Francisco García-Blanco

Characterization of binary solvent mixtures: the water–acetonitrile mixture

Binary mixtures of water and acetonitrile (W/AN) were characterized in the light of the pure solvent scales, using suitable probe/homomorph couples. Various thermodynamic (vapour pressure, softness parameter, excess molar Gibbs energy and enthalpy of mixing) properties, surface tension, viscosity, and spectroscopic (IR, Raman and NMR) properties of the mixtures, and the distribution of molecular structures (viz. free CH3CN and CH3CN-H2O complexes) are described in terms of their polarity, acidity and basicity, and the descriptions examined in relation to a potential physical significance.

Photophysical Study of the Probes Acrylodan (1‐[6‐(Dimethylamino)naphthalen‐2‐yl]prop‐2‐en‐1‐one), ANS (8‐Anilinonaphthalene‐1‐sulfonate) and Prodan (1‐[6‐(Dimethylamino)naphthalen‐2‐yl]propan‐1‐one) in Aqueous Mixtures of Various Alcohols

In this work, we examined in depth the photophysical behavior of the probes acrylodan (1-[6-(dimethylamino)naphthalen-2-yl]prop-2-en-1-one), ANS (8-anilinonaphthalene-1-sulfonate), and prodan (1-[6-(dimethylamino)naphthalen-2-yl]propan-1-one) in several pure solvents and aqueous mixtures of MeOH, i-PrOH, EtOH, and 2,2,2-trifluoroethanol between 0 – 90% (v/v). Although these probes have been widely used to study biological systems, the properties that govern their max emission remain somewhat obscure. These alcohols are soluble in all proportions in H2O, and are frequently used in peptide and protein studies as denaturing agents or because of their ability to substantially increase secondary structure or alter the folding-unfolding kinetics of proteins. The aim here was to rationalize the interpretation of max emission of these probes in terms of specific and general effects of the solvent. To this end, we used the mixture parameters SA (solvent acidity), SB (solvent basicity), and SPP (solvent polarity/polarizability). The results suggest that it is incorrect to categorize these compounds purely as probes of polarity, when using these solvents.

Aggregation of antitumoral drug emodin on Ag nanoparticles: SEF, SERS and fluorescence lifetime experiments.

We have studied the fluorescence and Raman emission of the anthraquinone drug emodin immobilized molecules on nanostructured silver surfaces, prepared through two different methods. Two different pHs (pH = 10 and pH = 6) have been used. The dye aggregation favors SEF at pH = 6, whereas quenching of fluorescence is observed at pH = 10, due to the short distance between emodin and Ag particles. Along with these results, SERS spectra have given us information about the different species present in the solution. We have used two different reducing agents to obtain the Ag nanoparticles: hydroxylamine hydrochloride and sodium citrate. In all the cases analysed, the enhancement of both SEF and SERS spectra, is larger for hydroxylamine hydrochloride than that for sodium citrate. We have also measured fluorescence lifetime, observing a shorter lifetime for emodin molecules near Ag nanoparticles than that for emodin solved in pure water, thus corroborating the results obtained in fluorescence emission spectra.

The Schiff base between pyridoxal-5′-phosphate and hexylamine. Equilibria in solution

Electrochemical and spectroscopic studies of the pyridoxal-5′-phosphate-hexylamine Schiff base (PHSB) over a wide pH range have been carried out. This compound has been used as a simple model of the binding of pyridoxal-5′-phosphate (PLP) to protein. In this work different equilibria involved in the formation reaction have been considered and a quantitative distribution of the species in solution has been obtained. The method is based on the analysis of the reduction wave and absorption bands of the PHSB. Protonation equilibria constants (PHSB pK) have been obtained. Absorption spectra as a function of the medium composition have been resolved by a log normal distribution. Tautomeric equilibria have been considered and microscopic pK have been evaluated. Fluorescence results show that Schiff-base species bearing a protonated ring nitrogen are the most fluorescent. However, the fluorescence decreases in an acid medium due to an hydrolysis reaction. An important conclusion of this investigation is that the combined use of electrochemical and spectroscopic techniques is a valuable tool for the quantitative characterization of the pyridoxal-5′-phosphate Schiff bases.

Interactions of 2,2,2-trifluoroethanol with aqueous micelles of Triton X-100

The effect of 2,2,2-trifluoroethanol (TFE) on micellar properties of Triton X-100 (TX-100) in aqueous solutions was investigated by cloud point (CP), viscosity, surface tension, and fluorescence techniques. The critical micelle concentration (CMC) values of the corresponding mixtures were obtained by the pyrene 1:3 ratio method and by surface tension data using the pendant drop technique. All the techniques provided about the same values for the CMC. Up to 0.83 M TFE increased the CMC by 30%. The small increase in the CMC is consistent with a slight increase in the solubility of the TX-100. Fluorescence measurements indicate that the TFE decreased the aggregation number by about 30%. The CP decrease and the intrinsic viscosity increase with TFE concentration are consistent with a preferential interaction of TFE with TX-100 micelles. TFE molecules form hydrophobic domains in the micellar layer palisade because they hydrogen bond with the oxyethylene group in TX-100. The intrinsic viscosity data are consistent with an increase in micelle hydrodynamic radius owing to the presence of TFE.

ELECTROCHEMICAL BEHAVIOUR OF THE SCHIFF BASE FROM PYRIDINE-4-ALDEHYDE AND N-HEXYLAMINE

Abstract A study of the Schiff base from pyridine-4-aldehyde and n-hexylamine (SBPYH) was carried out. Equilibrium in solution, between the aldehyde, amine and Schiff base, was reached in the time scale of the experiments. In dc and DP polarography one or two waves were obtained, depending on the experimental conditions. The effects of the pH, the amine concentration and the capillary characteristics were studied. The apparent constant of the SBPYH formation was obtained as a function of the pH. The current-time curves show the influence of the hydration-dehydration reactions of the aldehyde in the overall process. Polarographic parameters, Tafel slope, reaction orders and voltammetric results account for the electroreduction of the azomethine group in neutral and basic media.

Nucleotide-induced conformational transitions in the CBS domain protein MJ0729 of Methanocaldococcus jannaschii

Nucleotide-binding cystathionine β-synthase (CBS) domains function as regulatory motifs in several proteins distributed through all kingdoms of life. This function has been proposed based on their affinity for adenosyl-derivatives, although the exact binding mechanisms remain largely unknown. The question of how CBS domains exactly work is relevant because in humans, several genetic diseases have been associated with mutations in those motifs. In this work, we describe the adenosyl-ligand (AMP, ATP, NADP and SAM) properties of the wild-type CBS domain protein MJ0729 from Methanocaldococcus jannaschii by using a combination of spectroscopic techniques (fluorescence, FTIR and FRET). The fluorescence results show that binding to AMP and ATP occurs with an apparent dissociation constant of ~10 µM, and interestingly enough, binding induces protein conformational changes, as shown by FTIR. On the other hand, fluorescence spectra (FRET and steady-state) did not change upon addition of NADP and SAM to MJ0729, suggesting that tryptophan and/or tyrosine residues were not involved in the recognition of those ligands; however, there were changes in the secondary structure of the protein upon addition of NADP and SAM, as shown by FTIR (thus, indicating binding to the nucleotide). Taken together, these results suggest that: (i) the adenosyl ligands bind to MJ0729 in different ways, and (ii) there are changes in the protein secondary structure upon binding of the nucleotides.

A New Approach to the Study Of Pyridoxal-5′-Phosphate Schiff Bases

Fluorescence, UV spectroscopic and DC and DP Polarographie studies of the Schiff base of pyridoxal-5′-phosphate with n-hexylamine were carried out. The most fluorescent species of this model compound are those bearing a protonated ring nitrogen. The true distribution of the molecular species of the Schiff base as a function of the pH was obtained.

A study of the Schiff base formed between pyridoxal-5′-phosphate and poly-L-lysine of low polymerization degree

The interaction between pyridoxal-5′-phosphate (PLP) and poly-L-lysine of low polymerization degree has been studied in aqueous solutions. Formation of a Schiff base by a reversible reaction is observed according to absorption spectra and electrochemical results. The apparent equilibrium constant has been calculated as a function of the pH under conditions which favour addition of one molecule of the coenzyme to the polypeptide. A comparison of the poly-L-lysine : PLP Schiff base and Schiff bases from PLP and amino acids or amines is considered. From the study of the influence of the lysine : PLP ratio a characterization of the binding of PLP to the matrix is obtained in the pH interval 5–10. The response of the Schiff base to changes in pH or poly-L-lysine concentration point to an important role of the supporting polypeptide in the environment of the coenzyme favouring, under determined conditions, stabilization of the enolimine form or a carbinolamine intermediate.