RESUMEN
The local valence orbital structure of solid glycine, diglycine, and triglycine is studied using soft X-ray emission spectroscopy (XES), resonant inelastic soft X-ray scattering (RIXS) maps, and spectra calculations based on density-functional theory. Using a building block approach, the contributions of the different functional groups of the peptides are separated. Cuts through the RIXS maps furthermore allow monitoring selective excitations of the amino and peptide functional units, leading to a modification of the currently established assignment of spectral contributions. The results thus paint a new-and-improved picture of the peptide bond, enhance the understanding of larger molecules with peptide bonds, and simplify the investigation of such molecules in aqueous environment.
Asunto(s)
Modelos Químicos , Péptidos/química , Dispersión Dinámica de Luz , Electrones , Glicina/química , Glicilglicina/química , Oligopéptidos/química , Teoría Cuántica , Agua/química , Difracción de Rayos XRESUMEN
The occupied and unoccupied electronic structure of imidazole (C3N2H4) and imidazolium (C3N2H5+) in aqueous solutions is studied by X-ray emission spectroscopy (XES) and resonant inelastic soft X-ray scattering (RIXS). Both systems show distinct RIXS fingerprints with strong resonant effects. A comparison with calculated X-ray emission spectra of isolated imidazole and imidazolium suggests only a small influence of hydrogen bonding in the aqueous solution on the electronic structure of imidazole and imidazolium, and allows the attribution of specific spectral features to the non-equivalent nitrogen and carbon atoms in the molecules. In the case of nitrogen, this can also be achieved by site-selective resonant excitation. Furthermore, we find spectator shifts and symmetry selectivity in the RIXS spectra, as well as indications for rapid proton dynamics on the femtosecond timescale of the RIXS process, and derive the HOMO-LUMO gaps for the two molecules in aqueous solution.
RESUMEN
Nonresonant N K, O K, C K, and S L2,3 X-ray emission spectra of the 20 most common proteinogenic amino acids in their solid zwitterionic form are reported. They represent a comprehensive database that can serve as a reliable basis for the X-ray absorption spectroscopy (XES) studies of peptides and proteins. At the most important N and O K edges, clear similarities and differences between the spectra of certain amino acids are observed and associated with the specific chemical structure of these molecules and their functional groups. Analysis of these spectra allows the generation of spectral fingerprints of the protonated amino group, the deprotonated carboxylic group, and, using a building block approach, the specific nitrogen- and oxygen-containing functional groups in the side chains of the amino acids. Some of these fingerprints are compared to the spectra of reference compounds with the respective functional groups; they exhibit reasonable similarity, underlining the validity of the spectral fingerprint approach. The C K and S L2,3 XES spectra are found to be specific for each amino acid, in accordance with the different local environments of the involved C and S atoms, respectively.
Asunto(s)
Aminoácidos/química , Proteínas/química , Estructura Molecular , Espectrometría por Rayos XRESUMEN
Understanding the molecular structure of the hydration shells and their impact on the hydrogen bond (HB) network of water in aqueous salt solutions is a fundamentally important and technically relevant question. In the present work, such hydration effects were studied for a series of representative salt solutions (NaCl, KCl, CaCl2, MgCl2, and KBr) by soft X-ray emission spectroscopy (XES) and resonant inelastic soft X-ray scattering (RIXS). The oxygen K-edge XES spectra could be described with three components, attributed to initial state HB configurations in pure water, water molecules that have undergone an ultrafast dissociation initiated by the X-ray excitation, and water molecules in contact with salt ions. The behavior of the individual components, as well as the spectral shape of the latter component, has been analyzed in detail. In view of the role of ions in such effects as protein denaturation (i.e., the Hofmeister series), we discuss the ion-specific nature of the hydration shells and find that the results point to a predominant role of anions as compared to cations. Furthermore, we observe a concentration-dependent suppression of ultrafast dissociation in all salt solutions, associated with a significant distortion of intact HB configurations of water molecules facilitating such a dissociation.
RESUMEN
The electronic structure of gas-phase methanol molecules (H3COH, H3COD, and D3COD) at atmospheric pressure was investigated using resonant inelastic soft X-ray scattering (RIXS) at the O K and C K edges. We observe strong changes of the relative emission intensities of all valence orbitals as a function of excitation energy, which can be related to the symmetries of the involved orbitals causing an angularly anisotropic RIXS intensity. Furthermore, all observed emission lines are subject to strong spectator shifts of up to -0.9 eV at the O K edge and up to -0.3 eV at the C K edge. At the lowest O K resonance, we find clear evidence for dissociation of the methanol molecule on the time scale of the RIXS process, which is illustrated by comparing X-ray emission spectra of regular and deuterated methanol.
RESUMEN
The electronic structure of the amino acid L-cysteine in an aqueous environment was studied using resonant inelastic soft X-ray scattering (RIXS) in a 2D map representation and analyzed in the framework of a "building block" approach. The element selectivity of RIXS allows a local investigation of the electronic structure of the three functional groups of cysteine, namely, the carboxyl, amino, and thiol groups, by measuring at the O K, N K, and S L2,3 edges, respectively. Variation of the pH value allows an investigation of molecules with protonated and deprotonated functional groups, which can then be compared with simple reference molecules that represent the isolated functional groups. We find that such building blocks can provide an excellent description of X-ray emission spectroscopy (XES) and RIXS spectra, but only if all nearest-neighbor atoms are included. This finding is analogous to the building block principle commonly used in X-ray absorption spectroscopy. The building blocks show a distinct spectral character (fingerprint) and allow a comprehensive interpretation of the cysteine spectra. This simple approach opens the path to investigate the electronic structure of more complex biological molecules in aqueous solutions using XES and RIXS.
Asunto(s)
Cisteína/química , Electrones , Concentración de Iones de Hidrógeno , Modelos Químicos , Teoría Cuántica , Espectrometría por Rayos X , Agua/químicaRESUMEN
We present a novel gas cell designed to study the electronic structure of gases and gas/solid interfaces using soft x-ray emission and absorption spectroscopies. In this cell, the sample gas is separated from the vacuum of the analysis chamber by a thin window membrane, allowing in situ measurements under atmospheric pressure. The temperature of the gas can be regulated from room temperature up to approximately 600 °C. To avoid beam damage, a constant mass flow can be maintained to continuously refresh the gaseous sample. Furthermore, the gas cell provides space for solid-state samples, allowing to study the gas/solid interface for surface catalytic reactions at elevated temperatures. To demonstrate the capabilities of the cell, we have investigated a TiO2 sample behind a mixture of N2 and He gas at atmospheric pressure.
RESUMEN
Creatinine-specific antibodies have been generated and used for highly sensitive and specific immunochemical creatinine determinations. Creatinine was derivatized at N3 and coupled to KLH carrier protein. On the basis of this immunogen, monoclonal antibodies were developed by hybridoma technology. Antibodies from various clones have been characterized with BIAcore 2000 with respect to the dissociation constant and specificity. Antibodies of clone B90-AH5 exhibited the lowest dissociation constant (0.74 microM) and the highest specificity for creatinine and were chosen for the development of a competitive ELISA and an amperometric creatinine sensor. The creatinine sensor was constructed by fixing a creatinine-modified membrane on the top of a platinum working electrode which was then incorporated into a stirred electrochemical measuring cell. For creatinine determination the creatinine-containing sample was incubated with B90-AH5 and anti-IgG(mouse)-glucose oxidase conjugate and applied to the measuring cell. After a washing step glucose was added and the produced hydrogen peroxide was registered at Eappl = +600 mV vs Ag/AgCl. The measuring range was 0.01-10 microg/mL. The highest sensitivity for creatinine was achieved at 330 ng/mL (3 microM) and the lower detection limit at 4.5 ng/mL (40 nM). This is far below the relevant clinical range, which is 5-17 microg/mL (44-150 microM) and allows a reliable determination of very low creatinine concentrations in serum, where standard methods cannot be applied. After each measurement the sensor was regenerated with 10 mM HCl without any loss in binding activity.
Asunto(s)
Técnicas Biosensibles , Creatina/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Monoclonales/inmunología , Creatina/inmunología , Electroquímica , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Sensibilidad y EspecificidadRESUMEN
Immunoassays (IA) use the specific antigen antibody complexation for analytical purposes. Radioimmunoassays (RIA), fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are well established in clinical diagnostics. For the development of hand-held devices which can be used for point of care measurements, electrochemical immunoassays are promising alternatives to existing immunochemical tests. Moreover, for opaque or optically dense matrices electrochemical methods are superior. Potentiometric, capacitive and amperometric transducers have been applied for direct and indirect electrochemical immunoassays. However, due to their fast detection, broad linear range and low detection limit, amperometric transducers are preferred. Competitive and noncompetitive amperometric immunoassays have been developed with redox compounds or enzymes as labels. This review will give an overview of the most frequently applied principles in electrochemical immunoassays. The potential of an indirect competitive amperometric immunoassay for the determination of creatinine within nanomolar range and the circumvention of the most serious problem in electrochemical immunoassays, namely regeneration, will be discussed.
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Anticuerpos/metabolismo , Electroquímica , Inmunoensayo/métodos , Animales , Anticuerpos/inmunología , Técnicas Biosensibles/instrumentación , Creatinina/análisis , Electrodos , Humanos , Oxidación-ReducciónRESUMEN
A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.