Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 100
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Biochem Biophys Res Commun ; 495(2): 1858-1863, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29246762

RESUMO

Human hemoglobin (HbA) transports molecular oxygen (O2) from the lung to tissues where the partial pressure of O2 is lower. O2 binds to HbA at the heme cofactor and is stabilized by a distal histidine (HisE7). HisE7 has been observed to occupy opened and closed conformations, and is postulated to act as a gate controlling the binding/release of O2. However, it has been suggested that HbA also contains intraprotein oxygen channels for entrances/exits far from the heme. In this study, we developed a novel method of crystal immersion in liquid oxygen prior to X-ray data collection. In the crystals immersed in liquid oxygen, the heme center was oxidized to generate aquomethemoglobin. Increases of structural flexibility were also observed in regions that are synonymous with previously postulated oxygen channels. These regions also correspond to medically relevant mutations which affect O2 affinity. The way HbA utilizes these O2 channels could have a profound impact on understanding the relationship of HbA O2 transport within these disease conditions. Finally, the liquid oxygen immersion technique can be utilized as a new tool to crystallographically examine proteins and protein complexes which utilize O2 for enzyme catalysis or transport.


Assuntos
Cristalização/métodos , Hemoglobinas/química , Hemoglobinas/ultraestrutura , Simulação de Dinâmica Molecular , Oxigênio/química , Sítios de Ligação , Difusão , Porosidade , Ligação Proteica , Conformação Proteica
2.
Nat Commun ; 8: 16099, 2017 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-28665412

RESUMO

With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way. These cameras are superior to previous detectors in coping with the intrinsically low contrast and beam-induced motion of radiation-sensitive organic materials embedded in amorphous ice, and hence they have enabled the structure determination of many macromolecular assemblies to atomic or near-atomic resolution. Nevertheless, there are still limitations and one of them is the size of the target structure. Here, we report the use of a Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 Å. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.


Assuntos
Microscopia Crioeletrônica/métodos , Hemoglobinas/ultraestrutura , Humanos , Estrutura Molecular
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 179: 188-193, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28242448

RESUMO

Aggregation of proteins is a physiological process which contributes to the pathophysiology of several maladies including diabetes mellitus, Huntington's and Alzheimer's disease. In this study we have reported that aloe emodin (AE), an anthroquinone, which is one of the active components of the Aloe vera plant, acts as an inhibitor of hemoglobin (Hb) aggregation. Hb was thermally aggregated at 60°C for four days as evident by increased thioflavin T and ANS fluorescence, shifted congo red absorbance, appearance of ß sheet structure, increase in turbidity and presence of oligomeric aggregates. Increasing concentration of AE partially reverses the aggregation of the model heme protein (hemoglobin). The maximum effect of AE was observed at 100µM followed by saturation at 125µM. The results were confirmed by UV-visible spectrometry, intrinsic fluorescence, ThT, ANS, congo red assay as well as transmission electron microscopy (TEM). These results were also supported by fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) which shows the disappearance of ß sheet structure and appearance of α helices. This study will serve as baseline for translatory research and the development of AE based therapeutics for diseases attributed to protein aggregation.


Assuntos
Aloe/química , Antraquinonas/farmacologia , Hemoglobinas/química , Agregados Proteicos/efeitos dos fármacos , Temperatura Ambiente , Naftalenossulfonato de Anilina/química , Animais , Antraquinonas/química , Benzotiazóis , Bovinos , Dicroísmo Circular , Vermelho Congo/química , Hemoglobinas/ultraestrutura , Nefelometria e Turbidimetria , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Tiazóis/química
4.
Proc Natl Acad Sci U S A ; 114(7): 1474-1479, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28087691

RESUMO

Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.


Assuntos
Holografia/métodos , Proteínas/ultraestrutura , Imagem Individual de Molécula/métodos , Animais , Bovinos , Citocromos c/ultraestrutura , Elétrons , Grafite , Hemoglobinas/ultraestrutura , Holografia/instrumentação , Soroalbumina Bovina/ultraestrutura , Imagem Individual de Molécula/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Eletricidade Estática , Vácuo
5.
J Struct Biol ; 194(2): 171-9, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26882843

RESUMO

In the course of their intraerythrocytic development, malaria parasites incorporate and degrade massive amounts of the host cell cytoplasm. This mechanism is essential for parasite development and represents a physiological step used as target for many antimalarial drugs; nevertheless, the fine mechanisms underlying these processes in Plasmodium species are still under discussion. Here, we studied the events of hemoglobin uptake and hemozoin nucleation in the different stages of the intraerythrocytic cycle of the murine malaria parasite Plasmodium chabaudi using transmission electron tomography of cryofixed and freeze-substituted cells. The results showed that hemoglobin uptake in P. chabaudi starts at the early ring stage and is present in all developmental stages, including the schizont stage. Hemozoin nucleation occurs near the membrane of small food vacuoles. At the trophozoite stage, food vacuoles are found closely localized to cytostomal tubes and mitochondria, whereas in the schizont stage, we observed a large food vacuole located in the central portion of the parasite. Taken together, these results provide new insights into the mechanisms of hemoglobin uptake and degradation in rodent malaria parasites.


Assuntos
Hemeproteínas/metabolismo , Hemoglobinas/metabolismo , Estágios do Ciclo de Vida/fisiologia , Plasmodium chabaudi/metabolismo , Vacúolos/metabolismo , Animais , Transporte Biológico , Tomografia com Microscopia Eletrônica , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Eritrócitos/ultraestrutura , Hemeproteínas/ultraestrutura , Hemoglobinas/ultraestrutura , Processamento de Imagem Assistida por Computador , Malária/parasitologia , Masculino , Camundongos , Plasmodium chabaudi/crescimento & desenvolvimento , Plasmodium chabaudi/ultraestrutura , Proteólise , Vacúolos/ultraestrutura
6.
Biosens Bioelectron ; 77: 894-900, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26520252

RESUMO

Molecularly imprinted polymer (MIP) was prepared on the surface of Au electrode by electrochemically mediated atom transfer radical polymerization (eATRP) with hemoglobin (Hb) both as catalyst and template molecule. Firstly, the condition for eATRP such as the potential, time and Hb concentration were selected and determined to be -0.51 V, 120 min and 20mg/mL, respectively. Further, the electrode modified with MIP (MIP/Au) was carefully examined by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscope (SEM). Finally, the MIP/Au electrode was used as a biosensor and successfully applied for the determination of Hb by differential pulse voltammetry (DPV) measurement. The results of experiments showed that the proposed biosensor displayed a broader linear range and a lower detection limit for Hb determination when it was compared to those Hb sensors based on MIP. The linear range was from 1.0 × 10(-10) to 1.0 × 10(1)mg/L with a detection limit of 7.8 × 10(-11)mg/L (S/N=3.3). In a word, the work of this paper established a useful way for the preparation and application of biosensor based on protein imprinted polymers.


Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Hemoglobinas/análise , Hemoglobinas/química , Impressão Molecular/métodos , Polímeros/química , Adsorção , Catálise , Eletrodos , Desenho de Equipamento , Análise de Falha de Equipamento , Hemoglobinas/ultraestrutura , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Eur J Mass Spectrom (Chichester) ; 21(3): 221-31, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26307702

RESUMO

Native mass spectrometry (MS) has become an invaluable tool for the characterization of proteins and noncovalent protein complexes under near physiological solution conditions. Here we report the structural characterization of human hemoglobin (Hb), a 64 kDa oxygen-transporting protein complex, by high resolution native top-down MS using electrospray ionization and a 15-Tesla Fourier transform ion cyclotron resonance mass spectrometer. Native MS preserves the noncovalent interactions between the globin subunits, and electron capture dissociation (ECD) produces fragments directly from the intact Hb complex without dissociating the subunits. Using activated ion ECD, we observe the gradual unfolding process of the Hb complex in the gas phase. Without protein ion activation, the native Hb shows very limited ECD fragmentation from the N-termini, suggesting a tightly packed structure of the native complex and therefore a low fragmentation efficiency. Precursor ion activation allows a steady increase in N-terminal fragment ions, while the C-terminal fragments remain limited (38 c ions and four z ions on the α chain; 36 c ions and two z ions on the ß chain). This ECD fragmentation pattern suggests that upon activation, the Hb complex starts to unfold from the N-termini of both subunits, whereas the C-terminal regions and therefore the potential regions involved in the subunit binding interactions remain intact. ECD-MS of the Hb dimer shows similar fragmentation patterns as the Hb tetramer, providing further evidence for the hypothesized unfolding process of the Hb complex in the gas phase. Native top-down ECD-MS allows efficient probing of the Hb complex structure and the subunit binding interactions in the gas phase. It may provide a fast and effective means to probe the structure of novel protein complexes that are intractable to traditional structural characterization tools.


Assuntos
Hemoglobinas/química , Hemoglobinas/ultraestrutura , Modelos Químicos , Modelos Moleculares , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ciclotrons , Humanos , Subunidades Proteicas
8.
J Biomed Opt ; 19(8): 087002, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25121481

RESUMO

We have investigated the dependence of the Raman spectrum of an optically trapped red blood cell (RBC) on the orientation of the cell, relative to the polarization direction of the Raman excitation beam. The Raman scattered light polarized parallel to the polarization direction of the excitation beam was observed to depend upon the orientation of the cell. In particular, the heme bands at ∼754 cm⁻¹ and in the 1500 to 1700 cm⁻¹ region were observed to become maximum when the cells' equatorial plane was parallel to the excitation beam polarization direction and minimum when the cells' plane was normal to the polarization direction. In contrast, no significant orientational dependence was seen in the Raman scattered light polarized orthogonal to the polarization direction of the excitation beam. Theoretical simulations carried out to investigate these observations suggest that inside the RBCs, the hemoglobin molecules must be present in an ordered arrangement, such that heme-porphyrin planes become preferentially orientated parallel to the RBCs' equatorial plane.


Assuntos
Polaridade Celular/fisiologia , Eritrócitos/citologia , Eritrócitos/fisiologia , Hemoglobinas/metabolismo , Hemoglobinas/ultraestrutura , Refratometria/métodos , Análise Espectral Raman/métodos , Células Cultivadas , Simulação por Computador , Humanos , Modelos Cardiovasculares , Modelos Químicos , Modelos Moleculares , Pinças Ópticas , Conformação Proteica
9.
Biochem Biophys Res Commun ; 450(1): 36-41, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24858681

RESUMO

Hemoglobin (Hb) is in equilibrium between low affinity Tense (T) and high affinity Relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express 'high' and 'low' oxygen affinity Hbs. Although Hbs from former group have been studied extensively, a limited number of structural studies have been performed for the low oxygen affinity Hbs. Here, the crystal structure of low oxygen affinity sheep methemoglobin (metHb) has been determined to 2.7 Å resolution. Even though sheep metHb adopts classical R state like quaternary structure, it shows localized quaternary and tertiary structural differences compared with other liganded Hb. The critical group of residues in the "joint region", shown as a major source of quaternary constraint on deoxyHb, formed unique interactions in the α1ß2/α2ß1 interfaces of sheep metHb structure. In addition, the constrained ß subunits heme environment and the contraction of N-termini and A-helices of ß subunits towards the molecular dyad are observed for sheep metHb structure. These observations provide the structural basis for a low oxygen affinity and blunt response to allosteric effector of sheep Hb.


Assuntos
Hemoglobinas/química , Hemoglobinas/ultraestrutura , Modelos Químicos , Modelos Moleculares , Oxigênio/química , Ovinos/sangue , Animais , Sítios de Ligação , Simulação por Computador , Conformação Molecular
10.
J Struct Biol ; 183(1): 19-32, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23711417

RESUMO

Accurate modeling of image formation in cryo-electron microscopy is an important requirement for quantitative image interpretation and optimization of the data acquisition strategy. Here we present a forward model that accounts for the specimen's scattering properties, microscope optics, and detector response. The specimen interaction potential is calculated with the isolated atom superposition approximation (IASA) and extended with the influences of solvent's dielectric and ionic properties as well as the molecular electrostatic distribution. We account for an effective charge redistribution via the Poisson-Boltzmann approach and find that the IASA-based potential forms the dominant part of the interaction potential, as the contribution of the redistribution is less than 10%. The electron wave is propagated through the specimen by a multislice approach and the influence of the optics is included via the contrast transfer function. We incorporate the detective quantum efficiency of the camera due to the difference between signal and noise transfer characteristics, instead of using only the modulation transfer function. The full model was validated against experimental images of 20S proteasome, hemoglobin, and GroEL. The simulations adequately predict the effects of phase contrast, changes due to the integrated electron flux, thickness, inelastic scattering, detective quantum efficiency and acceleration voltage. We suggest that beam-induced specimen movements are relevant in the experiments whereas the influence of the solvent amorphousness can be neglected. All simulation parameters are based on physical principles and, when necessary, experimentally determined.


Assuntos
Chaperonina 60/ultraestrutura , Microscopia Crioeletrônica/métodos , Hemoglobinas/ultraestrutura , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Chaperonina 60/química , Hemoglobinas/química , Processamento de Imagem Assistida por Computador , Distribuição de Poisson , Complexo de Endopeptidases do Proteassoma/química , Software , Eletricidade Estática
11.
Ann Hematol ; 92(7): 899-906, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23494204

RESUMO

The aim of the present work was to evaluate the redox and oligomeric effects associated with the human hemoglobin of stored red blood cells that had been previously submitted to gamma radiation. Whole blood was collected from healthy donors and irradiated with 25 Gy of γ-radiation within 24 h of collection. At days 3, 5, 7, 9, 11, 14, and 28 postirradiation, fractions were removed and centrifuged, and the levels of methehemoglobin and oxyhemoglobin were measured. Hb was isolated to measure the denaturation and UV-vis spectra. The results from electrophoresis demonstrated that there was no fragmentation or cross-linking of the hemoglobin. However, ferrous center oxidation was identified as a very significant process. This mechanism is likely through an autoxidation process of the ferrous heme center, which has a maximal intensity between 5 and 7 days of storage. Interestingly, a subsequent reduction of the oxidized heme species was observed, and after 9 days of storage, the difference between the ferric species present in the control and irradiated samples was not representative. This interesting fact suggests a type of "protective action" by the blood to control the oxidative stress generated by the gamma irradiation. The UV-vis measurements demonstrated that the oxidized species was predominantly formed by hemichrome species (bis-histidine ferric heme species), which are usually associated with Heinz bodies. After 28 days of storage, evidence from the UV-vis measurements indicated that the oxidation of the irradiated sample was much higher than that observed in the control sample. These results demonstrate that despite the minimal polypeptide changes observed in the hemoglobin of stored red blood cells after gamma irradiation, the oxidation of the heme metallic center is not irrelevant and must be controlled to improve the hematological clinical procedures associated with the storage of red blood cells.


Assuntos
Preservação de Sangue , Eritrócitos/efeitos da radiação , Raios gama/efeitos adversos , Hemoglobinas/efeitos da radiação , Procedimentos de Redução de Leucócitos/métodos , Eletroforese das Proteínas Sanguíneas , Heme/efeitos da radiação , Hemoglobinas/ultraestrutura , Humanos , Metemoglobina/análise , Oxirredução , Estresse Oxidativo , Oxiemoglobinas/análise , Conformação Proteica/efeitos da radiação , Desnaturação Proteica , Sulfa-Hemoglobina/análise , Fatores de Tempo
12.
Arch Biochem Biophys ; 529(2): 99-104, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23232081

RESUMO

Methylglyoxal (MG) reacts with proteins to form advanced glycation end products (AGEs). Although hemoglobin modification by MG is known, the modified protein is not yet characterized. We have studied the nature of AGE formed by MG on human hemoglobin (HbA(0)) and its effect on structure and function of the protein. After reaction of HbA(0) with MG, the modified protein (MG-Hb) was separated and its properties were compared with those of the unmodified protein HbA(0). As shown by MALDI-mass spectrometry, MG converted Arg-92α and Arg-104ß to hydroimidazolones in MG-Hb. Compared to HbA(0), MG-Hb exhibited decreased absorbance around 280nm, reduced tryptophan fluorescence (excitation 285nm) and increased α-helix content. However, MG modification did not change the quaternary structure of the heme protein. MG-Hb appeared to be more thermolabile than HbA(0). The modified protein was found to be more effective than HbA(0) in H(2)O(2)-mediated iron release and oxidative damages involving Fenton reaction. MG-Hb exhibited less peroxidase activity and more esterase activity than HbA(0). MG-induced structural and functional changes of hemoglobin may enhance oxidative stress and associated complications, particularly in diabetes mellitus with increased level of MG.


Assuntos
Hemoglobinas/química , Hemoglobinas/ultraestrutura , Aldeído Pirúvico/química , Sítios de Ligação , Humanos , Ligação Proteica , Relação Estrutura-Atividade
13.
Acta Pharm ; 62(2): 201-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22750818

RESUMO

Laser light scattering and scanning electron microscopy (SEM) are used to study hemoglobin in the aqueous phase. The impact that salts [NaCl, Ca3(PO4)2] and iron oxide nanoparticles have on the hemoglobin size are also studied. The first set of experiments examined hemoglobin aggregates in the aqueous phases in the presence of salts and nanoparticles. Aqueous phase samples were then dehydrated and examined using SEM. The resulting structures resemble those observed in nanobacteria studies conducted in other labs. This study demonstrates that aggregates of hemoglobin and various salts found in a physiological environment can produce structures that resemble nanobacteria.


Assuntos
Hemoglobinas/química , Nanopartículas Metálicas/química , Nanopartículas Calcificantes/química , Fosfatos de Cálcio/química , Precipitação Química , Compostos Férricos/química , Hemoglobinas/ultraestrutura , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Varredura , Concentração Osmolar , Tamanho da Partícula , Cloreto de Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
J Biomech ; 45(11): 1947-51, 2012 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-22673758

RESUMO

Sickle cell disease (SCD) is caused by a single point mutation in the beta-chain hemoglobin gene, resulting in the presence of abnormal hemoglobin S (HbS) in the patients' red blood cells (RBCs). In the deoxygenated state, the defective hemoglobin tetramers polymerize forming stiff fibers which distort the cell and contribute to changes in its biomechanical properties. Because the HbS fibers are essential in the formation of the sickle RBC, their material properties draw significant research interests. Here, a solvent-free coarse-grain molecular dynamics (CGMD) model is introduced to simulate single HbS fibers as a chain of particles. First, we show that the proposed model is able to efficiently simulate the mechanical behavior of single HbS fibers. Then, the zippering process between two HbS fibers is studied and the effect of depletion forces is investigated. Simulation results illustrate that depletion forces play a role comparable to direct fiber-fiber interaction via Van der Waals forces. This proposed model can greatly facilitate studies on HbS polymerization, fiber bundle and gel formation as well as interaction between HbS fiber bundles and the RBC membrane.


Assuntos
Anemia Falciforme/metabolismo , Eritrócitos/química , Hemoglobinas/química , Hemoglobinas/ultraestrutura , Modelos Químicos , Modelos Moleculares , Simulação por Computador , Humanos , Tamanho da Partícula , Conformação Proteica
15.
ACS Nano ; 6(8): 6897-904, 2012 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-22732258

RESUMO

Seeking safe and effective artificial blood substitutes based on hemoglobin (Hb) as oxygen carriers is an important topic. A significant challenge is to enhance the loading content of Hb in a well-defined structure. Here we report a facile and controllable avenue to fabricate Hb spheres with a high loading content by templating decomposable porous CaCO(3) particles in collaboration with covalent layer-by-layer assembly technique. The surface of the Hb spheres was further chemically modified by biocompatible polyethylene glycol to protect and stabilize the system. Multiple characterization techniques were employed to reveal the loading and density of Hb in an individual CaCO(3) particle. The results demonstrate that the strategy developed in this work is effective and flexible for construction of the highly loaded Hb spheres. More importantly, such Hb spheres retain their carrying-releasing oxygen function. It may thus have great potential to develop Hb spheres with highly loaded content as realistic artificial blood substitutes in the future.


Assuntos
Substitutos Sanguíneos/síntese química , Hemoglobinas/química , Hemoglobinas/ultraestrutura , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Oxigênio/química , Cristalização/métodos , Teste de Materiais , Tamanho da Partícula , Ligação Proteica , Propriedades de Superfície
16.
Opt Express ; 20(9): 10295-309, 2012 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-22535119

RESUMO

In this paper, we present an automated approach to quantify information about three-dimensional (3D) morphology, hemoglobin content and density of mature red blood cells (RBCs) using off-axis digital holographic microscopy (DHM) and statistical algorithms. The digital hologram of RBCs is recorded by a CCD camera using an off-axis interferometry setup and quantitative phase images of RBCs are obtained by a numerical reconstruction algorithm. In order to remove unnecessary parts and obtain clear targets in the reconstructed phase image with many RBCs, the marker-controlled watershed segmentation algorithm is applied to the phase image. Each RBC in the segmented phase image is three-dimensionally investigated. Characteristic properties such as projected cell surface, average phase, sphericity coefficient, mean corpuscular hemoglobin (MCH) and MCH surface density of each RBC is quantitatively measured. We experimentally demonstrate that joint statistical distributions of the characteristic parameters of RBCs can be obtained by our algorithm and efficiently used as a feature pattern to discriminate between RBC populations that differ in shape and hemoglobin content. Our study opens the possibility of automated RBC quantitative analysis suitable for the rapid classification of a large number of RBCs from an individual blood specimen, which is a fundamental step to develop a diagnostic approach based on DHM.


Assuntos
Eritrócitos/metabolismo , Hemoglobinas/análise , Hemoglobinas/ultraestrutura , Holografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imagem Tridimensional/métodos , Microscopia/métodos , Células Cultivadas , Interpretação Estatística de Dados , Humanos
17.
Nano Lett ; 12(3): 1555-60, 2012 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-22324311

RESUMO

Hemoglobin nanocrystals were analyzed with tip-enhanced Raman scattering (TERS), surface-enhanced resonance Raman scattering (SERRS) and conventional resonance Raman scattering (RRS) using 532 nm excitation. The extremely high spatial resolution of TERS enables selective enhancement of heme, protein, and amino acid bands from the crystal surface not observed in the SERRS or RRS spectra. Two bands appearing at 1378 and 1355 cm(-1) assigned to the ferric and ferrous oxidation state marker bands, respectively, were observed in both TERS and SERRS spectra but not in the RRS spectrum of the bulk sample. The results indicate that nanoscale oxidation changes are occurring at the hemoglobin crystal surface. These changes could be explained by oxygen exchange at the crystal surface and demonstrate the potential of the TERS technique to obtain structural information not possible with conventional Raman microscopy.


Assuntos
Hemoglobinas/química , Hemoglobinas/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Sítios de Ligação , Teste de Materiais/métodos , Oxirredução , Ligação Proteica , Propriedades de Superfície
18.
19.
Eur J Pharm Sci ; 43(1-2): 57-64, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21443949

RESUMO

In the present work polymeric nanoparticles based on Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with m-PEG (2000) and 95% grafted with 2-methoxyethanol (VAM41-PEG) were loaded with human hemoglobin (Hb) and characterized from a physicochemical point of view. The assessment of structural and functional features of the loaded Hb was performed and the effect of the introduction of different reducing agents as aimed at minimizing Hb oxidation during the nanoparticles formulation process, was also investigated. Nanoparticles possessing an average diameter of 138±10 nm and physicochemical features suitable for this kind of application were successfully obtained. Although the oxidation of the protein was not avoided during its loading into nanoparticles, the presence of acidic moieties in the polymeric structure is proposed to be directly involved in the protein inactivation mechanism.


Assuntos
Materiais Biocompatíveis/química , Substitutos Sanguíneos/química , Hemoglobinas , Nanopartículas , Portadores de Fármacos/química , Éteres/química , Etilenoglicóis/química , Hemoglobinas/química , Hemoglobinas/ultraestrutura , Humanos , Anidridos Maleicos/química , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Polietilenoglicóis/química , Compostos de Vinila/química
20.
Biosens Bioelectron ; 25(9): 2009-15, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20167467

RESUMO

Highly porous hemoglobin (Hb) microbelts based biosensor was developed by directly electrospinning Hb onto the glassy carbon (GC) electrode surface without using immobilization matrix, offering an excellent electrochemical sensing platform. Ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy were performed to demonstrate that Hb still kept its native structure in the as-electrospun microbelts. The electrocatalytic property of Hb microbelts modified GC electrode was investigated using hydrogen peroxide (H2O2) and nitrite as model compounds. The cyclic voltammetry results have demonstrated that the Hb microbelts modified electrode shows enhanced activity in the electrochemical reduction of H2O2 and nitrite, which offers a number of attractive features and is explored to develop an amperometric biosensor. The Hb microbelts based amperometric biosensor has fast responses to H2O2 and nitrite, good dynamic response ranges, excellent detection limits of 0.61 microM for H2O2 and 0.47 microM for nitrite (S/N=3), and superior K(M,app) values of 0.093 mM for H2O2 and 0.713 mM for nitrite, respectively. These results demonstrate that the electrospun Hb microbelts can significantly enhance the direct electrochemistry of Hb and has great potential application in mediator-free biosensor applications.


Assuntos
Técnicas Biossensoriais , Hemoglobinas/química , Peróxido de Hidrogênio/análise , Nitritos/análise , Animais , Carbono , Bovinos , Técnicas Eletroquímicas , Hemoglobinas/ultraestrutura , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Espectrofotometria , Espectroscopia de Infravermelho com Transformada de Fourier
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA