Sílvia M. B. Costa

Spectroscopic Studies on the Interaction of a Water Soluble Porphyrin and Two Drug Carrier Proteins

The interaction of meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) sodium salt to human serum albumin and beta-lactoglobulin was studied by steady-state and dynamic fluorescence at different pH of aqueous solutions. The formation of TSPP J-aggregates and a noncovalent TSPP-protein complex was monitored by fluorescence titrations, which depend on pH and on the protein nature and concentration. The complex between TSPP and protein displays a heterogeneous equilibrium with large changes in the binding strength versus pH. The large reduction of the effective binding constant from pH 2 to 7 suggests that electrostatic interactions are a major contribution to the binding of TSPP to the aforementioned proteins. TSPP aggregates and TSPP-protein complex exhibit circular dichroism induced by the presence of the protein. Circular dichroism spectra in the ultraviolet region show that the secondary structure of both proteins is not extensively affected by the TSPP presence. Protein-TSPP interaction was also examined by following the intrinsic fluorescence of the tryptophan residues of the proteins. Fluorescence quenching by acrylamide and TSPP itself also point to small changes on the protein tertiary structure and a critical distance R(0) approximately 56 A, between tryptophan and bound porphyrin, was estimated using the long distance Förster-type energy transfer formalism.

Complexation of polymethine dyes with human serum albumin: a spectroscopic study

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.

Thermal unfolding of proteins at high pH range studied by UV absorbance.

This work describes a methodology to monitor protein unfolding by using the well known changes in tyrosine absorbance with the ionization of the side chain phenol group. It can be applied to proteins that are functionally active at pH values higher than 9.0 where the current UV differential spectroscopy technique can not be used. The simplicity and facility of the proposed methodology (only two absorbance measurements have to be acquired) can make it very useful namely for technological applications. Thermal unfolding of cutinase and alpha-chymotrypsin were followed using this methodology and the thermodynamic stability data were obtained assuming a two-state mechanism. The transition from the folded to the unfolded state was further confirmed by fluorescence maxima for both proteins proving the validity of the methodology based on UV measurements.

Photochemistry on surfaces: solvent–matrix effect on the swelling of cellulose. An emission and absorption study of adsorbed auramine O

Auramine O exhibits a significant fluorescence emission at room temperature when adsorbed on microcrystalline cellulose. This emission is about two to four orders of magnitude higher than the previously reported emission in different solvents, also at room temperature. The fluorescence quantum yield, ϕF, varies with the residual degree of humidity of the sample, as alterations in the rigidity of the environment greatly affect the non-radiative de-excitation of auramine O. A limiting upper value of ϕF≈ 0.35 was obtained after prolonged evacuation of the samples.A matrix-isolation mechanism was evaluated by using room-temperature fluorescence measurements of powdered samples of auramine O adsorbed on microcrystalline cellulose. Using polar protic and aprotic solvents, different degrees of swelling of the microcrystalline cellulose were obtained, resulting in changes of the ground-state reflectance spectra of auramine O trapped in the natural polymer chains, as shown by remission function data. The amount of dye adsorbed or entrapped is reflected in both the fluorescence quantum yields and lifetimes.When auramine O was adsorbed on porous silica, the fluorescence quantum yield was much lower, indicating a higher mobility of dye molecules on the silica surface. Oxygen does not affect the room-temperature fluorescence emission of auramine O in silica or in cellulose.

Intramolecular excited state interactions in amino-esters

Abstract N,N-Dimethylaminoalkyl and N-methyl-N-phenylaminoalkyl esters of 1- and 2-naphthoic acid, 9-anthroic acid and 3-pyrenoic acid exhibit excited intramolecular charge transfer interactions which lead to quenching of the fluorescence of the ester. In these bichromophoric systems excitation of either the aromatic amine or the hydrocarbon group leads to fluorescent exciplex formation, the ratio of exciplex-to-monomer intensities being greater when the amine is excited. The exciplex emission was also detected at 77 K, both in polar and non-polar solvents, suggesting that the interaction in these systems is of a dynamic type but also has a static contribution. In these systems transfer of amine excitation to the ester competes with direct exciplex formation from the aromatic amine.

Effects of normal and reverse micellar environment on the spectral properties, isomerization and aggregation of a hydrophilic cyanine dye

Abstract The spectral properties, cis → trans isomerization and aggregation of the anionic hydrophilic cyanine 1 , were studied in isooctane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water, cyclohexane–hexanol/Triton X-100/water reverse micelles and in aqueous solutions in the presence of surfactants. The incorporation of 1 into micelles leads to dimer→monomer decomposition and cis → trans isomerization. In AOT reverse micelles with w 0 ⩾5 ( w 0 =[H 2 O]/[AOT]) 1 forms J aggregates at concentrations much lower than one dye molecule per one micelle; at w 0 =50 the most probable aggregation number is 3. Premicellar J aggregates of 1 are observed with both cationic and anionic surfactants, but not with Triton X-100.

Fluorescence quantum yield evaluation of strongly absorbing dye solutions as a function of the excitation wavelength

Abstract A simple method to evaluate fluorescence quantum yields based on corrected fluorescence emission spectra of dyes in dilute and strongly absorbing solutions using different excitation wavelengths is presented. The method is supported by a detailed knowledge of the apparatus geometry and energy profile of excitation. Several recommended quantum counters were used (9,10-diphenylanthracene, rhodamines 101 and B, cresyl, violet, oxazine 1 and 1-ethyl-4-(4-p-dimethylaminophenyl)-1,3 butadienyl)-quinolinium perchlorate, (LDS 798) to cover the emission range from the UV to the visible and near-IR A curve of correction factor f vs. the optical density of the samples was obtained enabling an accurate determination to be made of the fluorescence quantum yield as a function of concentration and excitation wavelength. A case study of two squaraines (bis[4-(dimethylamino)phenyl]squaraine (HSQ) and bis[4-(dimethylamino)-2-methylphenyl]squaraine (MeSQ)) is presented. The quantum yields of these squaraines were obtained in saturated solutions in dichloromethane and in dilute samples at room temperature. The quantum yields determined are the same for high and low concentrations of the compounds indicating that no aggregation effects occur.

Denaturation of a Recombinant Cutinase from Fusarium solani in AOT-iso-Octane Reverse Micelles: a Steady-State Fluorescence Study

Abstract— Near UV absorbance and fluorescence spectroscopy show conformational changes of a recombinant cutinase from Fusarium solani incorporated in sodium‐di‐2‐ethylhexyl sulfosuccinate (AOT)‐iso‐octane reversed micelles with W0= [H2O]/[AOT] = 20. Excitation spectra were used to decompose cutinase absorbance in its Trp and Tyr components, showing that the latter absorb red‐shifted in the native cutinase in aqueous solution as compared to free Tyr, whereas in reverse micelles and denatured cutinase no shift is detected. Emission maxima variations (λmax 303, 311 and 335 nm, respectively in aqueous, reverse micelles and thermally denatured cutinase) reflect progressive changes in the micropolarity of the environment and exposure of Trp residues at the protein surface. The encapsulation of cutinase in AOT‐iso‐octane reversed micelles induces a time‐dependent denaturation measured by fluorescence intensity changes at 330 nm, which match the profile of enzyme activity loss in this media.

Photophysical and aggregation properties of a long-chain squarylium indocyanine dye

Abstract The photophysical properties of a long-chain squarilium indocyanine dye, bis[(1-octadecyl-3,3-dimethylindol-2-ylidene)methyl]squaraine (SQI1), were studied in various solvents and in normal and reverse micelles. The dependence of these properties on different solvent parameters is compared with the literature data on other squaraines. The absorption and fluorescence maxima of SQI1 and similar heterocyclic squarilium cyanines exhibit a good linear correlation with the Lorentz–Lorenz function. The rate of nonradiative deactivation of the SQI1 fluorescent state increases with the solvent polarity, but the viscosity effect is very small. A reversible photoisomerization process leading to the formation of a transient photoisomer with a long-wavelength absorption band was detected in SQI1. The lifetime of the photoisomer varies within a wide range, depending on the solvent polarity. SQI1 forms nonfluorescent aggregates in aqueous medium, which dissociate into monomeric molecules upon addition of TX100 above the critical micelle concentration.

Non-covalent dendrimer–porphyrin interactions: the intermediacy of H-aggregates?

The interaction of anionic meso-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) with poly(amidoamine) (PAMAM) dendrimers of generations 2 and 4 in aqueous solution was investigated with steady-state absorption and fluorescence techniques. At low dendrimer concentrations, the formation of low absorptive/emissive species occurs in these systems. With an increase of dendrimer concentration, the porphyrins rearrange at the dendritic outer shell and the absorption and emission spectra suggest the presence of H-aggregates of TSPP. These aggregates dissociate to yield monomeric complexed species in the limit of high dendrimer concentration, which presents very similar spectra for both generations studied. On the other hand, the emission of the same systems at pH 2 shows significant differences between dendrimer generations. While for generation 2 the fluorescence spectra practically coincide with that of the diacid TSPP and its J-aggregate, for generation 4 the spectra obtained are similar to that at high pH. This was interpreted according to a morphologic transition in PAMAM dendrimers around generation 3, to give a more compact structure, which provides a hydrophobic environment for the associated TSPP. At low pH, an increase in J-aggregation is observed in the dendrimers presence. Aging effects were observed, in particular for the systems where different aggregated forms of TSPP coexist, showing that for intermediate dendrimer concentrations these are thermodynamically labile systems.