Rose bengal (RB) readily binds to human serum albumin (HSA). At low RB concentrations, 90% of the dye is associated to the protein (5 microM), This association takes place in specific binding sites I and/or II. At higher RB concentrations, unspecific binding takes place with up to 10 RB molecules bound per protein molecule. The behavior of excited RB molecules bound to HSA is widely different to that observed in aqueous solution. Furthermore, the data also show that the behavior of bound RB molecules changes with the average number of dye molecules per protein (n). In particular, when n is large, the fluorescence yield is significantly reduced and no measurable long-lived triples and free singlet oxygen formation from bound dyes is detected. These results are related to self-quenching of the singlet and, most likely, excited triplets. All results point to the relevance of intra-protein generated singlet oxygen. However, when the dye is bound to the protein, at low oxygen concentrations such as those prevailing in vivo, trapping by oxygen of the triplet becomes inefficient and type I processes could contribute to the observed photoprocesses.
The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.
The photochemical and photophysical properties of 4-(3-hydroxy-2-methyl-4-quinolinoyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl free radical (QT) have been studied as a prefluorescent probe to monitor free radical processes in polymer films. This methodology takes advantage of the efficient intramolecular quenching of the fluorescence of quinoline by the paramagnetic nitroxide, which is disabled when TEMPO reacts with carbon-centered radicals. The fluorescence intensity-time profile observed in the thermal decomposition of 2,2′-azobis(isobutyronitrile) (AIBN) in poly(methyl methacrylate) (PMMA) films showed initial increments in the fluorescence with time, according to the trapping of carbon-centered radicals by QT in the polymer films. Comparison of data under nitrogen and oxygen saturation conditions suggests that oxygen trapping of the carbon-centered radicals at 90 °C is about 20 times faster than reaction with nitroxides. The activation energy for AIBN decomposition in PMMA was measured as 34.1 kcal/mol. Analysis of the fluorescence lifetime distribution establishes the involvement of both static and dynamic fluorescence quenching of the diamagnetic reaction product by AIBN.
The capacity of hydroxycinnamic acid derivatives to trap peroxyl radicals was evaluated by competitive kinetics and oxygen radical absorbance capacity (ORAC) indexes, using c-phycocyanin and pyranine as target molecules. The pattern of results is similar in all the systems, with the reactivity of the compound determined by the bond dissociation energy (BDE) of the hydrogen atom of the phenolic moiety. However, differences in the relative reactivity are observed depending upon the employed methodology (initial rate of consumption or ORAC-type methodology) and target molecule employed. These differences are explained in terms of the role played by secondary reactions of the initially formed phenoxyl radicals. This emphasizes the need for performing a complete kinetic analysis of the results in order to obtain meaningful evaluations of the relative reactivity of the tested compounds.
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