RESUMO
Humification is a ubiquitous natural process of biomass degradation that creates multicomponent systems of nonliving organic matter, including dissolved organic matter (DOM) and humic substances (HS) in water environments, soils, and organic rocks. Despite significant differences in molecular composition, the optical properties of DOM and HS are remarkably similar, and the reason for this remains largely unknown. Here, we employed fluorescence spectroscopy with (sub)picosecond resolution to elucidate the role of electronic interactions within DOM and HS. We revealed an ultrafast decay component with a characteristic decay lifetime of 0.5-1.5 ps and spectral diffusion originating from excitation energy transfer (EET) in the system. The rate of EET was positively correlated to the fraction of aromatic species and tightness of aromatic species packing. Diminishing the number of EET donor-acceptor pairs by reduction with NaBH4 (decrease of the acceptor number), decrease of pH (decrease of the electron-donating ability), or decrease of the average particle size by filtration (less donor-acceptor pairs within a particle) resulted in a lower impact of the ultrafast component on fluorescence decay. Our results uncover the role of electronic coupling among fluorophores in the formation of DOM and HS optical properties and provide a framework for studying photophysical processes in heterogeneous systems of natural fluorophores.
Assuntos
Substâncias Húmicas , Solo , Biomassa , Transferência de Energia , Substâncias Húmicas/análise , Espectrometria de FluorescênciaRESUMO
Endogenous autofluorescence of biological tissues is an important source of information for biomedical diagnostics. Despite the molecular complexity of biological tissues, the list of commonly known fluorophores is strictly limited. Still, the question of molecular sources of the red and near-infrared excited autofluorescence remains open. In this work we demonstrated that the oxidation products of organic components (lipids, proteins, amino acids, etc.) can serve as the molecular source of such red and near-infrared excited autofluorescence. Using model solutions and cell systems (human keratinocytes) under oxidative stress induced by UV irradiation we demonstrated that oxidation products can contribute significantly to the autofluorescence signal of biological systems in the entire visible range of the spectrum, even at the emission and excitation wavelengths higher than 650 nm. The obtained results suggest the principal possibility to explain the red fluorescence excitation in a large class of biosystems-aggregates of proteins and peptides, cells and tissues-by the impact of oxidation products, since oxidation products are inevitably presented in the tissue. The observed fluorescence signal with broad excitation originated from oxidation products may also lead to the alteration of metabolic imaging results and has to be taken into account.
Assuntos
Fluorescência , Imagem Molecular , Imagem Óptica , Oxirredução , Biomarcadores , Citometria de Fluxo , Humanos , Queratinócitos/metabolismo , Microscopia Confocal , Imagem Molecular/métodos , Imagem Óptica/métodos , Processos Fotoquímicos , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
Blood cell analysis is one of the standard clinical tests. Despite the widespread use of exogenous markers for blood cell quantification, label-free optical methods are still of high demand due to their possibility for in vivo application and signal specific to the biochemical state of the cell provided by native fluorophores. Here we report the results of blood cell characterization using label-free fluorescence imaging techniques and flow-cytometry. Autofluorescence parameters of different cell types - white blood cells, red blood cells, erythrophagocytic cells - are assessed and analyzed in terms of molecular heterogeneity and possibilities of differentiation between different cell types in vitro and in vivo.
RESUMO
Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (â¼350â¯nm excitation; â¼450â¯nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.
Assuntos
Amiloide/química , Insulina/química , Muramidase/química , Agregados Proteicos , Aminoácidos/química , Animais , Benzotiazóis/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Cinética , Oxirredução , Espectrometria de FluorescênciaRESUMO
Amyloid fibrils formation is the well-known hallmark of various neurodegenerative diseases. Thioflavin T (ThT)-based fluorescence assays are widely used to detect and characterize fibrils, however, if performed in bioliquids, the analysis can be biased due to the presence of other, especially abundant, proteins. Particularly, it is known that albumin may bind ThT, although the binding mechanism remains debatable. Here the role of low-order albumin oligomers in ThT binding is investigated using time-resolved fluorometry and size-exclusion chromatography. Under conditions used, the fraction of dimers in human (HSA) and bovine (BSA) serum albumin solutions is as low as â¼7%, however, it is responsible for â¼50% of ThT binding. For both albumins, the binding affinity was estimated to be â¼200 and â¼40µM for monomeric and dimeric species, respectively. Molecular docking suggested that ThT preferentially binds in the hydrophobic pocket of subdomain IB of albumin monomer in a similar position but with a variable torsion angle, resulting in a lower fluorescence enhancement (â¼40-fold) compared to amyloid fibrils (â¼1000-fold). Dimerization of albumin presumably creates an extra binding site at the subunit interface. These results demonstrate the underestimated role of low-order albumin oligomers that can be highly relevant when analyzing drugs binding using fluorescence spectroscopy.
Assuntos
Albumina Sérica/química , Tiazóis/química , Benzotiazóis , Sítios de Ligação , Cromatografia em Gel , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Albumina Sérica/metabolismo , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Tiazóis/metabolismoRESUMO
The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.
Assuntos
Capilares/anatomia & histologia , Colágeno Tipo I/análise , Derme/anatomia & histologia , Derme/química , Elastina/análise , Imagem Óptica/métodos , Colágeno Tipo III/análise , Voluntários Saudáveis , HumanosRESUMO
Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10-5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.
Assuntos
Fenômenos Biofísicos , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Luz , Processos Fotoquímicos , Proteínas de Bactérias/metabolismo , Fenômenos Biofísicos/efeitos da radiação , Carotenoides/metabolismo , Fluorescência , Cinética , Processos Fotoquímicos/efeitos da radiação , Synechocystis/metabolismo , Synechocystis/efeitos da radiaçãoRESUMO
We studied the interaction between bovine serum albumin (BSA) and lanthanide ions in aqueous solution in the 4.0÷9.5pH range. A strong increase of the solution turbidity was observed at pH values exceeding 6, which corresponds to the formation of Ln(OH)3 nanoparticles, while no changes were observed near the isoelectric point of BSA (pH 4.7). The results of the dynamic light scattering and protein adsorption measurements clearly demonstrated that the observed turbidity enhancement was caused by albumin sorption on the surface of Ln(OH)3 and colloid particles bridging via adsorbed protein molecules. Upon pH increase from 4.5 to 6.5, albumin adsorption on lanthanide colloids was observed, while the following increase of pH from 6.5 to 9.5 led to protein desorption. The predominant role of the electrostatic interactions in the adsorption and desorption processes were revealed in the zeta-potential measurements. No reversibility was observed upon decreasing pH from 9.5 to 4.5 that was suggested to be due to the other interaction mechanisms present in the system. It was shown that while for all lanthanide ions the interaction mechanism with BSA was similar, its manifestation in the optical properties of the system was significantly different. This was interpreted as a consequence of the differences in lanthanides hydrolysis constants.
Assuntos
Coloides/química , Elementos da Série dos Lantanídeos/química , Nanopartículas/química , Soroalbumina Bovina/química , Adsorção , Animais , Bovinos , Coloides/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Elementos da Série dos Lantanídeos/metabolismo , Nanopartículas/metabolismo , Soroalbumina Bovina/metabolismo , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3'-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the ß-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.
Assuntos
Proteínas de Bactérias/química , Proteínas Luminescentes/química , Simulação de Dinâmica Molecular , Absorção de Radiação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cianobactérias/química , Corantes Fluorescentes/farmacologia , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Tyrosine fluorescence in native proteins is known to be effectively quenched, whereas its emission increases upon proteins' unfolding. This suggests that tyrosine fluorescence could be exploited for probing structural rearrangements of proteins in addition to the extensively used tryptophan emission. We studied the possibility of using tyrosine fluorescence as an indicator of surfactant-induced conformational changes in albumins. It was shown that fluorescence of tyrosine residues, which are uniformly distributed all over the protein molecules, allows the detection of subtle structural rearrangements of proteins upon surfactant binding, which do not influence the properties of a single tryptophan residue buried in the inner hydrophobic region of human serum albumin. Tyrosine fluorescence properties, including its fluorescence lifetime, revealed the multistage character of surfactant binding to albumin, consistent with the data provided by other methods. The obtained results demonstrate the possibility of probing conformational changes in proteins using tyrosine photophysical parameters as indicators.
Assuntos
Fluorescência , Albumina Sérica/química , Tensoativos/química , Tirosina/química , Animais , Bovinos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Micelas , Conformação Proteica , Desdobramento de Proteína , Análise Espectral , Triptofano/químicaRESUMO
Intrinsic fluorescence quenching of bovine serum albumin (BSA) and europium(III) luminescence in BSA complexes were investigated. The number of BSA binding sites (n) and equilibrium constant (Keq) values were determined from both measurements provided qualitatively different results. While the modified Stern-Volmer relation for BSA fluorescence quenching gave n = 1 at pH 4.5 and pH 6, two sets of binding sites were determined from Eu(3+) luminescence with n1 = 2, n2 = 4 at pH 6 and n1 = 1, n2 = 2 at pH 4.5. The model explaining the discrepancy between the results obtained by these fluorescent approaches was suggested, and the limitations in application of the "log-log" Stern-Volmer plots in analysis of binding processes were discussed.
Assuntos
Európio/química , Soroalbumina Bovina/química , Animais , Sítios de Ligação , Bovinos , Európio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Soroalbumina Bovina/metabolismo , Espectrometria de FluorescênciaRESUMO
We use an advanced fluorescence method of Nonlinear Laser Fluorimetry in combination with Fluorescence Induction and Relaxation technique to study the influence of excess-light conditions on the physiological state of the green alga Chlorella pyrenoidosa. We demonstrate that zeaxanthin-dependent non-photochemical quenching leads to a significant increase in the rate constant of singlet-singlet annihilation of chlorophyll a excited state, which suggests profound conformational changes in the light-harvesting complexes of photosystem II.
Assuntos
Chlorella/metabolismo , Chlorella/efeitos da radiação , Fluorometria/métodos , Lasers , Luz/efeitos adversos , Chlorella/citologia , Escuridão , Cinética , Estresse Fisiológico/efeitos da radiação , Xantofilas/metabolismo , ZeaxantinasRESUMO
High light poses a threat to oxygenic photosynthetic organisms. Similar to eukaryotes, cyanobacteria evolved a photoprotective mechanism, non-photochemical quenching (NPQ), which dissipates excess absorbed energy as heat. An orange carotenoid protein (OCP) has been implicated as a blue-green light sensor that induces NPQ in cyanobacteria. Discovered in vitro, this process involves a light-induced transformation of the OCP from its dark, orange form (OCP(o)) to a red, active form, however, the mechanisms of NPQ in vivo remain largely unknown. Here we show that the formation of the quenching state in vivo is a multistep process that involves both photoinduced and dark reactions. Our kinetic analysis of the NPQ process reveals that the light induced conversion of OCP(o) to a quenching state (OCP(q)) proceeds via an intermediate, non-quenching state (OCP(i)), and this reaction sequence can be described by a three-state kinetic model. The conversion of OCP(o) to OCP(i) is a photoinduced process with the effective absorption cross section of 4.5 × 10(-3)Ų at 470 nm. The transition from OCP(i) to OCP(q) is a dark reaction, with the first order rate constant of approximately 0.1s(-1) at 25°C and the activation energy of 21 kcal/mol. These characteristics suggest that the reaction rate may be limited by cis-trans proline isomerization of Gln224-Pro225 or Pro225-Pro226, located at a loop near the carotenoid. NPQ decreases the functional absorption cross-section of Photosystem II, suggesting that formation of the quenched centers reduces the flux of absorbed energy from phycobilisomes to the reaction centers by approximately 50%.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Luz , Modelos Químicos , Fotossíntese/fisiologia , Proteínas de Bactérias/química , Cianobactérias/química , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
We investigate photophysical processes that take place in macromolecules of a fluorescent protein mRFP1 under UV excitation [when the energy transfer in a localized donor-acceptor (LDA) pair, which is presented in the molecules of the protein, becomes apparent]. We used a special approach based on the fluorescence laser spectroscopy technique. The energy transfer rates in LDA pairs and photophysical parameters of fluorophores (chromophores) of three spectral forms, which coexist in the ensemble of the macromolecules of this protein, were determined under pulse UV laser excitation.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Algoritmos , Transferência de Energia , Transferência Ressonante de Energia de Fluorescência/instrumentação , Lasers , Modelos Teóricos , Fatores de Tempo , Proteína Vermelha FluorescenteRESUMO
We discuss the problems with and the real possibilities of determining oil pollution in situ in coastal marine waters with fluorescence spectroscopy and of using artificial neural networks for data interpretation. In general, the fluorescence bands of oil and aquatic humic substance overlap. At oil concentrations in water from a few to tens of micrograms per liter, the intensity of oil fluorescence is considerably lower than that of humic substances at concentrations that typically are present in coastal waters. Therefore it is necessary to solve the problem of separating the small amount of oil fluorescence from the humic substance background in the spectrum. The problem is complicated because of possible interactions between the components and variations in the parameters of the fluorescence bands of humic substances and oil in water. Fluorescence spectra of seawater samples taken from coastal areas of the Black Sea, samples prepared in the laboratory, and numerically simulated spectra were processed with an artificial neural network. The results demonstrate the possibility of estimating oil concentrations with an accuracy of a few micrograms per liter in coastal waters also in cases in which the contribution from other organic compounds, primarily humic substances, to the fluorescence spectrum exceeds that of oil by 2 orders of magnitude and more.