Suzana M. Andrade

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.

The Influence of Water on the Photophysical and Photochemical Properties of Piroxicam in AOT/iso-octane/Water Reversed Micelles

The photophysical properties of Piroxicam, a nonsteroidal anti‐inflammatory drug (NSAID), were investigated at different pHext values in reversed micelles of Aerosol‐OT (AOT) in iso‐octane, using both steady‐state and picosecond time‐resolved fluorescence spectroscopy. In contrast with the very complex data obtained in aqueous media, where several prototropic species are in equilibrium, the reversed micellar system essentially favors two species. The absorption spectra shows only one isosbestic point at λ= 348 nm. Excited‐state intramolecular proton transfer (ESIPT), also detected in water, is promoted at low water pool contents measured by ω0= [H2O]/[AOT]. A strongly shifted (λem= 470 nm) tautomeric emission is found. Upon the gradual increase of ω0, striking differences with pHext are found. At pHext= 4, the drug preferentially locates itself in the interfacial region partitioning between a hydrophobic and a hydrophilic domain. Global analysis was applied to the decay data and the results were interpreted by the “two‐state excited‐state” formalism. At pHext= 7, the anionic species is prevalent and the probe locates itself deeper inside the water core of the reversed micelles. Thus, a strong dependence on water content is detected, approaching a behavior similar to that observed in free aqueous solutions.

J-aggregate formation in bis-(4-carboxyphenyl)porphyrins in water : pH and counterion dependence

The self aggregation behaviour of meso-tetraarylporphyrins containing two carboxyphenyl units in adjacent and opposite positions, respectively, 5,10-bis(4-carboxyphenyl)-15,20-diphenylporphyrin (DiCPP-adj) and 5,15-bis(4-carboxyphenyl)-10,20-diphenylporphyrin (DiCPP-opp), was investigated at different pH values. Ordered porphyrin architectures are obtained at pH = 0.8 and pH = 12 for both compounds studied, through an easy self assembly approach. The type of architecture and the extent of aggregation are related with the relative positions of the 4-carboxyphenyl groups attached to the porphyrin core and with the counterions present. The aggregates obtained exhibit spontaneous chirality, resonance light scattering, short fluorescence lifetimes and low fluorescence quantum yields. The data gives evidence that at pH = 0.8 with NO3− the preferred aggregation is more favoured for DiCPP-adj than for DiCPP-opp, whereas at pH = 12 the aggregate of DiCPP-opp is induced by Na+ and Li+ cations and stabilized by hydrophobic interactions. Deposition of aqueous solutions at key pHs on glass surfaces enables the detection of circular and ring aggregates viewed by fluorescence lifetime imaging microscopy (FLIM) with dimensions of a few micrometres.

Aggregation Kinetics of Meso-tetrakis(4-sulfonatophenyl) Porphine in the Presence of Proteins: Temperature and Ionic Strength Effects

The kinetics of J-aggregation was studied through UV/Vis spectroscopy for meso-tetrakis(4-sulfonatophenyl) porphine—TSPP at pH = 2.0 using the protein human serum albumin as template. The effect of protein concentration on the kinetics was monitored by the appearance of the J-aggregate band at 486 nm and the simultaneous decrease of the monomer absorption at 434 nm. A simple equation based on the X →k Y reaction, where k may be assumed as a pseudo-first-order rate, fits well both the J-aggregation formation and monomer transformation. Temperature dependence of the reaction rate follows an Arrhenius behavior up to T = 38°C, but above this value the dependence is inverted. This temperature seems to reflect the mid-point for the thermal denaturation of HSA. Ionic strength effect clearly exposes the prevalence of the electrostatic nature of this J-aggregation, and using a semi-empirical equation an estimate of the interaction length between TSPP-Na+ of 4.5. Å an was obtained in good agreement with crystallographic data. Absorption band shift and bandwidth were used to estimate the number of monomers in the J-aggregate unit that leads to spectral changes, and a number around 6–7 was found. There is no apparent growth of J-aggregate taking into account the invariance of the bandwidth of J-aggregate band with time at any of the temperatures studied.

Reorganization of self-assembled dipeptide porphyrin J-aggregates in water-ethanol mixtures.

The self-assembly of a neutral meso-methoxyphenylporphyrin functionalized with a dipeptide glycilglycine substituent (MGG) in water and in water-ethanol mixtures was studied by absorption and fluorescence spectroscopy. In water, hydrophobic interactions and the noncovalent intermolecular hydrogen bonding between the terminal carboxylate group of one porphyrin and the hydrogen atoms of the pyrrolic nitrogens of another porphyrin originate nonspecific disorganized H- and J-aggregates. The addition of ethanol (0.1-25% v/v) to the water creates small clusters within which porphyrin J-aggregates reorganize as revealed by a narrow intense band detected by the Rayleigh light scattering (RLS) at 443 nm. Similar phenomenology is detected in SDS premicellar aggregates. Computational DFT calculations of a model dimer formation stabilized via intermolecular hydrogen bonding estimate an energy gain of -22 kJ mol(-1) and a center-to-center and interplane distances between porphyrin moieties of 16.8 and 3.7 Å, respectively. The kinetics of the J-aggregate formation could be fitted with a time-dependent model, and an activation energy of 96 kJ mol(-1) was estimated. The aggregates morphology of MGG was followed by transmission electron microscopy (TEM) which showed rod-type structures of 5-8 μm evolving to spherical particles with increased ethanol content. Similar images and sizes were obtained in analogous samples using fluorescence lifetime imaging microscopy (FLIM) and dynamic light scattering (DLS). The formation of excitonically coupled supramolecular MGG structures of brickwork or staircase types is proposed in these water-ethanol mixtures.

The Location of Tryptophan, N-acetyltryptophan and α-Chymotrypsin in Reverse Micelles of AOT: A Fluorescence Study¶

Abstract The spectroscopic properties of α-chymotrypsin (α-Chym), l-tryptophan (Trp) and N-acetyl-l-tryptophan (NAT) solubilized in hydrated reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate in iso-octane were followed by fluorescence as a function of the amount of intramicellar water and initial pH. The lack of pH dependence observed for Trp in these systems, as opposed to what occurs in bulk water, and the similarities found for the protein in both media foresee different locations of these probes. In reverse micelles, fluorescence quenching studies using acrylamide emphasize the existence of structural alterations within the protein when its global charge changes from positive (pH = 7) to negative (pH = 10). The ensemble of the data points to an interfacial location of the zwitterionic Trp, an intermediate region of less tightly bound water for the location of the anionic Trp and NAT and an almost bulk water environment for α-Chym.

Incorporation of β-lactoglobulin in monolayers of dioctadecyldimethylammonium bromide studied by Brewster angle microscopy

The deposition of pure bovine β-lactoglobulin (βLG) monolayer or the incorporation in a dioctadecyldimethylammonium bromide (DODAB) monolayer, were studied by π–A measurements at the air–water interface and by direct visualization of the interface by BAM. The co-spreading of the monolayer material dissolved in a mixed volatile solvent (chloroform+ethanol) was selected based on reproducible data and minimum consumption of protein. Conformational changes induced by the mixed solvent on the native structure of βLG, investigated by circular dicroism, disappear after the solvent evaporation at the interface. The π–A isotherms suggest and BAM images confirm, that, at low surface pressures, βLG is incorporated in the liquid expanded monolayer of DODAB, while at the plateau near 30 mN m−1 the protein is squeezed out into micro-domains partially immersed in the subphase and strongly adsorbed under the DODAB layer. The topography of DODAB/yβLG mixed films changes with surface pressure and number of protein residues, y, incorporated per DODAB molecule. BAM observation shows that at low y DODAB molecules dominate the topography of monolayer at the interface.

Structural changes of α-chymotrypsin in reverse micelles of AOT studied by steady state and transient state fluorescence spectroscopy

Abstract Time-resolved fluorescence of α-chymotrypsin (α-chym) solubilised in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles was studied as a function of the amount of encapsulated water and initial pH. In reverse micelles the anionic interface leads to some conformational rearrangements at “pH ext ” =10 due to electrostatic interactions with the ionised groups of α-chym. The data point to the proteins location in a bulk water environment, which accounts for its high stability in these reverse micelles and allows the distinction of three emitting classes of tryptophan residues within the proteins matrix. Fluorescence quenching results show that these residues are differently accessed to the quencher molecules used (acrylamide, succinimide and iodide) and are consistent with a mechanism of penetration for the interaction with the protein.

Optical spectroscopy and photochemistry of porphyrins and phthalocyanines

Studies of excited singlet and triplet states of porphyrins and phthalocyanines in organized media of reverse micelles, vesicles, monolayers, and Langmuir-Blodgett films along with more complex supramolecular organizates with proteins and dendrimers, are reported. Self-assembly in these systems was followed by imaging and temporal fluorescence techniques.

Fluorescence quenching of Acridine Orange in microemulsions induced by the non-steroidal anti-inflammatory drug Piroxicam

The singlet excited-state quenching of Acridine Orange (AO) by methyl viologen (MV2+) and the non-steroidal anti-inflammatory drug Piroxicam (Prx), incorporated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane/water and Triton X-100 (Trx-100)/cyclohexane-hexanol/water (w/o) microemulsions, was followed by steady- and transient-state fluorescence. The water content was varied by using different values of omega 0 (omega 0 = [H2O]/[S]) at fixed AOT (0.1 M) and Trx-100 (0.2 M) concentrations. In AOT, MV2+ resides at the interface, while Prx partitions between the interface and bulk water, but considerably biased towards the latter compared to AO. The quenching process efficiency increases with increasing omega 0, but reaches a diffusional value similar to that of free water only for the case of Prx, underlining the electrostatic effect of the AOT interface. The quenching process in Trx-100 microemulsions is more efficient for Prx than for MV2+, pointing to a similar polyoxyethylene intra-chain location for the former and AO. In both cases, data obtained allowed the microviscosity of the aqueous interior at different omega 0 to be extrapolated and indicate an increase in eta w values with water content, reflecting changes in the shape of Trx-100 microemulsions, which occur at omega 0 = 8.