Quenching of the triplet state of Safranine-O by aliphatic amines in AOT reverse micelles studied by transient absorption spectroscopy

The photophysics of Safranine-O (3,6-diamino-2,7-dimethyl-5 phenyl phenazinium chloride) (SfH(+)Cl(-)) was investigated in reverse micelles (RMs) of AOT (sodium bis(2-ethylhexyl)sulfosuccinate) with special emphasis on the triplet state processes. The triplet is formed in its monoprotonated form, independently of the pH of the water used to prepare the RMs. While the intersystem crossing quantum yields in RMs are similar to those in organic solvents, the triplet lifetime is much longer. Since the pH in the water pool of AOT RMs is close to 5 and the triplet state of the dye is subjected to proton quenching, the long lifetime indicates that the dye resides in a region where it cannot be reached by protons during its lifetime. All the measurements indicate that the dye is localized in the interface, sensing a medium of micropolarity similar to EtOH : water (3:1) mixtures. The quenching by aliphatic amines was also investigated. While the quenching by the hydrophobic tributylamine is similar to that in methanol, the hydro-soluble triethanolamine is one order of magnitude more effective in RMs than in homogeneous solution. In the latter case the quenching process is interpreted by a very fast intramicellar quenching, the overall kinetics being controlled by the exchange of amine molecules between RMs. Semireduced dye is formed in the quenching process in RMs in the di-protonated state with a comparable quantum yield to the monoprotonated state formed in homogeneous solvents. The results point to the advantage of the reverse micellar system for the generation of active radicals for the initiation of vinyl polymerization, since a much lower concentration of amine can be employed with similar quantum yields.     

UVC induced oxidation of chloropurines: excited singlet and triplet pathways for the photoreaction

The phototransformation of 2-chloro, 6-chloro and 2,6-dichloropurines under UVC excitation (254 nm) has been studied and the major photoproducts have been identified using absorption spectroscopy, HPLC and mass spectrometry. It was shown that hydroxypurines were formed as the main products for all three investigated compounds both in the presence and absence of oxygen. In the case of 6-chloro- and 2,6-dichloropurine, a photodimer is also formed as a minor photoproduct in the absence of oxygen but is efficiently quenched in the presence of oxygen. Nanosecond photolysis experiments also revealed significant intersystem crossing to the triplet state of the chloropurines which has been characterized (transient absorption spectra, triplet formation quantum yields and rate constants of quenching by oxygen, Mn(2+) ions and ground state). Experimental evidence allows to conclude that the triplet state is involved in photodimer formation whereas the hydroxypurine is formed from the reaction of the excited singlet state of chloropurines with the solvent (water addition) through heterolytic C-Cl bond rupture. Mass spectrometry and (1)H NMR results allowed to propose a chemical pathway for dimer formation in the case of 2,6-dichloropurine in a two-step process: first a homolytic rupture of C-Cl bond in the triplet state of the molecule with the formation of purinyl radicals, which subsequently react with an excess of ground state molecules and/or hydroxypurine primarily formed.     

Triplet state population study in relation to the aggregation of proflavine in frozen solutions

The evolution of the proflavine triplet state population with the extent of aggregation is examines, by ESRmeasurements, as a function of organic solvents and mineral salts, well known for their ability to influence the aggregation process. The intensity of the triplet state population has also been investigated as a function of pH. The insertion of aromatic molecules like DNA's nucleotides between dye molecules is shown to increase the intensity of the triplet state. It is put forward that the observed triplet state is characteristic of the singly protonated proflavine which seems to be the active species in in vivo experiments.     

Fate of flavins in sensitized photodegradation of isohumulones and reduced derivatives: studies on formation of radicals via EPR combined with detailed product analyses.

Photodegradation of isohumulones accounts for formation of the lightstruck flavor in beer. The reactions involved are mediated by riboflavin, a natural photosensitizer present in beer in ppb quantities. The results of an investigation of this sensitized degradation process are presented herein. Product analyses and electron paramagnetic resonance spectroscopy, in steady-state as well as in time-resolved mode, offer extensive insight into the photophysical and photochemical details of the degradation mechanism. In contrast to energy transfer and Norrish type I alpha-cleavage reactions that take place on direct irradiation of isohumulones, the sensitization pathway proceeds via one-electron redox chemistry involving the excited triplet state of riboflavin and derivatives. The flavin semiquinone radical thus formed could be readily detected, either by steady state or by time-resolved electron paramagnetic resonance spectroscopy. Superimposed signals in the spectra revealed the presence of radical fragments derived from isohumulones or tetrahydroisohumulones, which, on recombination with riboflavin semiquinone radicals, produced stable reaction products that were identified by HPLC-MS. However, no superimposed signals were observed on sensitized irradiation of dihydroisohumulones.     

Photoreactions of p-benzo-, p-naphtho- and p-anthraquinones with ascorbic acid.

The photoreduction of 1,4-benzoquinone (BQ), 1,4-naphthoquinone (NQ), 9,10-anthraquinone (AQ) and several derivatives, e.g. dimethylBQ, trimethylBQ, duroquinone, bromoNQ, methoxyNQ, methylAQ and dimethylAQ in acetonitrile-water by ascorbate was studied by time-resolved UV-vis spectroscopy using 20 ns laser pulses at 308 nm and continuous 254 nm irradiation. The semiquinone radical (*QH/Q*(-)) is formed after H-atom transfer from ascorbate to the quinone triplet state. The rate constant for quenching is k(q)=(2-9) x 10(9) M(-1) s(-1). Termination of the radicals takes place in the micros-ms range. The results are compared with those initiated by electron transfer from DABCO under similar conditions, where the k(q) values are similar, but the termination of Q*(-) takes place by electron back transfer not yielding hydroquinones. Specific properties of the quinone triplet state, e.g. self-quenching, nucleophilic water addition and the effects of structure are discussed.     

Singlet-triplet excitation fission in light-harvesting complexes of photosynthetic bacteria and in isolated carotenoids

Time-resolved electron paramagnetic resonance was used to study the properties of carotenoid triplet states populated in LH2 light-harvesting complexes of phototrophic bacteria Allochromatium minutissimum, Rhodopseudomonas palustris, and in carotenoid films free of bacteriochlorophyll. The study was performed on purified LH2 preparations not contaminated by reaction centers, and under selective pigment excitation. The obtained results enable a conclusion that the carotenoid triplet states, both in LH2 complexes and films, are populated in the process of homofission of singlet excitation into two triplets, which involves only carotenoid molecules. It is observed that the fission process in magnetic field leads to predominant population of the T₀ spin sublevel of the triplet. One molecular spin sublevel of the triplet is demonstrated to possess an increased probability of intersystem crossing to the ground state, independent of the carotenoid configuration. Pigment composition of the LH2 protein heterodimers is discussed, and a conclusion of the possible presence of two interacting carotenoid molecules is made.     

Reaction of the m-THPC triplet state with the antioxidant Trolox and the anesthetic Propofol: modulation of photosensitization mechanisms relevant to photodynamic therapy?

Antioxidants may affect the outcome of photodynamic therapy (PDT) through the inactivation of reactive oxygen species. Their direct interaction with photosensitizers excited at the triplet state is also worthy of interest. This process is investigated by laser flash photolysis of m-THPC (meso-tetra(3-hydroxyphenyl)chlorin, Foscan) hydroalcoholic solutions added with Trolox (TrOH), a standard antioxidant or Propofol (PfOH, Diprivan(?)), a common anesthetic agent also characterized for its antioxidant properties. Transient UV-visible absorption spectra, kinetics at selected wavelengths and final spectra after extensive laser irradiation show that both compounds react with the m-THPC triplet state, (3)m-THPC, to ultimately restore the photosensitizer in its ground state. For PfOH, this process mainly appears as a single step obeying pseudo-first order kinetics. The bimolecular rate constant for the quenching of (3)m-THPC by PfOH is around 2 × 10(6) M(-1) s(-1), a value increased to some extent by the water content of the solution. A bimolecular reaction between (3)m-THPC and TrOH is observed with a rate constant of similar magnitude and dependence upon water. However, the reaction leads, at least partly, to intermediate species assigned to the TrO˙ radical and the m-THPC anion radical. Within a few ms, these species back react to yield m-THPC in its ground state. A general mechanism involving an intermediate activated complex with some charge transfer character is proposed. Depending on the redox potentials for the oxidation of the antioxidant, this complex evolves predominantly either toward the formation of radicals (TrOH) or back to the photosensitizer ground state (PfOH). Notably, the kinetics data suggest that Propofol may quench (3)m-THPC at concentrations relevant of clinical situation in PDT involving anesthesia.     

The formation and reaction of methyl radicals produced by direct photolysis of aqueous solutions of N-acetyl substituted aliphatic amino acids at 77K.

Photolysis of amino acids, peptides and their derivatives leads to the formation of free radicals in these substances. The electron-spin-resonance spectra obtained directly after irradiation at 77 K are not very well resolved. They are recognizable as the superposition of the spectra of different types of photoproduced radicals. CH3 radicals are formed by U.V. irradiation if methyl groups are present in the molecule. These radicals are easily detectable because of their four line spectrum. In this paper the formation of methyl radicals and their reaction with undamaged molecules of N-acetyl-substituted amino acids in investigated. The number of CH3 radicals present after a 30 min U.V. irradiation is higher if preceding U.V. irradiations and heat treatments are performed. The overall concentration of radicals is reduced only partially during this heat-treatment, while the CH3 radicals decay completely. Other experiments show that the rate of and the yield of CH3 radicals by U.V. irradiation increase with the dose of a preceding gamma-irradiation. The results suggest that there are substances present which are responsible for the higher production rate of methyl radicals after a preceding treatment. It is assumed that radicals are precursors of the fast-formed CH3 radicals     

Photothermal Spectra of Thylakoids Isolated from Cucumber Cotyledons at Various Stages of Greening

The character of interaction between carotenoids (Cars) and chlorophylls (Chls) in thylakoids isolated from cucumber cotyledons at three stages of greening (3, 6, and 24 h of irradiation with 120 µmol m^–2 s^–1) was studied. The shapes of the steady state photoacoustic spectra were changed with the change in time of greening and with the frequency of radiation modulation. The shapes show that changes not only in the contents of various pigments but also in pigment interactions with surrounding occur and that processes of thermal deactivation characterised by different kinetics take place. Slow processes of thermal deactivation are in most cases due to deactivation of triplet states. Long living triplet states are very often engaged in photochemical reactions that can destroy the tissue. Analysis of the time-resolved photothermal spectra shows that at later stage of greening, the chlorophyll (Chl) molecules are better shielded against photo-destruction because Cars more efficiently quench their triplet states. The yield of formation of the pigment triplet states measured by the time resolved photothermal method, always at the same energy absorbed by pigment mixture, declined during sample greening. The decay time of the slow component of pigment thermal deactivation, due predominantly to deactivation of the triplet state of Chl, decreases with the increase of time of greening from 6.2 µs for the 3-h sample to 1.5 µs for the 24 h sample. The energy taken by Cars from Chls is dissipated into heat, therefore the steady state and quick thermal deactivation values increased during the greening process. The Cars/Chls ratio in the thylakoids decreased during greening approximately 2 fold. Hence at a later phase of greening the Cars can quench the triplet states of Chls more efficiently than at an earlier phase of greening.     

Photoinduced electron transfer reaction from N-formyl-L-kynurenine and L-kynurenine to cytochrome C

The reduction of cytochrome c was found in the presence of N-formyl-L-kynurenine (NFK) and L-kynurenine (KN) during irradiation, suggesting electron transfer to cytochrome c. The reaction occurred both under aerobic and anaerobic conditions. In the former case, oxygen molecules may act mainly as a quencher of excited NFK and KN, and superoxide anion produced by electron transfer may partially contribute to the reduction. The reaction proceeded via the excited triplet state of NFK and KN. The actual reductive chemical species might be an intermediate from excited state NFK or KN, which is assumed to be ketyl radical type species.     

Effect of photosensitizers in chemical and biological processes: the MTO mechanism in photodynamic therapy.

It is suggested that in the course of the application of photosensitizers irradiated for cancer therapy a real competition occurs between the singlet oxygen mediated effects and the interactions between the triplet photosensitizers and doublet radicals formed in tumor cells while processes between photosensitizer radicals and molecules are negligible. This hypothesis is strongly supported by experimental findings and reveals a novel approach for the synthesis of new photosensitizers.     

Origin of the heterogeneous distribution of the yield of guanyl radical in UV laser photolyzed DNA

Oxidative guanine lesions were analyzed, at the nucleotide level, within DNA exposed to nanosecond ultraviolet (266 nm) laser pulses of variable intensity (0.002-0.1 J/cm(2)). Experiments were carried out, at room temperature, in TE buffer (20 mM Tris-HCl, pH 7.5; 1 mM EDTA) containing 35 mM NaCl, on 5'-end radioactively labeled double-stranded and single-stranded oligomer DNA at a size of 33-37 nucleobases. Lesions were analyzed on polyacrylamide gel electrophoresis by taking advantage of the specific removal of 8-oxodG from DNA by the formamidopyrimidine DNA glycosylase (Fpg protein) and of the differential sensitivity of 8-oxodG and oxazolone to piperidine. The quantum yields of lesions at individual sites, determined from the normalized intensities of bands, were plotted against the irradiation energy levels. Simplified model fitting of the experimental data enabled to evaluate the spectroscopic parameters characterizing excitation and photoionization processes. Results show that the distribution of guanine residues, excited to the lowest triplet state or photoionized, is heterogeneous and depends on the primary and secondary DNA structure. These findings are generalized in terms of excitation energy and charge-migration mediated biphotonic ionization. On the basis of the changes in the yield of the guanyl radical resulting from local helical perturbations in the DNA pi-stack, it can be assessed that the distance range of migration is <6-8 bp.     

Effects of gamma radiation on beta-lactoglobulin: oligomerization and aggregation

The conformational changes and aggregation process of beta-lactoglobulin (beta-LG) subjected to gamma irradiation are presented. Beta-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (a(w)) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of beta-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated beta-LG in solid state at different a(w) and dose level was essentially the same as the nonirradiated beta-LG. The scattering data also showed that the irradiation of beta-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of beta-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS-PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric beta-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of beta-LG dimers subunits, while the samples irradiated in the solid state were not modified.     

A study of solvent effects on the phosphorescence properties of flavins.

A combination of zero field triplet state techniques are used to study the excited electronic states of a series of flavin and flavin related molecules both in single crystals and glass matrices. Particular attention is given to the effects of solvent interaction on the triplet state properties of the flavin molecules. The total phosphorescence decay rate constants at 1.4 degrees K are reported for the flavin molecules in polar and nonpolar solvents. The rate constants are then correlated to the degree of solvent interaction. Results indicate possible complex formation between the isoalloxazine and adenine groups in FAD. Finally, the results and possible interpretation on the study of a flavoenzyme, L-amino acid oxidase are presented.     

Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy.

The investigation in this report aimed at providing photophysical evidence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.     

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ～1.9, ～1.5, and 1.1–1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments — carotenoids, Chl b, and phycobilins — were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.     

New approaches for study of mechanisms of bone demineralization due to microgravity

Changes in nano-scale subsystems of rat femurs due to the axial unloading of hindlimbs are studied by means of electron paramagnetic resonance (EPR). After irradiation by 60Co isotopes, the results indicate that weightlessness simulation leads to formation of free radicals in tropocollagen molecules and to a reduction in the amount of CO2 radicals, located on the surface of bioapatite nanocrystals.     

Phototransformation of radicals appearing in the result of peptide decarboxylation

Photoreactions of peptides Ala-Gly-Gly and Gly-Gly-Gly-Gly in the presence of potassium ferricyanide at 77 K were studied by modeling of ESR spectra. There were obtained parameters of ESR spectra of primary radicals of the peptide glycine residue appearing after decarboxylation, and of the secondary acyl radicals. Dependence of the second integrals of the experimental and triplet and singlet ESR spectra on irradiation time within wave lengths 410-510, 330-390 and greater than 530 nm was measured and discussed.     

Photophysical properties of quinones and their interaction with the photosynthetic reaction centre

Photophysical properties of tetramethyl-1,4-benzoquinone (TMBQ) and 2,6-dimethoxy-1,4-benzoquinone (DMOBQ) in solution and their interactions with the photosynthetic reaction centre (RC) isolated from the photosynthetic bacterium Rhodobacter sphaeroides have been investigated in this work. For these two benzoquinone derivatives an efficient ISC process which leads to the population of the lowest triplet state of the molecules upon direct excitation was observed. The presence of RC does not alter the properties of the triplet state of DMOBQ suggesting that interactions are negligible; on the other side RC efficiently quenched the T1 state of TMBQ. The behavior is rationalized in terms of redox potentials of quinones and kinetic characteristics of their transients.     

Kinetics of a collagen-like polypeptide fragmentation after mid-IR free-electron laser ablation

Tissue ablation with mid-infrared irradiation tuned to collagen vibrational modes results in minimal collateral damage. The hypothesis for this effect includes selective scission of protein molecules and excitation of surrounding water molecules, with the scission process currently favored. In this article, we describe the postablation infrared spectral decay kinetics in a model collagen-like peptide (Pro-Pro-Gly)₁₀. We find that the decay is exponential with different decay times for other, simpler dipeptides. Furthermore, we find that collagen-like polypeptides, such as (Pro-Pro-Gly)₁₀, show multiple decay times, indicating multiple scission locations and cross-linking to form longer chain molecules. In combination with data from high-resolution mass spectrometry, we interpret these products to result from the generation of reactive intermediates, such as free radicals, cyanate ions, and isocyanic acid, which can form cross-links and protein adducts. Our results lead to a more complete explanation of the reduced collateral damage resulting from infrared laser irradiation through a mechanism involving cross-linking in which collagen-like molecules form a network of cross-linked fibers.