Alimentos/métodos , Interações Hidrofóbicas e Hidrofílicas , Gelo , Músculos Paraespinais/química , Carne Vermelha/análise , Espectrometria de Fluorescência , Triptofano/química , Água/química
12.
J Am Soc Mass Spectrom ; 30(8): 1385-1388, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31286443

RESUMO

Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35 kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation. ᅟ.


Assuntos
Metilaminas/química , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Gases/química , Humanos , Íons/química , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína
13.
Food Funct ; 10(7): 4432-4439, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31287454

RESUMO

The objective of this study was to evaluate the physicochemical, textural, sensory and microbiological stability of cupcakes during storage after the addition of different proportions of quinoa protein isolate modified by heat denaturation (QPI-HD) and freezing-lyophilization (QPI-FL). The cupcakes containing modified quinoa protein exhibited greater firmness and water activity than the control cupcake. The texture profile analysis (TPA) revealed that the cupcakes with modified quinoa protein were statistically different from those with unmodified protein isolate and the control cupcake. Moreover, cupcakes with quinoa protein modified by either heat or freezing had greater acceptance and preference on the part of consumers. In addition to this, these cupcakes showed lesser growth of molds after 10 days of storage; this indicated that the abovementioned additive could extend the shelf life of cupcakes. These results showed that the addition of modified quinoa protein led to cupcakes with better sensory and textural properties and greater stability during storage.


Assuntos
Pão/análise , Chenopodium quinoa/química , Análise de Alimentos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Antifúngicos , Pão/microbiologia , Culinária , Farinha , Microbiologia de Alimentos , Liofilização , Congelamento , Temperatura Alta , Desnaturação Proteica , Estabilidade Proteica , Sementes/química , Paladar , Água/química
14.
Nutrients ; 11(7)2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31262028

RESUMO

Raw cow's milk was previously shown to suppress allergic symptoms in a murine model for food allergy. In the present study, we investigated the contribution of fat content and heat-sensitive milk components to this allergy-protective effect. In addition, we determined the potency of alkaline phosphatase (ALP), a heat-sensitive raw milk component, to affect the allergic response. C3H/HeOuJ mice were treated with raw milk, pasteurized milk, skimmed raw milk, pasteurized milk spiked with ALP, or phosphate-buffered saline for eight days prior to sensitization and challenge with ovalbumin (OVA). Effects of these milk types on the allergic response were subsequently assessed. Similar to raw milk, skimmed raw milk suppressed food allergic symptoms, demonstrated by a reduced acute allergic skin response and low levels of OVA-specific IgE and Th2-related cytokines. This protective effect was accompanied by an induction of CD103+CD11b+ dendritic cells and TGF-ß-producing regulatory T cells in the mesenteric lymph nodes. Pasteurized milk was not protective but adding ALP restored the allergy-protective effect. Not the fat content, but the heat-sensitive components are responsible for the allergy-protective effects of raw cow's milk. Adding ALP to heat-treated milk might be an interesting alternative to raw cow's milk consumption, as spiking pasteurized milk with ALP restored the protective effects.


Assuntos
Fosfatase Alcalina/imunologia , Dermatite Atópica/prevenção & controle , Manipulação de Alimentos/métodos , Hipersensibilidade Alimentar/prevenção & controle , Proteínas do Leite/imunologia , Pasteurização , Animais , Basófilos/imunologia , Basófilos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Dermatite Atópica/imunologia , Dermatite Atópica/metabolismo , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/metabolismo , Imunoglobulinas/sangue , Lipídeos/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos Endogâmicos C3H , Ovalbumina , Desnaturação Proteica , Pele/imunologia , Pele/metabolismo , Baço/imunologia , Baço/metabolismo
15.
Int J Biol Macromol ; 138: 958-965, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325504

RESUMO

Proteins are prone to unfolding and subsequent denaturation by changes in temperature, pH and other harsh conditions. Nanoparticles act as artificial 'chaperones' due to favourable orientation of the proteins on their scaffold which prevents aggregation and reconfigures denatured proteins into their native functional state. In the present study, thermal denaturation of Cholesterol oxidases from Pseudomonas aeruginosa PseA, Rhodococcus erythropolis MTCC 3951 and Streptomyces sp. were studied at temperatures 50-70 °C. Further, these thermally denatured proteins were refolded using functionalized Magnetic Iron (II, III) oxide nanoparticles which was confirmed using DLS, Zeta Potential Measurements, fluorescence and CD spectroscopy. The refolded proteins were found to regain their secondary structure and activity to a great extent.


Assuntos
Colesterol Oxidase/química , Nanopartículas de Magnetita/química , Desnaturação Proteica , Redobramento de Proteína , Ativação Enzimática , Compostos Férricos/química , Tamanho da Partícula , Análise Espectral , Temperatura Ambiente , Termodinâmica
16.
J Photochem Photobiol B ; 198: 111558, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31357173

RESUMO

Facile and low cost garlic clove extract based silver nanoparticles was synthesized and its broad spectrum of therapeutic activity including antibiofilm, antiparasitic and anti-breast cancer activity was evaluated. The synthesized garlic­silver nanoparticles (G-AgNPs) were characterized by various physico-chemical techniques. G-AgNPs showed good optical property, highly crystalline nature, spherical shape and uniformly dispersed with size measuring between 10 and 50 nm. G-AgNPs have shown greater anti-bacterial and antibiofilm activity on clinically important pathogens methicillin-resistant S. aureus and P. aerigunosa at 100 µg ml-1. The efficacy of G-AgNPs against earthworm evidenced its effectiveness as anti-helminthic agent in treating intestinal parasites. The significant inhibition of BSA protein denaturation proves its anti-inflammatory property. In addition, G-AgNPs have shown remarkable anticancer effect and significantly inhibited the human breast cancer cell (MCF-7) viability at 100 µg ml-1 after 24 h. A noticeable change in the morphology of MCF-7 cells was also noticed. G-AgNPs were non-toxic to human HEK293 embryonic cells. Also, the non-toxic nature of G-AgNPs to C. cornuta and no morphological, physiological changes proved its safety to the environment. It is concluded that G-AgNPs have a broad range of biological applications and it can be used as an eco-friendly material without having negative effects in the environment.


Assuntos
Anti-Helmínticos/química , Antibacterianos/química , Anti-Inflamatórios/química , Antineoplásicos/química , Alho/química , Nanopartículas Metálicas/química , Prata/química , Animais , Anti-Helmínticos/farmacologia , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Biofilmes/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Alho/metabolismo , Química Verde , Células HEK293 , Humanos , Nanopartículas Metálicas/toxicidade , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oligoquetos/efeitos dos fármacos , Tamanho da Partícula , Extratos Vegetais/química , Desnaturação Proteica/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia
17.
Phys Chem Chem Phys ; 21(24): 12806-12817, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31165827

RESUMO

We present a study of the combined effects of natural cosolvents (TMAO, glycine, urea) and pressure on the activity of the tetrameric enzyme lactate dehydrogenase (LDH). To this end, high-pressure stopped-flow methodology in concert with fast UV/Vis spectroscopic detection of product formation was applied. To reveal possible pressure effects on the stability and dynamics of the enzyme, FTIR spectroscopic and neutron scattering measurements were carried out. In neat buffer solution, the catalytic turnover number of the enzyme, kcat, increases up to 1000 bar, the pressure range where dissociation of the tetrameric species to dimers sets in. Accordingly, we obtain a negative activation volume, ΔV# = -45.3 mL mol-1. Further, the enzyme substrate complex has a larger volume compared to the enzyme and substrate in the unbound state. The neutron scattering data show that changes in the fast internal dynamics of the enzyme are not responsible for the increase of kcat upon compression. Whereas the magnitude of kcat is similar in the presence of the osmolytes, the pressure of deactivation is modulated by the addition of cosolvents. TMAO and glycine increase the pressure of deactivation, and in accordance with the observed stabilizing effect both cosolvents exhibit against denaturation and/or dissociation of proteins. While urea does not markedly affect the magnitude of the Michaelis constant, KM, both 1 M TMAO and 1 M glycine exhibit smaller KM values of about 0.07 mM and 0.05 mM below about 1 kbar. Such positive effect on the substrate affinity could be rationalized by the effect the two cosolutes impose on the thermodynamic activities of the reactants, which reflect changes in water-mediated intermolecular interactions. Our data show that the intracellular milieu, i.e., the solution conditions that have evolved, may be sufficient to maintain enzymatic activity under extreme environmental conditions, including the whole pressure range encountered on Earth.


Assuntos
L-Lactato Desidrogenase/química , Solventes/química , Glicina/química , Cinética , Metilaminas/química , Modelos Moleculares , Pressão , Desnaturação Proteica , Dobramento de Proteína , Multimerização Proteica , Termodinâmica , Ureia/química , Água/química
18.
Food Chem ; 293: 103-111, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151589

RESUMO

Defects with textures and flavors are a common problem, causing many economic losses in the dry-cured ham industry. To obtain a better understanding of the defects of dry-cured ham, texture, protein denaturation, protein structure, and water distribution of normal and defective hams were investigated. Compared with normal ham, more than 1.5-fold values in adhesiveness and bitterness, and less than 0.8-fold values in hardness were found in defective ham. The intense denaturation of sarcoplasmic proteins and actin, and the dramatic transformation of α-helix to ß-sheet were the key modification of proteins; a high proportion (92.39%) of immobile water contributed to the excessive softness and adhesiveness of defective hams. Furthermore, high denaturation of proteins could accelerate the degradation of proteins, which further developed the bitterness and adhesiveness of defective hams. Partial least squares regression demonstrated that the discrepancies in protein denaturation, protein structure and water distribution were related with bitterness and adhesiveness of Jinhua ham.


Assuntos
Produtos da Carne/análise , Proteínas/química , Água/química , Adesividade , Animais , Análise de Componente Principal , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Desnaturação Proteica , Suínos , Paladar
19.
Eur J Pharm Biopharm ; 142: 506-517, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31175923

RESUMO

In biotherapeutic protein research, an estimation of the studied protein's thermal stability is one of the important steps that determine developability as a function of solvent conditions. Differential Scanning Fluorimetry (DSF) can be applied to measure thermal stability. Label-free DSF measures amino acid fluorescence as a function of temperature, where conformational changes induce observable peak deformation, yielding apparent melting temperatures. The estimation of the stability parameters can be hindered in the case of multidomain, multimeric or aggregating proteins when multiple transitions partially coincide. These overlapping protein unfolding transitions are hard to evaluate by the conventional methodology, as peak maxima are shifted by convolution. We show how non-linear curve fitting of intrinsic fluorescence DSF can deconvolute highly overlapping transitions in formulation screening in a semi-automated process. The proposed methodology relies on synchronous, constrained fits of the fluorescence intensity, ratio and their derivatives, by combining linear baselines with generalized logistic transition functions. The proposed algorithm is applied to data from three proteins; a single transition, a double separated transition and a double overlapping transition. Extracted thermal stability parameters; apparent melting temperatures Tm,1, Tm,2 and melting onset temperature Tonset are obtained and compared with reference software analysis. The fits show R2 = 0.94 for single and R2 = 0.88 for separated transitions. Obtaining values and trends for Tonset in a well-described and automated way, will aid protein scientist to better evaluate the thermal stability of proteins.


Assuntos
Proteínas/química , Varredura Diferencial de Calorimetria/métodos , Fluorescência , Fluorometria/métodos , Desnaturação Proteica , Estabilidade Proteica , Desdobramento de Proteína , Temperatura Ambiente
20.
J Biosci Bioeng ; 128(5): 630-635, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31196790

RESUMO

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to analyze the purity of a protein. However, it is necessary to denature (via boiling) the samples before subjecting them to electrophoresis. In the case of protease-containing samples, autolysis of the protease can occur, affecting the accuracy of results. In this study, we investigated the methods for analyzing the purity of Dispase I, a thermolysin-like neutral protease. When we analyzed D protease, a neutral metalloprotease component of Dispase I and highly purified Dispase I using the conventional SDS-PAGE method, a large number of bands were detected in both cases. These bands (putative D protease fragments) were assumed to result from autolysis. To inactivate D protease (optimal pH 7-8), 0.05 M sulfuric acid was utilized (pH 0.7-2.5). Using a conventional sample preparation solution, acid-treated Dispase I samples (without boiling) were made, and SDS-PAGE (15% w/v gel) was carried out. Our findings show that autolysis was inhibited under strong acidic conditions, and protein denaturation was achieved by treatment with sulfuric acid and SDS without boiling. Using this modified SDS-PAGE method, the purities of Dispase I and the purified enzyme were determined to be approximately 80% and 98%, respectively. Furthermore, we demonstrated that this method can be applied for the analysis of other samples including non-acidic proteases (e.g., thermolysin, subtilisin, and trypsin) and protease-contaminated samples (a mixed solution of albumin and D protease).


Assuntos
Endopeptidases/análise , Eletroforese em Gel de Poliacrilamida , Desnaturação Proteica/efeitos dos fármacos , Ácidos Sulfúricos/farmacologia , Trometamina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA