Time-resolved Phosphorescence Detection Research Articles

Singlet oxygen lifetime changes in dying glioblastoma cells.

Time-resolved phosphorescence detection was employed to determine the lifetime of singlet oxygen in live cells. Using hypericin as a photosensitizer, singlet oxygen was generated in U87MG glioblastoma cells. The phosphorescence of singlet oxygen was detected in aqueous cell suspensions following pulsed laser excitation. Our goal was to eliminate or reduce the problems associated with lifetime measurements in water-based cell suspensions. The apparatus enabled simultaneous singlet oxygen phosphorescence and transient absorption measurements, reducing uncertainty in lifetime estimation. The changes in singlet oxygen lifetime were observed during early and late apoptosis induced by photodynamic action. Our findings show that the effective lifetime of singlet oxygen in the intracellular space of the studied glioblastoma cells is 0.4 μs and increases to 1.5 μs as apoptosis progresses. Another group of hypericin, presumably located in the membrane blebs and the plasma membrane of apoptotic cells, generates singlet oxygen with a lifetime of 1.9 μs.

Spiro‐OMeTAD as Redox Mediator and Singlet Oxygen Scavenger in Lithium‐Oxygen Batteries

AbstractLithium‐oxygen batteries (LOBs) are considered one of the new alternatives to lithium‐ion batteries mainly due to its high energy density. Several strategies are pursued in order to avoid parasitic reactions that fade the battery capacity. In this work, it was carried out a photochemical and electrochemical characterization of Spiro‐OMeTAD working as a redox mediator (RM) in the LOBs. By using the Time Resolved Phosphorescence Detection (TRPD) technique together with Rose Bengal, we demonstrate that RM can deactivate the singlet oxygen produced in the cell, decreasing its performance. A concentration of 5 mM Spiro‐OMeTAD in the electrolyte avoids solubility problems, maximizes its catalytic effects during the charge, and is still enough to act as a singlet oxygen scavenger. Finally, lithium nitrate was added to the electrolyte to passivate the metallic lithium anode with the aim to prevent the RM decomposition and the increase of LOBs performance.

Revisiting the Graphitized Nanodiamond-Mediated Activation of Peroxymonosulfate: Singlet Oxygenation versus Electron Transfer.

Graphitized nanodiamonds (ND) exhibit outstanding capability in activating peroxymonosulfate (PMS) for the removal of aqueous organic micropollutants (OMPs). However, controversial observation and interpretation regarding the effect of graphitization degree on ND's activity and the role of singlet oxygen (1O2) in OMP degradation need to be clarified. Herein, we investigated graphitized ND-mediated PMS activation. Experiments show that the activity of ND increases first and then decreases with the monotonically increased graphitization degree. Further experimental and theoretical studies unveil that the intensified surface graphitization alters the degradation mechanism from singlet oxygenation to an electron-transfer pathway. Moreover, for the first time, we applied a self-constructed, time-resolved phosphorescence detection system to provide direct evidence for 1O2 production in the PMS-based system. This work not only elucidates the graphitization degree-dependent activation mechanism of PMS but also provides a reliable detection system for in situ analysis of 1O2 in future studies.

Sensitized photo-oxidation of gadusol species mediated by singlet oxygen

Gadusols are efficient nature UV sunscreens with antioxidant capacity. The kinetics of the quenching reactions of singlet oxygen O2(1∆g) by gadusol species was evaluated in aqueous solution as well as in the presence of direct charged micelles. Time-resolved phosphorescence detection of O2(1∆g) indicated that gadusolate, the main species under biological pH, is a more efficient quencher than the enol form with a rate constant of ca. 1.3 × 108 L mol−1 s−1. The deactivation proceeds via a collisional mechanism with clear dominance of chemical pathways, according to the rates of gadusol and oxygen consumptions, and typical photooxidation quantum yields of ca. 7%. The relative contributions of the chemical and physical quenching steps were not affected by the presence of anionic or cationic micelles emulating simple pseudo-biological environments. The products of the photo-oxidative quenching support a type II mechanism initiated by the addition of O2(1∆g) to the C-C double bond of gadusolate. These results point to the relevance of considering the role of sacrifice antioxidant along with the UV-screening function for gadusol, particularly in the context of potential biotechnological applications of this natural molecule.

Antioxidant capacity of (+)-catechin visible-light photoirradiated in the presence of vitamin B2

ABSTRACTObjectives: Catechins are important components of human diet and have received special attention due to their antioxidant capacity. The purpose of this paper was to study the antioxidant action of (+)-catechin (CTQ) in the presence of vitamin B2 (riboflavin) as light-absorbing agent. Furthermore, two model compounds, catechol (CTC) and resorcinol (RSC), were selected in order to elucidate the reactive target of the CTQ molecule. The influence of pH-medium was investigated.Methods: Stationary photolysis, polarographic detection of dissolved oxygen, reactive oxygen species (ROS)-scavengers, time-resolved near-IR phosphorescence detection, stationary, and time-resolved fluorescence and laser flash photolysis techniques were employed.Results: CTQ interacts with riboflavin under visible-light photoirradiation as well as with different ROS which are generated in this mechanism. Radical-scavenging activity increases with increasing of pH-medium.Discussion: pH-effect of the medium on radical-scavenging activity comes from the increased electron-donating ability of CTQ upon deprotonation. These results are very interesting due to the fact that the pH of the food products displays important variations.The -scavenging ability of CTQ, would be equal to the additive contribution of each reactive center, CTC, and RSC, present at the molecule of CTQ. However, CTQ would have a moderate ability to removal of -species at pH 7.

On the photooxidation of the multifunctional drug niclosamide. A kinetic study in the presence of vitamin B2 and visible light

Objectives: The multifunctional drug niclosamide (NSD), extensively employed therapeutically, is a frequent pollutant of surface waters. Considering the environmental importance of photodegradative processes for this type of contaminant, the kinetic and mechanistic aspects of the possible visible-light-mediated photooxidation of NSD were studied under naturalistic conditions.Methods: The visible-light absorber riboflavin (vitamin B2) was employed as a photosensitizer. The vitamin can usually be found in natural waters and is the most common endogenous photosensitizer in mammals.The interaction of NSD with electronically excited states of Rf and with photogenerated reactive oxygen species (ROS) was evaluated through conventional UV spectroscopy, laser flash photolysis, time-resolved phosphorescence detection of singlet molecular oxygen (O2(1Δg)), and polarographic dosage of dissolved oxygen.Results: Ground state NSD quenched the long-lived triplet excited state of Rf (3Rf*) and the photogenerated ROS (O2(1Δg)) and superoxide radical anion . As a result, NSD was photooxidized. The rate constants for the interaction NSD–O2(1Δg) are particularly low, in the order of 106/M/s, although the whole interaction is attributable to a pure reactive process. The O2(1Δg) quenching was faster in alkaline medium, favored by the ionization of the NSD phenolic group.Under Rf-photosensitization, NSD was degraded very much more rapidly than phenol, the latter being considered a paradigmatic water-contaminant model compound.NSD may behave as an antioxidant in bio-environments, as demonstrated employing the photooxidizable amino acid tryptophan as a relevant biological target.Discussion: The results indicate that a -mediated process is the main route for the Rf-sensitized photooxidation of NSD. Photodegradation of the biocide in the presence and absence of phenol and tryptophan was quantitatively evaluated, discussed, and interpreted in terms of competitive quenching processes of 3Rf*, O2(1Δg), and by the substrates.

Sensitized phosphorescence as detection method for the enantioseparation of bupropion by capillary electrophoresis

A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.

Characterization and Singlet Oxygen Quenching Capacity of Spray-Dried Microcapsules of Edible Biopolymers Containing Antioxidant Molecules

Microcapsules of gum arabic or maltodextrin 20DE containing antioxidant molecules (AOx), for example, carotenoids and tocopherol derivatives, were prepared by the spray-drying technique. The properties of these microcapsules were evaluated by several techniques, such as dynamic light scattering, scanning electronic microscopy, and steady-state and time-resolved fluorescence spectroscopy of microencapsulated pyrene. The quenching of photochemically generated singlet molecular oxygen ((1)O(2)) by the AOx in homogeneous solvents as well as in microcapsule solutions was evaluated using time-resolved phosphorescence detection of (1)O(2). The quenching rate constant of the process, k(Q)(AOx), was strongly dependent on the type of the AOx. These results are explained by compartmentalization effects of the AOx in the core of the microcapsules and the accessibility of (1)O(2). The contribution of the biopolymer as quencher of (1)O(2) was also investigated. The present results can be applied to the design of edible antioxidant microcapsules within the food and cosmetic industries.

Photodegradation of bisphenol A and related compounds under natural-like conditions in the presence of riboflavin: Kinetics, mechanism and photoproducts

The aerobic riboflavin (Rf)-sensitized photodegradation of the endocrine disruptor 4,4′-isopropylidenebisphenol (bisphenol A, BPA), and of the related compounds 4,4′-isopropylidenebis(2,6-dibromophenol) and 4,4′-isopropylidenebis(2,6-dimethylphenol) has been studied in water and water–methanol mixtures through visible-light continuous photolysis, polarographic detection of oxygen uptake, stationary and time-resolved fluorescence spectroscopy, time-resolved near-IR phosphorescence detection and laser flash photolysis techniques. Bisphenols (BPs) quench excited singlet and triplet states of Rf, with rate constants close to the diffusion limit. BPs and dissolved molecular oxygen, employed in similar concentrations, competitively quench triplet excited Rf. As a consequence, superoxide radical anion and singlet molecular oxygen (O 2( 1Δ g )) are produced by electron- and energy-transfer processes, respectively, as demonstrated by auxiliary experiments employing selective quenchers of both oxidative species and the exclusive O 2( 1Δ g ) generator Rose Bengal. As a global result, the photodegradation of Rf is retarded, whereas BPs are degraded, mainly by an O 2( 1Δ g )-mediated mechanism, which constitutes a relatively efficient process in the case of BPA. Oxidation, dimerization and fragmentation products have been identified in the photooxidation of BPA. Results indicate that BPs in natural waters can undergo spontaneous photodegradation under environmental conditions in the presence of adequate photosensitizers.

Singlet oxygen quenching by anthocyanin's flavylium cations

The quenching of singlet molecular oxygen (1O2) by the flavylium cation form of six widespread anthocyanin derivatives: cyanidin 3-glucoside (CG), cyanidin 3-rutinoside (CR), cyanidin 3-galactoside (CGL), malvidin (M), malvidin 3-glucoside (MG) and malvidin 3,5-diglucoside (MDG) was studied in 1% HCl methanol solution by time-resolved phosphorescence detection (TRPD) of 1O2 and photostationary actinometry using perinaphthenone and methylene blue as sensitizers, respectively. The average value of the total (k0) and chemical (kc) quenching rate constants were ∼ 4×108 m−1 s−1 and 3×106 m−1 s−1, respectively, indicating the good performance of the studied anthocyanins as catalytic quenchers of 1O2. The quenching efficiency was larger for malvidin than for cyanidin derivatives, probably by the extra electron-donating methoxy group in ring B of the malvidin derivatives; and it was also dependent on the number and type of glycosylated substitution. As observed for other phenolic-like derivatives, the quenching of 1O2 by anthocyanins was mediated by a charge-transfer mechanism, which was modulated by the total number of –OR substituents that increases the electron-donating ability of these compounds.

Singlet molecular oxygen [O2(1Δg)]-mediated photodegradation of tyrosine derivatives in the presence of cationic and neutral micellar systems

The kinetics of rose bengal-sensitized photooxidation of tyrosine and several tyrosine-derivatives (tyr-D) named tyrosine methyl ester, tyrosine ethyl ester and tyrosine benzyl ester was studied in buffered pH 11 water, and buffered pH 11 micellar aqueous solutions of 0.01 M cetyltrimethylammonium chloride (CTAC) and 0.01 M-octylphenoxypolyethoxyethanol [triton X100 (TX100)]. Through time-resolved phosphorescence detection of singlet molecular oxygen (O(2)((1)Delta(g))) and polarographic determination of oxygen consumption, the respective bimolecular rate constants for reactive (k(r)) and overall (k(t)) quenching of the oxidative species by tyr-D were evaluated. Both rate constants behave in different fashion depending on the particular reaction medium. k(r)/k(t) values, increase in the sense CTAC<<TX100 < water, indicating, for the tyr-D family studied, an excellent degree of self-protection against O(2)((1)Delta(g))-attack in the CTAC micellar system and a high photooxidability level in water. Results were interpreted in terms of a competition between solvent polarity effects, local substrate concentration and electron donating capabilities of the substrates in the different media that can contribute to predict the extent of photodynamic damage in biological environments.

Scavenging of riboflavin-photogenerated oxidative species by uric acid, xanthine or hypoxanthine: A kinetic study

Air-equilibrated aqueous solutions of the purine bases (PBs) uric acid (URA), xanthine (XAN) and hypoxanthine (HXA) have been irradiated with visible light in the presence of riboflavin (Rf) as the only light-absorbing compound, to study to what extent the reactive oxygenated species (ROSs) singlet molecular oxygen, O 2( 1Δ g), and superoxide radical anion, O 2 −, are involved in the observed photodegradation processes. Both reactive species are generated from triplet excited riboflavin ( 3Rf*) by well-known processes: energy transfer from 3Rf* to ground state oxygen to yield O 2( 1Δ g), and electron transfer from each PB to 3Rf* to produce the radical anion Rf − that, after another electron transfer step to ground state oxygen, yields O 2 − and Rf. The kinetics of the involved processes have been studied by polarographic detection of oxygen uptake and time-resolved phosphorescence detection of O 2( 1Δ g), at concentrations in the range 0.05–0.5 mM in aqueous solutions at pH values 5, 7, 9, and 12, to compare the reactivity of the neutral and ionic species of each purine base. By means of Rose Bengal photosensitized reactions, it could be shown that O 2( 1Δ g) is quenched by the three PBs with an overall rate constant decreasing in the order URA > XAN > HXA, with values ranging from 1.16 × 10 9 M −1 s −1 (URA at pH 12) to 5 × 10 6 M −1 s −1 (HXA at pH 7). When Rf is used instead, the quenching process takes place mostly by a chemical mechanism that prevents the oxidation of Rf by O 2( 1Δ g). Experiments with Rf and superoxide dismutase indicate that the PBs also react with O 2 − by a mechanism which is not simple. With URA, largely the most efficient O 2( 1Δ g)-quencher, the major ROS generated is O 2 −, and the process is characterized by the largest rate of oxygen uptake, indicating efficient overall-scavenging and URA oxidation. With HXA, the less efficient O 2( 1Δ g)-quencher, the process shows a relatively high rate of oxygen consumption, especially in alkaline solution, likely as a result of the efficient generation of O 2 − from Rf − and its concomitant reaction with HXA. Finally, with XAN the quenching efficiency takes intermediate values. At physiological pH, URA is the most efficient antioxidant, showing the highest overall rate constant for O 2( 1Δ g) quenching, the lowest efficiency for the reaction with O 2( 1Δ g), and the highest relative rate of oxygen uptake.

Kinetic study of the photosensitized oxygenation of the flavanone naringin and its chalcone

The kinetic of the O 2( 1Δ g)-photosensitized oxidation of the flavanone naringin (7-rhamnoglucosyl-4′,5-dihydroxyflavanone, FL) and its chalcone isomer (4′-rhamnoglucosyl-2′,6′,4-trihydroxychalcone, CH) in neutral and 1 mM NaOH ethanol solutions was studied using Rose Bengal as photosensitizer. The rate constants for the chemical quenching of O 2( 1Δ g) by the flavonoids ( k r) were determined using either UV–vis absorption spectroscopy or HPLC techniques, and for the total (physical + chemical) quenching ( k t) were determined using time-resolved phosphorescence detection of O 2( 1Δ g) at 1270 nm. A larger reactivity towards O 2( 1Δ g) of CH than for FL in neutral ethanol solutions was observed, due to the extra conjugated double bond in CH. However, in alkaline media the reactivity of both isomers was larger than in neutral conditions. This behavior was associated to the increment of electron density by the formation of a carbanion in FL and to the presence of the extended conjugated π-system in the CH isomer by deprotonation of phenolic groups. In all cases, the formation of 4-hydroxybenzoic acid as ending product was detected by HPLC. Based on reported mechanisms of O 2( 1Δ g)-mediated oxidation of flavonoids, the formation of 7-rhamnoglucosyl-5,4′-dihydroxyflavonol as primary photo-oxidation product was proposed. The reactivity towards O 2( 1Δ g) of the aglycone of naringin (5,7,4′-trihydroxyflavanone or naringenin, NG) in alkaline conditions was lower than for the parent naringin, due to incapability of NG to form a carbanion species. These results are expected to have significance in biosynthesis, antioxidant properties and stability of naturally occurring flavonoids.

Scavenging of photogenerated singlet molecular oxygen and superoxide radical anion by sulpha drugs - Kinetics and mechanism

The ability of the sulfanilic antibiotics (SDs), dapsone (DAP), sulfisoxazole (SFX), sulfadiazine (SFD), and sulfanilic acid (SFNA) to act as scavengers of the visible-light-photogenerated species superoxide radical anion (O2·–) and singlet molecular oxygen (O2(1Δg)) was studied employing the natural pigment riboflavin (Rf) and the artificial dye Rose Bengal as photosensitisers. A complex mechanism, common to all the SDs studied, was elucidated through stationary photolysis, polarographic detection of oxygen uptake, fluorescence spectroscopy, time-resolved phosphorescence detection of O2(1Δg), and laser flash photolysis. Visible-light irradiation of aqueous and aqueous methanolic solutions of Rf (ca. 0.02 mmol/L) plus SD (ca. 0.5 mmol/L) photogenerated excited singlet and triplet Rf (1Rf* and 3Rf*). Under these experimental conditions, only 3Rf* is quenched either by oxygen, giving rise to O2(1Δg) by electronic energy transfer to dissolved ground-state oxygen, or by SD, yielding semireduced Rf through an electron-transfer process. Complementary experiments performed in pure water employing superoxide dismutase and sodium azide inhibition of oxygen uptake, in parallel with laser flash photolysis data, showed that O2·– is also formed, probably due to the reaction of the anion radical from Rf with dissolved oxygen, yielding also neutral, ground-state Rf. Both active oxygen species, namely, O2·– and O2(1Δg), are quenched by the SDs and, as a result, photodegradation of the SDs — each to a different extent — and photodegradation of the sensitiser itself were observed. The SDs that kinetically behave as the better physical quenchers of O2(1Δg), which are in principle good candidates as photoprotectors, namely, DAP and SFD, suffer photooxidation, exhibiting high to moderate oxygen consumption rates due to the O2·– oxidative pathway, whereas for SFNA and SFX, oxidation predominantly occurs through an O2(1Δg)-mediated mechanism. Microbiological results for SFX, taken as a representative SD, indicate that the photodegradation of the drug, upon visible-light Rf-sensitised irradiation, is accompanied by a net loss in bacteriostatic activity.Key words: photooxidation, singlet oxygen, superoxide radical anion, sulpha drugs.

Effect of reverse micelles on the Rose Bengal-sensitized photo-oxidation of 1- and 2-hydroxynaphthalenes

Benzylhexadecyl dimethylammonium chloride (BHDC) and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) water-in-oil micro-emulsions were employed to study the influence of medium heterogeneity on singlet molecular oxygen [O2(1Δg)]-mediated degradation of 1-hydroxynaphthalene (1-OHN) and 2-hydroxynaphthalene (2-OHN) in their naphthoate form. The kinetic study in the micellar system, that was considered as a closer model for the environment of contaminants in natural polluted waters, consisted of a comparative work with the process in homogeneous solution, by varying surfactant structure and water content of the micro-emulsion. While it is known that 1-OHN and 2-OHN are rapidly and efficiently photo-oxidised in aqueous medium, time-resolved phosphorescence detection of O2(1Δg) and stationary photolysis experiments demonstrate that both the values for the overall and reactive rate constants for the quenching of O2(1Δg) and the photo-oxidation efficiencies are lowered in BHDC micelles, whereas the photo-oxidative process in AOT micro-emulsions was totally inhibited. Results are interpreted and discussed on the basis of different locations of the probe in the micellar environment.

Micellar effect on the scavenging of singlet molecular oxygen by hydroxybenzenes

The antioxidative effectiveness of three hydroxyaromatic derivatives (OHAD), namely phenol, resorcinol and phloroglucinol, as reflected by their scavenging ability of the photochemically generated species singlet molecular oxygen [O2(1Δg)], was studied in aqueous solution and micellar media. Kinetic results, obtained through time-resolved phosphorescence detection of O2(1Δg) emission and polarographic methods, at pH 7 and 12 in homogeneous and CTAB micellar media, were compared. Calculated photooxidation quantum efficiencies (φr), ranging from very low values (~0.01) to relatively high ones (~0.3), depend on the preferential solubilization sites of the hydroxyaromatic derivatives either in the micellar or homogeneous media. OHAD in water pH 7 and in CTAB 0.02 M pH 12 exhibit their highest antioxidative efficiency as O2(1Δg) scavengers. In these conditions, they present the best degree of self protection against O2(1Δg)-mediated photooxidation with relatively high values for the overall quenching rate constant and, simultaneously, the lowest φr.

Monofunctional Derivatives of Coproporphyrins for Phosphorescent Labeling of Proteins and Binding Assays

p-Isothiocyanatophenyl derivatives of Pt(II)– and Pd(II)–coproporphyrin I are described as stable monofunctional reagents which enable simple covalent labeling of proteins and other biomolecules under mild conditions in aqueous solutions. Labeling procedure was optimized for antibodies, avidin, and neutravidin. Photophysical properties of resulting conjugates important for their use in binding assays based on time-resolved phosphorescence detection were studied. The functional activity and long-term storage stability of antibody conjugates were assessed in comparison with unmodified proteins. The new labels and their conjugates were evaluated in the solid-phase immunoassays using commercial time-resolved phosphorescence readers Victor2 and Arcus-1230 (Wallac). Potential applications of these reagents in in vitro diagnostics are discussed.

Kinetics of the dye-sensitized photooxidation of trihydroxybenzenes

The kinetics of the singlet molecular oxygen [O2(1Δg)]-mediated photooxidation of the three isomeric trihydroxybenzenes (THBs), compounds of potential environmental significance as aquatic contaminants, has been studied in water solution as a function of pH and ionic strength, as well as in benzene and acetonitrile. Rate constants for chemical and overall interactions with O2(1Δg), determined by time-resolved IR phosphorescence detection and polarographic methods, are in the range 0.05×107–24.0×107M−1s−1, depending on the medium and the particular compound. In water at pH 2, the photooxidation quantum efficiencies of 1,2,3-THB, 1,2,4-THB and 1,3,5-THB are 0.07, 0.07 and 0.19, respectively. The photooxidative processes are highly favored by the ionization of the OH groups, the increase of solvent polarity, and the presence of salts in the medium, strongly suggesting the participation of a polar encounter complex of the type [THB-O2(1Δg)]. This kinetic behavior is similar to that observed in simple phenols and dihydroxybenzenes, although THBs are much easier photooxidizable, even in non-ionized form. These results possess environmental relevance, because they demonstrate that any THB in aqueous media undergoes spontaneous and fast solar-promoted photooxidation under practically any field condition.

Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).

Monosubstituted naphthalenes as quenchers and generators of singlet molecular oxygen

The monosubstituted naphthalenes (MSN) 1-hydroxynaphthalene (1-OHN), 2-hydroxynaphthalene (2-OHN), 1-methoxynaphthalene, 2-methoxynaphthalene, 1-aminonaphthalene (1-AN), 2-aminonaphthalene (2-AN), 1-methylnaphthalene, 1-fluoronaphthalene and 1-cyanonaphthalene, and the parent compound naphthalene have been evaluated as physical and chemical quenchers and as generators of singlet molecular oxygen, [O 2( 1 Δ g )], in water and in organic solvents. Rate constant values for the overall ( k t) and chemical ( k r) quenching of O 2( 1 Δ g ), respectively, determined by time-resolved phosphorescence detection and by substrate or oxygen consumption- were from less than 10 4–3.92 × 10 8 M −1 s −1, depending on the solvent polarity and the pH of the solution. The derivatives 1-OHN, 2-OHN, 1-AN and 2-AN react with O 2( 1 Δ g ), generated with external sensitisers, with quantum efficiencies in the range 0.03–0.67. The photooxidation mechanism must include the formation of an encounter complex with partial charge-transfer character of the type [O 2( 1 Δ g ) δ− …MSN δ+ ], in accordance with the observed variation of k t with the global free energy change. All the herein studied MSN generate O 2( 1 Δ g ) upon direct irradiation, with fair to good quantum yields. The structure of the photooxidation products from 1-OHN and 2-OHN, two compounds with environmental interest, have been determined by combined GC/MS analysis. Benzoic acid, phthalic acid, 1,4-naphthoquinone and a dimer were identified in the reaction mixture from 1-OHN, while 2-OHN yielded phthalic acid and the dimer 2,2′-dihydroxy-1,1′-binaphthyl.