Triplet Probes Research Articles

Evaluation of Probes to Measure Oxidizing Organic Triplet Excited States in Aerosol Liquid Water.

Oxidizing triplet excited states of organic matter (3C*) drive numerous reactions in fog/cloud drops and aerosol liquid water (ALW). Quantifying oxidizing triplet concentrations in ALW is difficult because 3C* probe loss can be inhibited by the high levels of dissolved organic matter (DOM) and copper in particle water, leading to an underestimate of triplet concentrations. In addition, illuminated ALW contains high concentrations of singlet molecular oxygen (1O2*), which can interfere with 3C* probes. Our overarching goal is to find a triplet probe that has low inhibition by DOM and Cu(II) and low sensitivity to 1O2*. To this end, we tested 12 potential probes from a variety of compound classes. Some probes are strongly inhibited by DOM, while others react rapidly with 1O2*. One of the probe candidates, (phenylthiol)acetic acid (PTA), seems well suited for ALW conditions, with mild inhibition and fast rate constants with triplets, but it also has weaknesses, including a pH-dependent reactivity. We evaluated the performance of both PTA and syringol (SYR) as triplet probes in aqueous extracts of particulate matter. While PTA is less sensitive to inhibition than SYR, it results in lower triplet concentrations, possibly because it is less reactive with weakly oxidizing triplets.

Estimation of membrane activity of water-soluble polysubstituted fullerene derivatives by luminescence methods

Patterns of the interaction between water-soluble polysubstituted fullerene derivatives (PFDs) and the lipid bilayer of phosphatidylcholine liposomes were investigated by applying triplet and fluorescent probes. Objective quantitative criteria have been proposed for the evaluation of membranotropic action of chemical substances, notably, fullerene derivatives that quench fluorescent probes with different localizations within the membrane. Thus, the defined criteria are the rate constants for the quenching of the fluorescence of triplet probes and the equilibrium constants for PFD-probe complexes, which characterize their stability. The localization of PFDs in the membrane was determined by comparing rate constants for the quenching of eosin phosphorescence and equilibrium constants for PFD-chromophore complexes. In addition, the efficiency of the interaction of PFDs with various sites of the phospholipid membrane has been seen to depend on the charge of addends that are attached to the polysubstituted derivatives.

Luminescent techniques in investigation of the biological properties of fullerene-based hybrid nanostructures

It has been shown that amino acid derivatives of C60 fullerene (ADFs) are effective quenchers of phosphorescence of the triplet probes eosin and erythrosine. The rate constants of erythrosine phosphorescence quenching by ADFs in aqueous solutions and model membranes have been determined. Using the triplet probe technique, the ability of ADFs to be transported across the lipid bilayer into the inner space of liposomes have been revealed. By monitoring a change in the fluorescence intensity of a luminescent hybrid structure based on C60-proline and eosin, the distribution of the hybrid structure in animal organs was established. The stability of hybrid structures subjected to various chemical and biological impacts was studied by recording the fluorescence of a Gd@C82-based hybrid structure.

Study of rare encounters in a membrane using quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals

Measurements of active encounters between molecules in native membranes containing ingredients, including proteins, are of prime importance. To estimate rare encounters in a high range of rate constants (rate coefficients) and distances between interacting molecules in membranes, a cascade of photochemical reactions for molecules diffusing in multilamellar liposomes was investigated. The sensitised cascade triplet cis-trans photoisomerisation of the excited stilbene involves the use of a triplet sensitiser (Erythrosin B), a photochrome stilbene-derivative probe (4-dimethylamino-4'-aminostilbene) exhibiting the phenomenon of trans-cis photoisomerisation, and nitroxide radicals (5-doxyl stearic acid) to quench the excited triplet state of the sensitiser. Measurement of the phosphorescence lifetime of Erythrosin B and the fluorescence enhancement of the stilbene-derivative photochrome probe, at various concentrations of the nitroxide probe, made it possible to calculate the quenching rate constant k(q)= 1.1 x 10(15) cm2 M(-1) s(-1) and the rate constant of the triplet-triplet energy transfer between the sensitiser and stilbene probe k(T)= 1.0 x 10(12) cm2 M(-1) s(-1). These values, together with the data on diffusion rate constant, obtained by methods utilising various theoretical characteristic times of about seven orders of magnitude and the experimental rate constants of about five orders of magnitude, were found to be in good agreement with the advanced theory of diffusion-controlled reactions in two dimensions. Because the characteristic time of the proposed cascade method is relatively large (0.1 s), it is possible to follow rare collisions between molecules and free radicals in model and biological membranes with a very sensitive fluorescence spectroscopy technique, using a relatively low concentration of probes.

A fluorescence-based method for direct measurement of submicrosecond intramolecular contact formation in biopolymers: an exploratory study with polypeptides.

A fluorescent amino acid derivative (Fmoc-DBO) has been synthesized, which contains 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a small, hydrophilic fluorophore with an extremely long fluorescence lifetime (325 ns in H2O and 505 ns in D2O under air). Polypeptides containing both the DBO residue and an efficient fluorescence quencher allow the measurement of rate constants for intramolecular end-to-end contact formation. Bimolecular quenching experiments indicated that Trp, Cys, Met, and Tyr are efficient quenchers of DBO (k(q) = 20, 5.1, 4.5, and 3.6 x 10(8) M(-1) x s(-1) in D2O), while the other amino acids are inefficient. The quenching by Trp, which was selected as an intrinsic quencher, is presumed to involve exciplex-induced deactivation. Flexible, structureless polypeptides, Trp-(Gly-Ser)n-DBO-NH2, were prepared by standard solid-phase synthesis, and the rates of contact formation were measured through the intramolecular fluorescence quenching of DBO by Trp with time-correlated single-photon counting, laser flash photolysis, and steady-state fluorometry. Rate constants of 4.1, 6.8, 4.9, 3.1, 2.0, and 1.1 x 10(7) s(-1) for n = 0, 1, 2, 4, 6, and 10 were obtained. Noteworthy was the relatively slow quenching for the shortest peptide (n = 0). The kinetic data are in agreement with recent transient absorption studies of triplet probes for related peptides, but the rate constants are significantly larger. In contrast to the flexible structureless Gly-Ser polypeptides, the polyproline Trp-Pro4-DBO-NH2 showed insignificant fluorescence quenching, suggesting that a high polypeptide flexibility and the possibility of probe-quencher contact is essential to induce quenching. Advantages of the new fluorescence-based method for measuring contact formation rates in biopolymers include high accuracy, fast time range (100 ps-1 micros), and the possibility to perform measurements in water under air.

Substituted stilbenes: a new view on well-known systems: New applications in chemistry and biophysics

In this review, we present new areas of application of stilbene compounds in chemistry and biophysics. The first is the application of the linear free-energy relationships (LFER) to photochemistry of substituted stilbenes. The second is the development of the fluorescence-photochrome technique for investigation of different dynamical processes in biological membranes and surface systems. The various time-scale processes occurring with the stilbene molecules after irradiation were found to exhibit different sensitivity to intramolecular donor–acceptor effects of substituents and medium polarity. The sensitivity is quantitatively characterised by ρ-constant of the linear Hammett-like relationships. The obtained experimental data on LFER has independently confirmed the Saltiel mechanism of the photoisomerisation and established the quantitative basis for detailed mechanistic analysis of the different stages of this important photochemical reaction. The measurements of the direct and sensitised trans– cis and cis– trans photoisomerisation allowed investigation the rotational and translational diffusion of the stilbene labels and their aromatic moieties labelled proteins in a wide temporal range. The cascade photochemical system based on the combination of the stilbene, triplet and nitroxide-spin probes gained an additional advantage to measure the rate constant of the triplet state quenching and to estimate the local concentration of stable radicals. High sensitivity, validity and relatively low cost of conventional fluorescence facilities are the main advantages of these fluorescent-photochrome methods.

Triplet state solvation dynamics: Basics and applications

Applying solvation dynamics experiments to viscous liquids or glassy materials near their glass transition involves long lived triplet probes, whose time dependent phosphorescence signals depend upon the local dipolar orientational dynamics, mechanical responses, and polarities. The current understanding of experimental results regarding steady state and time dependent optical line shapes and positions is reviewed with emphasis on the relation to the macroscopic dielectric properties. Several applications are discussed in detail, where advantage is taken of the spatially local instead of ensemble averaging character of this technique. These examples include studies of dynamical heterogeneity, rotational solute/solvent coupling, secondary relaxations in the glassy state, as well as confinement and interfacial effects.

Labelled trinucleotides as quantitative probes to identify Bacillus spp. using fluorescent in situ hybridization

The number of nucleotide triplet repeats in 16 S rRNA sequences can be used for detection and identification of bacteria. Labelled TTT, GGG and ATA triplets were hybridized to the ribonucleic acid of Bacillus subtilis and Bacillus fusiformis whole-cells and the number of such triplets was quantified by synchronous fluorescence spectrometry. Each species was distinctly identified by specific ratios of labelled TTT, GGG and ATA triplets as well as characteristic fluorescence spectra. Notwithstanding the absence of intrinsec specificity, fluorescein-conjugated nucleotide triplet probes appear to be a useful tool for fluorescent spectrometric identification of micro-organisms through the quantitation of trinucleotide repeats.

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. A novel labeling method in study of membranes and surface systems.

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.

Using the Decay of Incorporated Photoexcited Triplet Probes to Study Unilamellar Phospholipid Bilayer Membranes

The decay kinetics of triplet excited probes (meso-tetraphenylporphyrin or anthracene) in unilamellar phospholipid bilayer membranes was studied using nanosecond laser-induced transient absorption ...

Quenching of the cascade reaction between triplet and photochrome probes by nitroxide radicals

A cascade of photochemical reactions between the triplet sensitizer Erythrosin B and the photochrome stilbene-derivative probe exhibiting the phenomenon of cis–trans photoisomerization has been investigated. The cascade triplet cis–trans photoisomerization of the excited stilbene chromophore is sensitized with the excited triplet Erythrosin B and depressed with nitroxide radicals quenching the excited triplet state of sensitizer. The rate of the cascade photoisomerization is sensitive to the molecular dynamics of media and radicals' concentration. The proposed method allows to measure the product of quenching rate constant and the sensitizer's triplet lifetime k q × τ ph. Calibration of the `triple' system permits the quantitative detection of the nitroxide radicals in the vicinity of the stilbene-derivative photochrome and sensitizing triplet probes. The experiment is carried out with the constant-illumination fluorescence technique. Sensitivity of the method is close to 10 −12 mole of fluorescence molecules per sample and the minimal local concentration of nitroxide radicals being detected is about 10 −5 M. This method allows to investigate any chemical, biological objects and surface processes of microscopic scale when the minimal volume is about 10 −3 μl.

A Comparative Study of Rotational Relaxations of an Eosin-protein Complex Using Delayed Fluorescence and Phosphorescence

Abstract The slow rotational diffusion of a macromolecule bovine serum albumin in solution has been investigated by measuring the decay of dichroism in laser-pulse induced transient emissions from triplet probes covalently bound to the protein. A comparison of the anisotropy parameters of phosphorescence and delayed fluorescence from eosin-bound bovine serum albumin is described. The results indicate that the measurement of delayed fluorescence is often the preferable option for investigating the rotational diffusion of the eosin-bound macromolecules.

Emission of triplet probes in polymer matrices; monoketones of benzophenone type

Emission spectra of monoketones of the benzophenone type were investigated in amorphous, glassy and microcrystalline polymer matrices at 77 K. The polarity of matrices of amorphous glassy polymers exerts a distinct influence on the vibrational resolution of emission spectra. Improvement in resolution of the emission spectra was achieved in non-polar microcrystalline matrices, such as polyethylene or isotactic polypropylene, for benzophenone derivatives substituted at position 4. The effect of the polarity on the life-time of monoketones of this type was smaller than on the vibrational resolution. Emission originating in the n, π * triplet state decayed mono-exponentially in the range from 1 to 10 msec in a polymer matrix. The origin of the emission with decay longer than 10 msec was not determined unequivocally and the decay was more complex.

Excited triplet states as probes in organized systems. An overview of recent results

Triplet molecules have been employed as probes in organized systems in solution and in the solid state. This article present an account of our recent work in this area. In particular, these probes are very useful when studying processes involving relatively slow mobility, where the short lifetime of singlet states would make fluorescence a poor monitoring technique. Among the molecules examined, xanthone and 4,4′-dimethylbenzophenone have proven useful in cyclodextrin complexes, leading to information on polarity, mobility and excited state reactivity. Other triplet probes based on the β-phenylpropiophenone moiety yield information on intracavity mobility, inclusion geometries and generally on the importance of steric factors in the formation and stability of cyclodextrin complexes. Finally, delayed fluorescence form pyrene derivatives has been employed as a probe for mobility and exchange in reversed micelles.

Rotational dynamics of type I Fc epsilon receptors on individually-selected rat mast cells studied by polarized fluorescence depletion

We report the first application of polarized fluorescence depletion (PFD), a technique which combines the sensitivity of fluorescence detection with the long lifetimes of triplet probes, to the measurement of membrane protein rotational diffusion on individually selected, intact mammalian cells. We have examined the rotation of type I Fc epsilon receptors (Fc epsilon RI) on rat mucosal mast cells of the RBL-2H3 line in their resting monomeric and differently oligomerized states using as probes IgE and three monoclonal antibodies (mAbs; H10, J17, and F4) specific for the Fc epsilon RI. PFD experiments using eosin (EITC)-IgE show that individual Fc epsilon RI on cells have a rotational correlation time (RCT) at 4 degrees C of 79 +/- 4 microseconds. Similarly, Fc epsilon RI-bound EITC-Fab fragments of the J17 Fc epsilon RI-specific mAb exhibit an RCT of 76 +/- 6 microseconds. These values agree with previous measurements of Fc epsilon RI-bound IgE rotation by time-resolved phosphorescence anisotropy methods. Receptor-bound EITC-conjugated divalent J17 antibody exhibits an increased RCT of 140 +/- 6 microseconds. This is consistent with the ability of this mAb to form substantial amounts of Fc epsilon RI dimers on these cell surfaces. The ratio of limiting to initial anisotropy in these experiments remains constant at about 0.5 from 5 degrees C through 25 degrees C for IgE, Fab, and intact mAb receptor ligands. Extensive cross-linking by second antibody of cell-bound IgE, of intact Fc epsilon RI-specific mAbs or of their Fab fragments, however, produced large fixed anisotropies demonstrating, under these conditions, receptor immobilization in large aggregates. PFD using the mAbs H10 and F4 as receptor probes yielded values for triplet lifetimes, RCT values, and anisotropy parameters essentially indistinguishable from those obtained with the mAb J17 clone. Possible explanations for these observations are discussed.

Oxygen gradients in mitochondria examined with delayed luminescence from excited-state triplet probes

Phosphorescent probes are described that are quenchable by dioxygen and that partition into membranes. These probes are derivatives of porphyrin, in which the central metal, either zinc or palladium, induces intersystem crossing to enhance the triplet yield. The location of the probe in a suspension of membranes depends upon the charge distribution of side groups on the porphyrins. Probes that partition into the membrane are sensitive to phase transitions in lecithin artificial membranes. In the mitochondria these membrane probes are within transfer distance from tryptophans in membrane proteins. Although absolute concentrations of oxygen in membranes cannot be determined by this method, by comparing the oxygen dependence of a probe in the aqueous phase with that in the membrane phase under actively respiring and inhibited conditions, it is possible to examine the extent of O2 depletion at the mitochondrial surface. We show that at oxygen tensions of 0.2 microM and higher these gradients are insignificant at usual oxygen consumption rates of mitochondria.

Structure and molecular dynamics of metalloenzymes studied by physical label methods

The physical principles of a new approach to the investigation of enzymes are discussed, including relaxation versions of the spin label method and also the methods of triplet, Mössbauer, electron-scattering, photochrome and radical pair labels and probes. The combined approach is demonstrated with examples from studies of the structure, dynamics and reaction mechanism of the metallo-enzyme nitrogenase, photosynthetic reaction centers, cytochrome P-450, etc. Some general concepts which emerge in connection with these data (the multielectron mechanism of redox reactions, long-distance electron transfer in proteins, the chemical mechanism of bioenergetic coupling, the cascade mechanism of charge photoseparation, and the crystalline-amorphous dynamical model of proteins) are considered.

Biomembrane structure and function: recent studies and new techniques.

The consensus view of biomembrane structure is outlined. The present model is built upon a fluid lipid matrix, usually two molecules in length, into which the proteins are embedded. The lipid bilayer organization is discussed, such as their phase transition and fluid character and the effect of cholesterol upon the chain organization. The non-lamellar arrangement which some lipids adopt is described. The use of new physical techniques for obtaining information about the structure and dynamics of membrane proteins are described. These techniques include electron diffraction, electron microscopy and FTIR spectroscopy. Models of the structures of the Ca2+-ATPase and the glucose transporter from erythrocytes are shown, indicating the putative helices embedded in the lipid bilayer and the groups of amino acids in the aqueous environment. These models are based upon biochemical methods to obtain amino acid sequences using DNA cloning techniques. Finally, an experimental method using triplet probes is described for the study of the rotational dynamics of membrane proteins. Labelled monoclonal antibodies for studying the dynamics of the glucose transporter have been used.

Haem localization in haemoproteins by spin and triplet tools

The rate constants of efficient exchange interaction (kex) of spin-labelled lysozyme and the triplet probes perylene, eosine and anthracene butanoic acid with the haemoproteins were measured in microsomes and in solution by electron paramagnetic resonance and by the registration of delayed annihilation fluorescence. Constants of efficient exchange interactions with the haem groups of myoglobin, haemoglobin, cytochrome c and b5 are 3-22 X 10(7) M-1 s-1 in solution. The experiments with membrane-bound cytochrome P-450 revealed no exchange interactions with the probes located in solution or in the membrane. These results can be accounted for by the deeper incorporation of cytochrome P-450 haem into the protein globule as compared to the other haemoprotein haems studied.

Methods of physical labels - a combined approach to the study of microstructure and dynamics in biological systems

The physical principles of several new approaches to the investigation of biological and model systems are discussed, including versions of the spin label method based on relaxation measurements, and also the methods of triplet, Mössbauer, electron-scattering and radical-pair labels and probes. It is shown that all these methods make it possible (1) to investigate molecular mobility of the medium with the correlation frequencies τ c −1 = 10 −3 − 10 11 s −1, (2) to measure the rate constants of collisions K tr = 10 3 − 10 10 M −1 s −1, (3) to measure the distance between centers up to 100 Å and finally, (4) to evaluate the immersion depths of paramagnetic and chromophore centers in matrices up to 40 Å. The combined approach is demonstrated with examples from studies of the structure of nitrogenase, the reaction centers of photosynthetic bacteria and sarcoplasmic reticulum membranes and from studies of the molecular dynamics of proteins and membranes.