Characterization of the solid phases of paracetamol and fenamates at equilibrium in saturated solutions

Differential scanning calorimetry (DSC), supported by hot stage microscopy, IR spectroscopy and X-ray powder diffractometry, was used to investigate the characteristics of the solid phases of mefenamic, niflumic, and flufenamic acids and of paracetamol, before and after equilibration with saturated solutions in different solvents. Mixtures of Lewis base (dioxane and ethyl acetate) and amphiprotic solvents (ethanol and water) were prepared for evaluating the influence of both nature and polarity of the solvents. Solid-state analysis performed on the original samples (commercial products) made it possible to establish that paracetamol, mefenamic acid and flufenamic acid were in their respective Form I. No polymorphic modifications are known for niflumic acid. Paracetamol, niflumic and mefenamic acids did not show any change after equilibration with the various solvents or solvent mixtures, regardless of their different chemical nature. In contrast, DSC, IR and X-ray analyses revealed the partial recrystallization of flufenamic acid into its polymorphic Form III in solid phases at equilibrium with ethanol, ethyl acetate and their blends, as well as in dioxane-water mixtures containing 30 to 100% dioxane and in ethanol-water mixtures with a water content less than 50%. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fluorescent behavior of B-phycoerythrin in microemulsions of aerosol OT/water/isooctane

Taking advantage of its unusual fluorescent properties, the incorporation of B-phycoerythrin (B-PE) in aerosol OT (AOT, sodium bis-(2-ethylhexyl) sulphosuccinate)/water/isooctane microemulsions was investigated by following their steady-state and time-resolved fluorescence as a function of the water-to-surfactant molar ratio, w(0). The fluorescent intensity at 575 nm increased continuously with increasing water content, saturating at a w(0) around 35 and staying practically constant at w(0)> or =40. The steady-state anisotropy showed an initial increase with increasing water content until w(0)=23 and then decreased strongly, staying practically constant when w(0)> or =40. The values of the fluorescent parameters, anisotropy and fluorescent intensity, were unchanged when the water content of the system increased in the range between w(0)=40 to 50. This implies the effective incorporation of B-PE in the microemulsion droplets with w(0)> or =40, as well as the equilibrium of the dispersion at these water/surfactant ratios, since higher water content does not affect the main surrounding microenvironment of the protein. The overall incorporation in the microemulsion droplets caused minor spectroscopic changes with respect to biliprotein in aqueous solution of 20 mM sodium phosphate buffer, pH 7.0, such as a blue absorption shift of 3 nm and an emission shift of 1.5 nm, as well as a slight increase in excitation anisotropy spectrum mainly caused by a decrease in protein mobility. Therefore, there are no important interactions between the chromophores and the AOT sulfonate head groups. Emission intensity decays followed complex kinetics in both aqueous and dispersion media. The stability with time and temperature of the biliprotein in the microemulsion was higher than in the aqueous solution. All the results can be explained in terms of B-PE inclusion in the water droplets of AOT microemulsions where the protein has similar configuration and conformation to that in aqueous solution but with the chromophores more protected.     

Evaluation of the complexity of charge recombination kinetics in photosynthetic bacteria

Abstract Improvements in instrumentation and methodology have allowed us to collect data of high signal to noise and reliability on the kinetics of recovery of both light-induced absorbance changes and ESR signals at 95 K. The results obtained by the two methods are identical and can not be fit with a single exponential curve. The decay kinetics can be fit well with three exponential components which represent 85, 9 and 6% of the total change with rate constants of 29 s⁻¹, 69 s⁻¹ and 2.3 s⁻¹, respectively. An interesting effect by molecular oxygen on the relaxation time of the donor cation radical was found by ESR measurements at low temperatures and higher microwave power. This interaction with oxygen could be blocked by addition of small amounts (e.g. 0.05%) of organic solvents such as ethanol. A variety of systems were examined including R. rubrum whole cells and chromatophores prepared from R. rubrum and Rps. sphaeroides. R. rubrum chromatophore samples were examined at high and low light intensities, at pH values from 6 to 10, in the presence and absence of air and after equilibration in D₂O media. In all cases, the same decay kinetics were observed. It seems possible that the observed complex decay may be a characteristic of phototraps of all photosynthetic material and reflect fundamental structural and functional features yet to be uncovered.     

Fluorescence-labelled DNA probes to detect complementary sequences in homogeneous media

Sensitive, safe and easy-to-use probes for the detection of nucleic acids are urgently called for. To this end we are in the process of developing a fluorescence-based technique to work in homogeneous assay media. We have examined pyrene and fluorescein as fluorescent labels for natural DNA probes. A fraction of the cytosine residues of a single-stranded cDNA was randomly labelled with either pyrene or fluorescein using the bisulfite-catalyzed diamine reaction. Both fluorophores showed fluorescence quenching when the labelled probe was hybridized with its complementary strand and we describe the changes in steady-state fluorescence intensity that occurred upon hybridization. Our results demonstrate that pyrene quenching is more efficient than fluorescein quenching and thus pyrene-labelled probes are more sensitive for detecting and quantifying DNA from natural sources.     

Three-state 2',7'-difluorofluorescein excited-state proton transfer reactions in moderately acidic and very acidic media

2',7'-Difluorofluorescein (Oregon Green 488, OG488) is a novel fluorescein dye derivative which presents important advantages for improving the fluorimetric applications in the biomedical and biochemical sciences. In aqueous solution it displays four prototropic forms, namely cation (C), neutral (N), monoanion (M), and dianion (D). In previous works, we found (J. Phys. Chem. A 2005, 109, 734-747, 2840-2846) that OG488 undergoes excited-state proton transfer reactions, which may affect the results from applications using this dye. We established that the excited-state proton transfer (ESPT) reactions between neutral, monoanionic, and dianionic forms of OG488 are promoted by acetate buffer, and we characterized the ground and excited species involved. We also solved the kinetics of the prototropic reactions using global compartmental analysis. In the present paper, we extend our study on the ESPT reactions of OG488 to acidic media, in which only the three prototropic species cation, neutral, and monoanion coexist. We have solved the kinetics of the three-state ESPT reaction by means of global three-compartmental analysis of a fluorescence decay surface in moderately acidic media (pH between 1.1 and 3.0), recovering the kinetic and spectral parameters of this three-state system. This system is one of the most complex solved to date, due to the strong overlap of the absorption and emission spectra of the neutral and monoanionic forms of OG488. We also found that the cation behaves as "super" photoacid, showing a very high deprotonation rate constant (1.04 x 10(11) s(-1)) and an enhanced acidity. Therefore, we also carried out experiments at very high perchloric acid concentrations, dealing with some other effects which become noteworthy at these [H(+)]. The presence of xanthylium cation quenching due to "free" water molecules, and the reduction in the amount of water clusters acting as proton acceptors, are processes which alter notably the time course of the excited-species in this high [H(+)] range.     

RECENT ADVANCES IN POLARIZATION SPECTROSCOPY: PERSPECTIVES OF THE EXTENSION TO THE SOFT X-RAY REGION

In this paper we present a general review of some of the new branches in the field of optical activity that have been developed during the last five years. Also, the conditions under which circular intensity differential scattering can be measurable in the soft X-ray region of the spectrum are established. It is found that the parameter which determines the strength of the preferential interaction of chiral molecules with opposite circular polarizations at these high energies is the anisotropy of the atomic polarizabilities in the molecule. The possibility of extending the other techniques discussed here to shorter wavelengths, is also discussed.     

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.     

2',7'-Difluorofluorescein excited-state proton reactions: correlation between time-resolved emission and steady-state fluorescence intensity

The presence of excited-state buffer-mediated proton exchange reactions influences the steady-state fluorescence signals from dyes in solution. Since biomolecules in general have some chemical groups that can act as proton acceptors/donors and are usually dissolved in buffer solutions which can also behave as appropriate proton acceptors/donors, the excited-state proton exchange reactions may result in distorted steady-state fluorescence signals. In a previous paper (J. Phys. Chem. A 2005, 109, 734-747), we evaluated kinetic and other pertinent parameters for the excited-state proton reactions of the prototropic forms of 2',7'-difluorofluorescein (Oregon Green 488, OG488), recording a fluorescence decay surface at different pH values and acetate buffer concentrations, analyzed by means of global compartmental analysis. In this article we use the rate constants and the corrected pre-exponential factors from the previously recorded fluorescence decay traces to simulate the decay times and associated pre-exponentials at different acetate buffer concentrations and constant pH and compare these theoretically calculated values with new experimental data. We also calculate the steady-state fluorescence intensity vs pH and vs acetate buffer concentration (at constant pH) and compare these calculated emission values with the experimental data previously published. The agreement between the experimental and simulated data is excellent.     

Influence of the automotive Start/Stop system on noise emission: Experimental study

One of the most common environmental impacts of road transportation is the traffic noise. Linked to this, Start/Stop is a technology which has demonstrated to save fuel by powering off the engine when the vehicle is stopped, such as in front of a traffic light, and restarting the engine instantly when the driver pushes back the pedal brake to proceed. The technology helps also to reduce the CO₂ emission, playing a key role in a way to accomplish stringent emission norms for vehicle manufacturer. However, we are not sure whether it reduces the noise emission and how much? Thus, the main aim of this work is to assess the engine noise emissions of a vehicle incorporating a Start/Stop system in urban traffic, and compare it with those radiated by the mean traffic stream. Experimental results demonstrate that there are no contributions of the Start/Stop system to reduce meaningfully the engine noise in urban traffic.     

Multivariate analysis in provenance studies: Cerrillos obsidians case, Peru

We present the preliminary results of a provenance study of obsidians samples from Cerrillos (ca. 800–100 b.c.) using Mössbauer Spectroscopy. The Cerrillos archaeological site, located in the Upper Ica Valley, Peru, is the only Paracas ceremonial center excavated so far. The archaeological data collected suggest the existence of a complex social and economic organization on the south coast of Peru. Provenance research of obsidian provides valuable information about the selection of lithic resources by our ancestors and eventually about the existence of communication routes and exchange networks. We characterized 18 obsidian artifacts samples by Mössbauer spectroscopy from Cerrillos. The spectra, recorded at room temperature using different velocities, are mainly composed of broad asymmetric doublets due to the superposition of at least two quadrupole doublets corresponding to Fe²⁺ in two different sites (species A and B), one weak Fe³⁺ doublet (specie C) and magnetic components associated to the presence of small particles of magnetite. Multivariate statistical analysis of the Mössbauer data (hyperfine parameters) allows to defined two main groups of obsidians, reflecting different geographical origins.