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1.
Food Chem ; 304: 125442, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31491714

RESUMO

In this study, the effects of moderate electric fields during thermal denaturation of ß-lactoglobulin were examined through an in situ circular dichroism approach, complemented by intrinsic extrinsic fluorescence analysis. Results have shown that the effects of electric fields in protein unfolding were linearly dependent on the applied electric field intensity (V/cm) and increased by the use of low electric frequencies - i.e. 50 to 200 Hz. These electric effects caused significant changes on ß-lactoglobulin melting temperature, unfolded conformation and subsequent intermolecular interactions, revealed by the increase of surface hydrophobicity (ANS affinity) and higher conservation of retinol binding. The obtained data provides a clear evidence that moderate electric fields contribute to distinct folding/unfolding of ß-lactoglobulin, resulting in structural modifications. These findings are relevant for (bio)-technological applications involving electric fields processing, bringing new insights for the development of innovative strategies to control protein function and tune production of functional protein systems.

2.
Res Microbiol ; 170(6-7): 256-262, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31419583

RESUMO

The gram-negative, obligate intracellular human pathogen, Chlamydia trachomatis has a bi-phasic developmental cycle. The histone H1-like C. trachomatis DNA binding protein, Hc2, is produced late during the developmental cycle when the dividing reticulate body transforms into the smaller, metabolically inactive elementary body. Together with Hc1, the two proteins compact the chlamydial chromosome and arrest replication and transcription. Hc2 is heterogeneous in length due to variation in the number of lysine rich pentamers. Six pentamers and one hexamer constitute a 36 amino acid long repetitive unit that, in spite of variations, is unique for Chlamydiaceae. Using synthetic peptides, the DNA-binding capacity of the 36 amino acid peptide and that of a randomized peptide was analyzed. Both peptides bound and compacted plasmid DNA, however, electron microscopy of peptide/DNA complexes showed major differences in the resulting aggregated structures. Fluorescence spectroscopy was used to analyze the binding. After complexing plasmid DNA with each of three different intercalating dyes, increasing amounts of peptides were added and fluorescence spectroscopy performed. The major groove binder, methyl green, was displaced by both peptides at low concentrations, while the minor groove binder, Hoechts, and the intercalating dye, Ethidium Bromide, were displaced only at high concentrations of peptides.

3.
PLoS One ; 11(10): e0165419, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27788212

RESUMO

The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm) showed a plasmon absorption band located within the near-infrared range (650-900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.


Assuntos
Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Fototerapia , Polímeros/química , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Ouro/toxicidade , Humanos , Ácido Hialurônico/química , Luz , Ácido Oleico/química , Estabilidade Proteica/efeitos da radiação , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Temperatura Ambiente
4.
PLoS One ; 10(12): e0144454, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26656259

RESUMO

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 µW and 0.3 µW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Muramidase/química , Fotoquímica , Raios Ultravioleta , Animais , Embrião de Galinha , Proteínas do Ovo , Fluorescência , Ácido Hialurônico/química , Ácido Oleico/química , Oxirredução , Temperatura Ambiente
6.
PLoS One ; 9(11): e111617, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25386651

RESUMO

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.


Assuntos
Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Terapia Ultravioleta , Receptores ErbB/metabolismo , Humanos , Ligação Proteica , Conformação Proteica/efeitos da radiação
7.
Nano Lett ; 13(9): 4299-304, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-23915079

RESUMO

Fully exploiting the capability of nano-optics to enhance light-matter interaction on the nanoscale is conditioned by bringing the nano-object to interrogate within the minuscule volume where the field is concentrated. There currently exists several approaches to control the immobilization of nano-objects but they all involve a cumbersome delivery step and require prior knowledge of the "hot spot" location. Herein, we present a novel technique in which the enhanced local field in the hot spot is the driving mechanism that triggers the binding of proteins via three-photon absorption. This way, we demonstrate exclusive immobilization of nanoscale amounts of bovine serum albumin molecules into the nanometer-sized gap of plasmonic dimers. The immobilized proteins can then act as a scaffold to subsequently attach an additional nanoscale object such as a molecule or a nanocrystal. This universal technique is envisioned to benefit a wide range of nano-optical functionalities including biosensing, enhanced spectroscopy like surface-enhanced Raman spectroscopy or surface-enhanced infrared absorption spectroscopy, as well as quantum optics.

8.
Nanoscale ; 5(19): 8874-8, 2013 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-23598462

RESUMO

In a 2D self-organized crystalline structure more than 1000 unit-cells can be observed in a single image. Here we exploit the benefits from having a large number of observations of the same unit cell utilizing an image processing methodology. We obtain sub-picometer resolution data from a 50 pm image of graphene, revealing a 1% axial elongation and a 3 fold symmetry, indicating a chair conformation.

9.
PLoS One ; 7(12): e50733, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23227203

RESUMO

In this work we report the effects of continuous UV-light (276 nm, ~2.20 W.m(-2)) excitation of human insulin on its absorption and fluorescence properties, structure and functionality. Continuous UV-excitation of the peptide hormone in solution leads to the progressive formation of tyrosine photo-product dityrosine, formed upon tyrosine radical cross-linkage. Absorbance, fluorescence emission and excitation data confirm dityrosine formation, leading to covalent insulin dimerization. Furthermore, UV-excitation of insulin induces disulphide bridge breakage. Near- and far-UV-CD spectroscopy shows that UV-excitation of insulin induces secondary and tertiary structure losses. In native insulin, the A and B chains are held together by two disulphide bridges. Disruption of either of these bonds is likely to affect insulin's structure. The UV-light induced structural changes impair its antibody binding capability and in vitro hormonal function. After 1.5 and 3.5 h of 276 nm excitation there is a 33.7% and 62.1% decrease in concentration of insulin recognized by guinea pig anti-insulin antibodies, respectively. Glucose uptake by human skeletal muscle cells decreases 61.7% when the cells are incubated with pre UV-illuminated insulin during 1.5 h. The observations presented in this work highlight the importance of protecting insulin and other drugs from UV-light exposure, which is of outmost relevance to the pharmaceutical industry. Several drug formulations containing insulin in hexameric, dimeric and monomeric forms can be exposed to natural and artificial UV-light during their production, packaging, storage or administration phases. We can estimate that direct long-term exposure of insulin to sunlight and common light sources for indoors lighting and UV-sterilization in industries can be sufficient to induce irreversible changes to human insulin structure. Routine fluorescence and absorption measurements in laboratory experiments may also induce changes in protein structure. Structural damage includes insulin dimerization via dityrosine cross-linking or disulphide bond disruption, which affects the hormone's structure and bioactivity.


Assuntos
Dimerização , Dissulfetos/metabolismo , Insulina/farmacologia , Insulina/efeitos da radiação , Fotólise/efeitos da radiação , Tirosina/análogos & derivados , Raios Ultravioleta , Absorção , Animais , Sítios de Ligação , Dicroísmo Circular , Reagentes para Ligações Cruzadas , Glucose/metabolismo , Cobaias , Humanos , Insulina/química , Cinética , Fotólise/efeitos dos fármacos , Estrutura Terciária de Proteína , Radioimunoensaio , Espectrometria de Fluorescência , Compostos de Sulfidrila/metabolismo , Tirosina/química , Tirosina/metabolismo
10.
PLoS One ; 7(7): e41322, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22848462

RESUMO

The protein structure is a cumulative result of interactions between amino acid residues interacting with each other through space and/or chemical bonds. Despite the large number of high resolution protein structures, the "protein structure code" has not been fully identified. Our manuscript presents a novel approach to protein structure analysis in order to identify rules for spatial packing of amino acid pairs in proteins. We have investigated 8706 high resolution non-redundant protein chains and quantified amino acid pair interactions in terms of solvent accessibility, spatial and sequence distance, secondary structure, and sequence length. The number of pairs found in a particular environment is stored in a cell in an 8 dimensional data tensor. When plotting the cell population against the number of cells that have the same population size, a scale free organization is found. When analyzing which amino acid paired residues contributed to the cells with a population above 50, pairs of Ala, Ile, Leu and Val dominate the results. This result is statistically highly significant. We postulate that such pairs form "structural stability points" in the protein structure. Our data shows that they are in buried α-helices or ß-strands, in a spatial distance of 3.8-4.3Å and in a sequence distance >4 residues. We speculate that the scale free organization of the amino acid pair interactions in the 8D protein structure combined with the clear dominance of pairs of Ala, Ile, Leu and Val is important for understanding the very nature of the protein structure formation. Our observations suggest that protein structures should be considered as having a higher dimensional organization.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Dobramento de Proteína , Proteínas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
11.
J Fluoresc ; 22(1): 323-37, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21997288

RESUMO

Continuous 295 nm excitation of whey protein bovine apo-α-lactalbumin (apo-bLA) results in an increase of tryptophan fluorescence emission intensity, in a progressive red-shift of tryptophan fluorescence emission, and breakage of disulphide bridges (SS), yielding free thiol groups. The increase in fluorescence emission intensity upon continuous UV-excitation is correlated with the increase in concentration of free thiol groups in apo-bLA. UV-excitation and consequent SS breakage induce conformational changes on apo-bLA molecules, which after prolonged illumination display molten globule spectral features. The rate of tryptophan fluorescence emission intensity increase at 340 nm with excitation time increases with temperature in the interval 9.3-29.9°C. The temperature-dependent 340 nm emission kinetic traces were fitted by a 1st order reaction model. Native apo-bLA molecules with intact SS bonds and low tryptophan emission intensity are gradually converted upon excitation into apo-bLA molecules with disrupted SS, molten-globule-like conformation, high tryptophan emission intensity and red-shifted tryptophan emission. Experimental Ahrrenius activation energy was 21.8 ± 2.3 kJ x mol(-1). Data suggests that tryptophan photoionization from the S(1) state is the likely pathway leading to photolysis of SS in apo-bLA. Photoionization mechanism(s) of tryptophan in proteins and in solution and the activation energy of tryptophan photoionization from S(1) leading to SS disruption in proteins are discussed. The observations present in this paper raise concern regarding UV-light pasteurization of milk products. Though UV-light pasteurization is a faster and cheaper method than traditional thermal denaturation, it may also lead to loss of structure and functionality of milk proteins.


Assuntos
Apoproteínas/química , Dissulfetos/química , Lactalbumina/química , Processos Fotoquímicos , Raios Ultravioleta , Animais , Bovinos , Modelos Moleculares , Conformação Proteica/efeitos da radiação , Espectrometria de Fluorescência , Compostos de Sulfidrila/química , Temperatura Ambiente
12.
PLoS One ; 6(12): e25638, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22174733

RESUMO

Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis.


Assuntos
Aminoácidos/química , Proteínas/química , Motivos de Aminoácidos , Aminoácidos/análise , Bases de Dados de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Solventes/química
13.
J Fluoresc ; 21(5): 1897-906, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21494846

RESUMO

Isomerization of trans-stilbenes is known to be induced by light. The two isomers have distinct absorption, fluorescence excitation and emission spectra. Resveratrol, 3,4',5-trihydroxystilbene, is a member of the stilbene family. The interest of the scientific community in resveratrol has increased over the last years due to its biomedical properties. Whereas there is a growing confidence that trans-resveratrol is non-toxic, very little is known about the pharmacology of cis-resveratrol. Of this very reason there is considerable interest in knowing the energetics of the trans-cis conversion. Cis-resveratrol is characterized by a large fluorescence quantum yield when compared to trans-resveratrol. In the present paper we report a detailed analysis of the spectral changes induced in trans-resveratrol upon 260 nm excitation for different time periods. Spectral changes have been monitored with UV-visible absorption and steady-state fluorescence spectroscopy at pH 4 at 20, 25, 30, 35, 40, 45 and 50 °C. Continuous 260 nm excitation induces a blue shift in the absorption and fluorescence excitation spectra of resveratrol and a 14 nm blue shift in its fluorescence emission. The photoisomerization yield is reported as a function of 260 nm excitation time. 330 min continuous excitation led to ~60% isomerization yield. The kinetics of trans-cis isomerization has been monitored following the increase in fluorescence quantum yield upon continuous 260 nm excitation of trans-resveratrol. The study was carried out at the above mentioned temperatures in order to obtain the Arrhenius activation energy of photoisomerization. Activation energy and pre-exponential factor were 3.7 ± 0.3 kcal.mol(-1) and 10.6 ± 1.6 s(-1), respectively. The activation energy is comparable with previously reported values for the photoisomerization of other stilbenes.


Assuntos
Estilbenos/química , Raios Ultravioleta , Fluorescência , Cinética , Estrutura Molecular , Resveratrol , Espectrometria de Fluorescência , Estereoisomerismo , Temperatura Ambiente , Fatores de Tempo
14.
J Fluoresc ; 21(2): 663-72, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21107664

RESUMO

Medical interest in nanotechnology originates from a belief that nanoscale therapeutic devices can be constructed and directed towards its target inside the human body. Such nanodevices can be engineered by coupling superparamagnetic nanoparticle to biomedically active proteins. We hereby report the immobilization of a PhEst, a S-formylglutathione hydrolase from the psychrophilic P. haloplanktis TAC125 onto the gold coated surface of modified superparamagnetic core-shell nanoparticles (Fe(3)O(4)@Au). The synthesis of the nanoparticles is also reported. S-formylglutathione hydrolases constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes. PhEst was originally annotated as a putative feruloyl esterase, an enzyme that releases ferulic acid (an antioxidant reactive towards free radicals such as reactive oxygen species) from polysaccharides esters. Dynamic light scattering, scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-visible absorption spectroscopy, fluorescence spectroscopy, magnetic separation technique and enzyme catalytic assay confirmed the chemical composition of the gold covered superparamagnetic nanoparticles, the binding and activity of the enzyme onto the nanoparticles. Activity data in U/ml confirmed that the immobilized enzyme is approximately 2 times more active than the free enzyme in solution. Such particles can be directed with external magnetic fields for bio-separation and focused towards a medical target for therapeutical as well as bio-sensor applications.


Assuntos
Equipamentos e Provisões , Magnetismo , Nanopartículas de Magnetita/química , Nanotecnologia/instrumentação , Análise Espectral , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ouro/química , Modelos Moleculares , Conformação Proteica , Pseudomonas/enzimologia , Propriedades de Superfície , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo
15.
Biotechnol Bioeng ; 108(5): 999-1010, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21125586

RESUMO

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.


Assuntos
Fótons , Soroalbumina Bovina/química , Sequência de Aminoácidos , Microscopia Eletrônica de Varredura , Modelos Moleculares , Dados de Sequência Molecular , Nanopartículas , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Raios Ultravioleta
16.
Protein Sci ; 19(9): 1751-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20665692

RESUMO

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.


Assuntos
Biomarcadores Tumorais/imunologia , Técnicas Biossensoriais/métodos , Proteínas Imobilizadas/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/diagnóstico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/química , Humanos , Proteínas Imobilizadas/análise , Proteínas Imobilizadas/química , Imunoensaio/métodos , Fragmentos Fab das Imunoglobulinas/química , Masculino , Microscopia de Fluorescência , Modelos Moleculares , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/química , Análise Serial de Proteínas/métodos , Espectrometria de Fluorescência , Raios Ultravioleta
17.
J Fluoresc ; 20(2): 483-92, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19943094

RESUMO

Modification of hyaluronic acid (HA) with aryl succinic anhydrides results in new biomedical properties of HA as compared to non-modified HA, such as more efficient skin penetration, stronger binding to the skin, and the ability to blend with hydrophobic materials. In the present study, hyaluronic acid has been derivatised with the anhydride form of phenyl succinic acid (PheSA). The fluorescence of PheSA was efficiently quenched by the HA matrix. HA also acted as a singlet oxygen scavenger. Fluorescence lifetime(s) of PheSA in solution and when attached to the HA matrix has been monitored with ps resolved streak camera technology. Structural and fluorescence properties changes induced on HA-PheSA due to the presence of singlet oxygen were monitored using static light scattering (SLS), steady state fluorescence and ps time resolved fluorescence studies. SLS studies provided insight into the depolymerisation kinetics of PheSA derivatised HA matrix in the presence of singlet oxygen. Time resolved fluorescence studies grave insight into the dynamics of the reaction mechanisms induced on HA-PheSA by singlet oxygen. These studies provided insight into the medical relevance of PheSA derivatised HA: its capacity of scavenging singlet oxygen and of quenching PheSA fluorescence. These studies revealed that HA-PheSA is a strong quencher of electronic excited state PheSA and acts as a scavenger of singlet oxygen, thus medical applications of this derivatised form of HA may protect tissues and organs, such as skin, against reactive oxygen species damage.


Assuntos
Ácido Hialurônico/química , Succinatos/química , Anidridos/química , Fluorescência , Cinética , Luz , Espalhamento de Radiação , Oxigênio Singlete/química , Fatores de Tempo
18.
J Nanosci Nanotechnol ; 9(7): 4333-7, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19916452

RESUMO

Our group has previously shown that biomolecules containing disulfide bridges in close proximity to aromatic residues can be immobilized, through covalent bonds, onto thiol derivatized surfaces upon UV excitation of the aromatic residue(s). We have also previously shown that our new technology can be used to print arrays of biomolecules and to immobilize biomolecules according to any specific pattern on a planar substrates with micrometer scale resolution. In this paper we show that we can immobilize proteins according to diffraction patterns of UV light. We also show that the feature size of the immobilized patterns can be as small as the diffraction limit for the excitation light, and that the immobilized patterns correspond to the diffraction pattern used to generate it. The flexibility of this new technology will in principle make it possible to create any pattern of biomolecules onto a substrate, which can be generated by a UV diffraction pattern. Such patterns can have sub-micron feature sizes and could therefore be of great relevance for present and future nanotechnological applications.


Assuntos
Biopolímeros/química , Cristalização/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Proteínas/química , Refratometria/métodos , Adsorção , Sítios de Ligação , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Ligação Proteica , Propriedades de Superfície
19.
Biophys J ; 97(1): 211-26, 2009 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-19580759

RESUMO

Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after approximately 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR *-. Additional mechanisms that may lead to the near disappearance of Trp(*) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.


Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/efeitos da radiação , Fotólise , Raios Ultravioleta , Elétrons , Proteínas Fúngicas/química , Proteínas Fúngicas/efeitos da radiação , Fusarium , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Muramidase/química , Muramidase/efeitos da radiação , Análise Espectral , Triptofano/química , Triptofano/efeitos da radiação
20.
Proteomics ; 9(15): 3945-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19637236

RESUMO

The technique of UV-light-assisted immobilization of disulfide containing proteins has been combined with the Fourier-transforming properties of lenses as well as with a simple millimeter scale feature size spatial mask. The result is a new simple and inexpensive way of creating high-density protein arrays with feature sizes down to a few hundred nanometers, which represents an improvement of tenfold over existing commercially available high-density protein arraying methods.


Assuntos
Dissulfetos/química , Proteínas Imobilizadas/análise , Análise Serial de Proteínas/métodos , Raios Ultravioleta , Análise de Fourier , Proteínas Imobilizadas/química , Óptica e Fotônica , Análise Serial de Proteínas/economia
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