Chromophoric Species Research Articles

A Tale of Two Chromophores: Investigating Two Distinct Isoforms of Cysteine Dioxygenase

Cysteine dioxygenase (CDO) is a mononuclear iron-containing enzyme that converts L-cysteine to L-cysteine sulfinic acid. The active site iron of CDO is coordinated by 3-His residues, and within 3.3Å of the iron center exists a unique thioether Cys-Tyr crosslink [1]. Wild-type CDO exists in a heterogeneous mixture of crosslinked and noncrosslinked species. Recent studies suggest the Cys-Tyr crosslink enhances catalytic activity, and plays a role in stabilizing the iron center through second-sphere interactions [2]. Two isoforms of CDO with distinct colors were identified in purifications of wild-type CDO. The appearance of the two CDO isoforms was observed in eluted fractions from an anion exchange column. Separate elution patterns for the two chromophoric isoforms suggest the species exist in different ionization states. The two isoforms had a similar amount of iron bound, and there was no significant difference in the steady-state kinetic parameters between the isoforms when monitoring oxygen consumption. The EPR spectra showed a high spin ferric signal for both isoforms, but there was a noticeable 4-fold decrease in the relative peak intensity for one chromophoric species. The results from the EPR studies suggest that the metal center of the two chromophoric species have different oxidations states due to altered reduction potentials generated by second-sphere charge-transfer interactions. Identifying the amino acid residues that participate in the charge transfer interactions will provide valuable insight regarding the diverging reduction potentials of the two chromophoric species.

Photophysics and Photochemistry of a DNA–Protein Cross-Linking Model: A Synergistic Approach Combining Experiments and Theory

The photophysical and photochemical properties of 5-benzyluracil and 5,6-benzyluracil, the latter produced by photocyclization of the former through irradiation with femtosecond UV laser pulses, are investigated both experimentally and theoretically. The absorption spectra of the two molecules are compared, and the principal electronic transitions involved are discussed, with particular emphasis on the perturbation induced on the two chromophore species (uracil and benzene) by their proximity. The photoproduct formation for different irradiation times was verified with high-performance liquid chromatography and nuclear magnetic resonance measurements. The steady-state fluorescence demonstrates that the fluorescence is a distinctive physical observable for detection and selective identification of 5- and 5,6-benzyluracil. The principal electronic decay paths of the two molecules, obtained through TDDFT calculations, explain the features observed in the emission spectra and the photoreactivity of 5-benzyluracil. The order of magnitude of the lifetime of the excited states is derived with steady-state fluorescence anisotropy measurements and rationalized with the help of the computational findings. Finally, the spectroscopic data collected are used to derive the photocyclization and fluorescence quantum yields. In obtaining a global picture of the photophysical and photochemical properties of the two molecules, our findings demonstrates that the use of 5-benzyluracil as a model system to study the proximity relations of a DNA base with a close-lying aromatic amino acid is valid at a local level since the main characteristics of the decay processes from the excited states of the uracil/thymine molecules remain almost unchanged in 5-benzyluracil, the main perturbation arising from the presence of the close-lying aromatic group.

New Insights into the Mechanisms of Photodegradation/Stabilization of P3HT:PCBM Active Layers Using Poly(3-hexyl-d13-Thiophene)

The photo-oxidation mechanism of thin-film blends based on poly(3-hexylthiophene):phenyl-C61-butyric acid methyl ester (P3HT:PCBM) upon irradiation with ultraviolet–visible light (UV-Vis) was studied. The use of deuterated P3HT, i.e., poly(3-hexyl-d13-thiophene) (P3HdT), permitted discrimination of carbon originating from the hexyl-d13 chain and carbon originating from PCBM and the nondeuterated thiophene unit. The photo-oxidation of both components of the blend was monitored using the combination of various analytical techniques to probe the bulk and the surface of the deposits. The results show that the stabilization of P3HT by PCBM is due to a morphological reorganization between P3HT and PCBM. This change occurs at a low temperature (ca. 42 °C) and increases the lifetime of the primary property, i.e., the ability of the active layer to absorb light. However, this is counterbalanced by the enhanced formation of oxidized PCBM molecules, which may act as electrons traps. It is shown that UV light is harmful for P3HT, PCBM, and P3HT:PCBM blend stabilities, even if PCBM provides a filter effect that is strongest at short wavelengths. It is proposed that the photochemical behavior of the chromophoric species involved in the chain radical oxidation of P3HT is a key characteristic in the underlying mechanism. The results obtained in this work advance the understanding of active layer stability and will help improve the design of long lifetime organic solar cells thanks to the use of cutoff filter in the substrate or encapsulation of the devices.

PH-dependent electronic surface spectra of chromophore species in the charged silica–water interface

The structure of the pH-dependent silica–water interface has been probed with two charged counter-ion chromophores Crystal Violet (CV) and Malachite Green (MG) by evanescent wave cavity ring-down electronic spectroscopy. The electronic spectra of these species in the charged interface have been measured directly over the wavelength range 570–680 nm. The effective CV interfacial counter-ion concentration is 185-fold larger than the bulk with a fully charged silica surface at pH 9, corresponding to a surface concentration of 0.26 molecules nm−2. The chromophore–silica surface association and dissociation kinetics indicate a binding energy at pH 9 >28 kJ mol−1 for each chromophore: a loosely bound molecular layer. The electronic spectrum of CV in the interface shows two maxima: one feature is blue-shifted 2 nm from the solution-phase maximum at 583 nm, and the second feature is red-shifted 24 nm. A Lorentzian deconstruction of the spectra indicates that they are consistent with two molecular environments: one closely associated with the surface sharing delocalised electrons and a second confined to the interface but randomly oriented.

An overview of using solid wastes for pigment industry

Abstract This work reports the use of wastes (Al-rich anodizing sludge, Cr/Ni galvanizing sludge, foundry sand, tionite and marble sawing mud) to formulate distinct inorganic pigment structures that were synthesized by the solid state reaction method. One of the tested formulations is a novel pigment based on hibonite (CaO·6Al2O3), that was found a suitable hosting structure to accommodate cobalt or nickel chromophore ions in tetrahedral coordination, then giving strong blue or turquoise hues, respectively. The use of Cr as chromophore was tested in three distinct pigment structures: (i) red-wine Cr–CaSnSiO5 and brown Cr–CaTiSiO5; (ii) Cr–CaTiO3; (iii) and Cr–TiO2. Developed phases were determined, and the colouring mechanisms of the chromophore species were defined based on diffuse reflectance data. Then, the pigments were added to a standard transparent glaze, and the hues which developed were estimated from L*a*b* colorimetric measurements under the oxygen partial pressure in the atmosphere, to define stable workability window.

Hydrogen Abstraction Followed by Nitroxide‐Mediated Polymerization: Synthesis of 2,7‐Dibromofluorene‐Labeled Polystyrene

AbstractChromophore end‐labeled polystyrene is synthesized using nitroxide‐mediated polymerization (NMP) by decomposing 2‐2′‐azoisobutyronitrile (AIBN) or benzoyl peroxide (BPO) in the presence of fluorene or fluorene derivatives. End‐labeling is dependent on the thermally produced radical species selectively abstracting a hydrogen atom from the 9‐position of the fluorene species prior to initiation of styrene. From gel permeation chromatography (GPC) data and UV–Vis analysis, it is found that AIBN initiation, compared to BPO, leads to a more controlled polymerization system, producing polymers with predictable molecular weights, narrower polydispersity index (PDI) values (<1.3), and higher amounts of fluorene end‐labeling. In terms of the reaction parameters, no consistent trend is observed as a function of the timing of styrene's addition or the temperature at which the hydrogen abstraction phase is performed. Analysis of the chromophore content by UV–Vis spectroscopy demonstrated that the presence of bromine atoms on the 2‐ and 7‐position of the fluorene species leads to higher percent labeling of the chromophore species, presumably due to a more facile abstraction of the hydrogen at the 9‐position.

Systemic Low-Dose UVB Inhibits CD8 T Cells and Skin Inflammation by Alternative and Novel Mechanisms

Exposure to UVB radiation before antigen delivery at an unirradiated site inhibits functional immunological responses. Mice treated dorsally with suberythemal low-dose UVB and immunized with ova in abdominal skin generated ova-specific CD8 T cells with a significantly decreased activation, expansion, and cytotoxic activity compared with unirradiated mice. UVB also impaired the delayed-type hypersensitivity (DTH) reaction to ova. Transfer of CD4⁺CD25⁺cells from UVB-exposed mice did not suppress the ova-specific CD8 T-cell response or DTH reaction in unexposed mice, confirming that systemic low-dose UVB does not induce long-lived functional regulatory CD4⁺CD25⁺ T cells. Repairing cyclobutane pyrimidine dimer-type DNA damage and blocking aryl hydrocarbon receptor signaling also did not reverse the immunosuppressive effect of UVB on ova-specific CD8 T cells and DTH, suggesting that cyclobutane pyrimidine dimers and the aryl hydrocarbon receptor are not required in systemic low-dose UVB-induced immunosuppression. The known UVB chromophore, cis-urocanic acid, and reactive oxygen species triggered the inhibition of DTH caused by UVB, but they were not involved in the modulation of CD8 T cells. These findings indicate that systemic low-dose UVB impedes the primary response of antigen-specific CD8 T cells by a novel mechanism that is independent of pathways known to be involved in systemic suppression of DTH.

Effects of Electrolyte Concentration on the Rotational Dynamics of Resorufin

We report on the rotational diffusion dynamics of the anionic chromophore resorufin in water and N-octyl-2-pyrrolidone (NOP) solutions as a function of solution electrolyte concentration. Our data show that resorufin exhibits a single exponential anisotropy decay in aqueous solutions containing up to 0.1 M lithium perchlorate (LiClO(4)). In contrast to the observed behavior of resorufin in pure NOP, where biexponential decay occurs, we also observe a single exponential anisotropy decay for resorufin in NOP with the addition of up to 0.1 M tetrabutylammonium bromide (TBAB) or tetraoctylammonium bromide (TOAB). For resorufin in NOP, the reorientation time constant increases with increasing electrolyte concentration, consistent with complexation between the resorufin anion and the electrolyte ammonium cation. We observe a qualitatively different trend in the aqueous resorufin solutions and understand these data for both solvent systems in the context of interactions between the chromophore and cationic species present.

Microfluidic “thin chips” for chemical separations

This paper describes the design, development and application of microfluidic "thin chips" fabricated from PDMS. Thin chips consist of multiple layers of PDMS chemically bonded onto each other. Unlike thicker PDMS chips that suffer from lack of sensitivity due to PDMS absorption in the VIS and UV range, the thinness of these chips allows for the detection of chromophoric species within the microchannel via an external fiber optics detection system. C18-modified reversed-phase silica particles are packed into the microchannel using a temporary taper created by a magnetic valve and separations using both pressure- and electrochromatographic-driven methods are detailed.

Color inhibition of phenolic antioxidants in Ziegler‐Natta polyethylene. II. In‐situ solution studies

AbstractPart I of this series of papers showed that color suppression (in some cases superior to that with established phosphites) of polyethylenes (PEs) containing a highly discoloring hindered phenolic antioxidant, Lowinox 22IB46 [2,2′‐isobutylidenebis(4,6‐dimethylphenol)] (L22IB46), and titanium catalyst residues could be achieved by using a range of relatively low cost multifunctional compounds, among which dipentaerythritol (DPE) and triisopropanolamine (TIPA) performed notably well. It was speculated that the latter compounds in some way blocked the formation of highly chromophoric species, which were thought to be titanium phenolates. The studies conducted in Part II further investigate this effect via a solution‐based model‐compound approach, with decahydronaphthalene and titanium and aluminum isopropoxides as models for the PE and Ti‐ and Al‐based catalyst residues, respectively. This approach enabled the use of second‐derivative UV spectroscopy of refluxing solutions for the detection of chromophoric species. The level of antioxidant consumption was determined by using UV spectroscopy, and L22IB46 – (color suppressant) reaction products were characterized by using infrared spectroscopy. Titanium(IV) was found to play a major role in discoloration (via production of titanium phenolates) and the consumption of L22IB46 relative to that of aluminum, which displayed little color formation activity. This observation shows that L22IB46 oxidation products are not necessarily the major cause of discoloration. The DPE and TIPA also performed well in this solution‐based study, though interesting chain‐length‐related (and possibly solubility‐related) effects were apparent with poly(ethylene glycol). J. VINYL ADDIT. TECHNOL., 2009. © 2009 Society of Plastics Engineers

Star-Shaped Tetrathiafulvalene-Fused Coronene with Large π-Extended Conjugation

A tristar shaped, planar TTF-fused coronene 1 was synthesized. Its electronic properties have been studied experimentally by the combination of electrochemistry and UV-vis-NIR spectroscopy. Thereby, a nanosized graphite fragment is largely extended in its size, supplemented with a multielectron donor functionality, and shaped to a strongly chromophoric species absorbing intensely in the visible part of the optical spectrum.

Efficient Photoconversion Distorts the Fluorescence Lifetime of GFP in Confocal Microscopy: A Model Kinetic Study on Mutant Thr203Val

Phototransformations of autofluorescent proteins are applied in high-resolution microscopy and in studying cellular transport, but they are detrimental when accidentally occurring in blinking or photobleaching (BL). Here, we investigate the kinetics of phototransformations of a photoactivatable green fluorescent protein (GFP) in confocal microscopy. Photoconversion (PC) is achieved by excitation of the barely present anionic chromophore state R(eq) (-) in the GFP mutant Thr203Val. Besides the shift of the equilibrium between the neutral chromophore state RH and R(eq) (-), the photoconverted anionic chromophore R(PC) (-) exhibits a reduced fluorescence lifetime tau(fl)=2.2 ns. In fluorescence lifetime imaging microscopy, tau(fl) is found to depend, however, on the excitation conditions and history. The underlying photochemistry is described by the kinetic scheme of consecutive reactions, R(eq) (-)-->R(PC) (-)-->P(dark), in which the anionic chromophore species and the dark protein P(dark) are coupled by PC and BL. Time-correlated single-photon-counting detection in a confocal geometry of freely diffusing species is used to compute the quantum yields for PC and BL, Phi(PC) and Phi(BL). The assessed values are Phi(PC)=5.5 x 10(-4) and Phi(BL)>1 x 10(-5). Based on these values, PC provokes misinterpretation in fluorescence resonance energy transfer experiments and is responsible for spectroscopic peculiarities in single-molecule detection.

Modeling Photoabsorption of the asFP595 Chromophore

The fluorescent protein asFP595 is a promising photoswitchable biomarker for studying processes in living cells. We present the results of a high level theoretical study of photoabsorption properties of the model asFP595 chromophore molecule in biologically relevant protonation states: anionic, zwitterionic, and neutral. Ground state equilibrium geometry parameters are optimized in the PBE0/(aug)-cc-pVDZ density functional theory approximation. An augmented version of multiconfigurational quasidegenerate perturbation theory (aug-MCQDPT2) following the state-averaged CASSCF/(aug)-cc-pVDZ calculations is used to estimate the vertical S0-S1 excitation energies for all chromophore species. An accuracy of this approach is validated by comparing the computed estimates of the S0-S1 absorption maximum of the closely related chromophore from the DsRed protein to the known experimental value in the gas phase. An influence of the CASSCF active space on the aug-MCQDPT2 excitation energies is analyzed. The zwitterionic form of the asFP595 chromophore is found to be the most sensitive to a particular choice and amount of active orbitals. This observation is explained by the charge-transfer type of the S0-S1 transition involving the entire conjugated pi-electron system for the zwitterionic protonation state. According to the calculation results, the anionic form in the trans conformation is found to possess the most red-shifted absorption band with the maximum located at 543 nm. The bands of the zwitterionic and neutral forms are considerably blue-shifted compared to those of the anionic form. These conclusions are at variance with the results obtained in the TDDFT approximation for the asFP595 chromophore. The absorption wavelengths computed in the aug-MCQDPT2/CASSCF theory are as follows: 543 (535), 470 (476), and 415 (417) nm for the anionic, zwitterionic, and neutral forms of the trans and cis (in parentheses) isomers of the asFP595 chromophore. These data can be used as a reference for further theoretical studies of the asFP595 chromophore in different media and for modeling photoabsorption properties of the asFP595 fluorescent protein.

Trapping of a Chromophoric Intermediate in the Pdx1‐Catalyzed Biosynthesis of Pyridoxal 5′‐Phosphate

I320 has it: The serendipitous trapping, by aromatization, of an enzyme-bound chromophoric intermediate (I320; see picture, blue box) involved in vitamin B6 formation using a common biological reducing agent provides new information regarding the structure of the chromophoric species.

Magnetically controlled valve for flow manipulation in polymer microfluidic devices

A simple, external in-line valve for use in microfluidic devices constructed of elastomer such as polydimethylsiloxane (PDMS) is described. The actuation of the valve is based on the principle that flexible polymer walls of a liquid channel can be pressed together by the aid of a permanent magnet and a small metal bar. In the presence of a small NdFeB magnet lying below the channel of interest, the metal bar is pulled downward simultaneously pushing the thin layer of PDMS down thereby closing the channel stopping any flow of fluid. The operation of the valve is dependent on the thickness of the PDMS layer, the height of the channel, the gap between the chip and the magnet and the strength of the magnet. The microfluidic channels are completely closed to fluid flows commonly used in microfluidic applications. The valve allows for fabrication of a “thin chip” that allows for detection of chromophoric species within the microchannel via an external fiber optics detection system. C18-Modified reverse phase silica particles are packed into the microchannel using a temporary taper created by the magnetic valve and separations using both pressure and electrochromatographic driven methods is detailed.

Concerning the color change of pyrromethene 650 dye in electron-donor solvents

This paper studies the change in the color from violet to green of pyrromethene 650 (PM650) dye observed in certain N, N′-dialkyl-amides characterized by their high electron-donor ability and low proton-donor capacity. For this purpose, the photophysical properties of PM650 are registered in diluted solutions of several amides, including dimethylformamide/dioxane mixtures. The color change of PM650 is discussed on the basis of the formation of new hypsochromic absorption and fluorescence bands, which are assigned to the formation of new chromophoric species as consequence of a specific reaction between PM650 and the amide solvents. The influence of the ageing time of the sample and the temperature on the color change is also discussed. In some cases the ageing leads to the bleaching of the dye.

Determination of Photo‐oxidation Products Within Photoyellowed Bleached Wool Proteins

Photo-oxidative processes occurring in wool can lead to significant photoyellowing of the fiber. In particular, wool that has been chemically bleached photoyellows more rapidly and to a greater degree than untreated wool. Direct identification of the chromophores responsible for such yellow discoloration in irradiated wool has proven to be elusive for many years. This article describes the characterization and location of yellow photo-oxidation products within the proteins of photoyellowed bleached wool fabric, using advanced protein chemistry techniques. The discolored fabric was enzymatically digested and chromatographed by high-pressure liquid chromatography, with monitoring at 400 nm, to select out fractions containing yellow chromophoric species. Thorough tandem mass spectrometric analysis was then used to sequence peptides and, in turn, to characterize modifications to key amino acid residues that had resulted in yellow chromophore formation. In total, 11 separate yellow chromophoric species were identified, ten derived from tryptophan residues and one from tyrosine. The tryptophan-derived modifications characterized included hydroxytryptophan, N-formylkynurenine, hydroxyformylkynurenine, kynurenine, hydroxykynurenine, carbolines, tryptophandiones and nitrotryptophan. The tyrosine-derived modification of tyrosine to dopa was also identified. The range of photomodifications we observed provides insight into the photo-oxidation pathways occurring within irradiated fibrous proteins leading to the formation of yellow chromophores.

Determination of chromophore charge states in the low pH color transition of the fluorescent protein Rtms5 H146S via time-dependent DFT

The red fluorescent protein Rtms5 H146S displays a transition from blue (absorbance λ max 590 nm) to yellow (absorbance λ max ∼453 nm) upon titration to low pH. The p K a of the reaction depends on the concentration of halide, offering promise for new expressible halide sensors. The protonation state involved in the low pH form of the chromophore remains, however, ambiguous. We report calculated excitation energies of different protonation states of an RFP chromophore model. These suggest that the relevant titration site is the phenoxy moiety of the chromophore, and the relevant base and conjugate acid are anionic and neutral chromophore species, respectively.

Thermal degradation of poly(vinyl chloride) synthesized with a titanocene catalyst

PVC was synthesized using a trichloroindenyltitanium–methylaluminoxane catalyst at room temperature, and its degradation was monitored along with a commercial sample at 160, 170 and 180 °C under air or nitrogen atmosphere. The process was followed by HCl evolution, yellowing index, colour formation and thermogravimetric analysis. The produced polymer had a lower molecular weight and higher surface area, compared with a commercial PVC, while 1H NMR and T g values show minimal differences between materials. The HCl evolution degradation studies indicate that produced PVC has a lower thermal resistance than commercial PVC, while TGA reveals the opposite behaviour. Yellowing index and colour evaluation give evidence that nitrogen atmosphere and high surface area in produced PVC allow the polyene growth, whereas low surface area and air atmosphere generate shorter polyenes and chromophoric species. Differences in degradation performance are thought to be due to chemical origin, inherent morphology and differences in instrumentation.

Cyanobacterial Phytochrome-like PixJ1 Holoprotein Shows Novel Reversible Photoconversion Between Blue- and Green-absorbing Forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.