Excited-State Dynamics of Fully Reduced Flavins and Flavoenzymes Studied at Subpicosecond Time Resolution

The photophysics of the fully reduced states of a number of flavins (flavin mononucleotide, flavin adenine dinucleotide and 3-N-methyllumiflavin) and flavoenzymes (glucose oxidase from Aspergillus niger and the flavodehydrogenase component isolated from flavocytochrome b₂) was studied using subpicosecond laser excitation at Λ. = 312 nm. The prompt transient absorption spectra (measured from 400 to 850 nm) were all closely similar in the case of the free flavins in aqueous solution. The decay of the transient absorbance obeyed biexponential kinetics with a fast component of lifetime ranging from 4 to 130 ps and a slower phase with a lifetime above 1 ns. The spectral structure changed appreciably during the rapid decay phase. In contrast, in the case of the enzymes only a very slight decay was apparent over the probed time interval (1 ns) and the shape of the spectrum remained unchanged. It is proposed that the two transient spectra appearing in the free flavins correspond to two conformations differing by their degree of nonplanarity, whereas in the flavoenzymes only one conformation is possible.     

Multisite topographic microfluorometry of intracellular and exogenous fluorochromes.

Abstract. Microspectro fluorometry combined with microinjection of metabolites allows the monitoring of pathways regulating the energy metabolism, biosynthetic or metabolizing activity of the intact living cell. The topographic scan of ? 100–150 adjacent regions within a cell or the spectral scan of fluorescence from a single cell region is completed within 60 ms. The in situ activity of intracellular enzymes and substrate concentration vs rate relationships are derived from the kinetic analysis of transients (e.g. NAD(P) ? NAD(P)H) elicited by sequential injections with increasing doses of substrate (e.g. glucose-6-P, 6-phosphogluconate). A method of approximation is used to predict the non-linear behavior of whole biochemical systems (e.g. the sum of reactions involved in NAD(P) reduction-reoxidation transients). Approximation to power law kinetics is validated by prediction of system behavior over a 10–100 fold variation in the input concentration of substrate. The NAD-dependent glycolytic chain (faster rise kinetics) and NADPH-dependent shunt pathway (slower rise kinetics) are triggered optionally, by enhanced demand for degradation, synthesis or metabo-lization of exogenous compounds (hydrocarbons, aflatoxins). Compartmentation of NAD(P) pathways is suggested by differences in the reoxidation of cytoplasmic vs nuclear NAD(P)H. The intercellular transfer of molecules can be evaluated by microinjection of fluorescent tracers. The difference spectra ΔI_F due to microinjection of substrate are resolved in terms of bound vs free NAD(P)H (with differentiation of other endogenous and exogenous fluorochromes). On the basis of the above, a tentative scheme of intra- and intercellular dynamics can be developed.     

EXCITED STATES OF SIX-MEMBERED N-HETEROCYCLES. FLUORESCENCE, PHOSPHORESCENCE AND ACID—BASE EQUILIBRIA OF ELECTRONICALLY EXCITED PHENAZINE

Abstract— An account of a systematic study of the acid-base equilibria of phenazine in the two lowest excited (π,π) states is presented. Pure electronic levels of the free base and of both its protonated forms have been located by spectroscopic methods. Fluorescence, phosphorescence and corresponding absorption spectra have been measured. The O-O energies of the free base, of the singly-protonated species and of the doubly protonated form in the lowest triplet state (³L_α(π, π)) are: 15, 475 cm^-1, 14, 175 cm^-1 and about 9300cm^-1, respectively. This last value has been estimated from the experimentally determined S-T splitting in the other two forms. Corresponding energies of the lowest singlet state (^IL_α(π,π)) are: 23,500 cm^-1, 21,250cm^-1 and 17,300 cm^-1. The fluorescence of the free base has been found in polar as well as in non-polar solvents and has been checked by the fluorescence excitation spectrum. Fluorescence quantum yields for the free base have been measured: 8.6 times 10^-4 and 3.0 × 10^-5 in ethanol and hexane solutions, respectively. Emission in ethanol has been ascribed to (π,π), that in hexane —to (π, π). fluorescence. The changes of pK_α's under excitation, calculated from the Forster's cycle, are equal: δpK_a¹=+2.8±0.3; δpK_a¹¹?+10±1.5 in the lowest (π, π) triplet state and δpK_a¹=+4.8±0.5; δpK_a¹¹=+8.4 ± 0.5 in the lowest (π,π) singlet state. The δpK_a¹¹ in the triplet state is at least as high as that in the ¹L_a(π, π) state. P P P calculations of the electronic levels and of the molecular diagrams have been performed. The energies obtained exceed experimental values by not more than 0.5 eV. An increase of the net charge on nitrogen δp under excitation has been found to be +50, +70 and +19 per cent in the ¹L_a, ¹L_b and ³L_a states, respectively. A good correlation has been found between δpK_a¹ and δp in both excited states, which have been studied experimentally.     

Ion Selective Electrode Determination of Free Versus Total Fluoride Ion in Simple and Fluoroligand Coordinated Hexafluoropnictate (PnF<Subscript>6</Subscript><Superscript>−</Superscript>, Pn = P,As, Sb,Bi) Salts

Interpretation of the results of determinations of free fluoride (F_f⁻) and total fluoride (F_t⁻) obtained with fluoride ISE while conducting elemental chemical analysis of bulk material of newly synthesized inorganic fluoride compounds is of crucial importance for the purpose of determination of purity and stoichiometry of these compounds. Knowledge of the properties and behavior of these compounds in aqueous media is therefore essential. Observations are presented on the determinations of the amounts of F_t⁻ and F_f⁻ in fluorinated compounds, in the particular hexafluoropnictate salts (PnF₆⁻, Pn = P, As, Sb, Bi) as found in aqueous media and in some compounds with XeF₂, AsF₃ ligands. A critical look at the determined amounts of F_f⁻, F_t⁻and calculated amounts of bound fluoride (F_b⁻) is provided.     

Very-low-pressure pyrolysis of nitroso- and pentafluoronitrosobenzene C?NO bond dissociation energies

The high-pressure absolute rate constants for the decomposition of nitrosobenzene and pentafluoronitrosobenzene were determined using the very-low-pressure pyrolysis (VLPP) technique. Bond dissociation energies of DH⁰(C₆H₅? NO) = 51.5 ± 1 kcal/mole and DH⁰ (C₆F₅? NO) = 50.5 ± 1 kcal/mole could be deduced if the radical combination rate constant is set at log k_r(M^?1·sec^?1) = 10.0 ± 0.5 for both systems and the activation energy for combination is taken as 0 kcal/mole at 298°K. δH_f⁰(C₆H₅NO), δH_f⁰(C₆F₅NO), and δH_f⁰(C₆F₅) could be estimated from our kinetic data and group additivity. The values are 48.1 ± 1, –160 ± 2, and – 130.9 ± 2 kcal/mole, respectively. C–X bond dissociation energies of several perfluorinated phenyl compounds, DH⁰(C₆F₅–X), were obtained from the reported values of δH_f⁰(C₆F₅X) and our estimated δH_f⁰(C₆F₅) [X = H, CH₃, NO, Cl, F, CF₃, I, and OH].     

Synthesis and Fluorescence Properties of Pyrimidine‐Based Diboron Complexes with Donor–π–Acceptor Structures

Pyrimidine‐based diboron complexes bearing β‐iminoenolate ligands and phenyl groups as bulky substituents on the boron atoms were synthesized as novel fluorescent dyes, and their fluorescence properties were investigated in solution and in the solid state. The diboron complexes with donor–π–acceptor structures showed positive solvatochromism in the fluorescence spectra. The cyano derivative exhibited the most dramatic redshift of the fluorescence maximum F_max with increasing solvent polarity (from 551 nm in n‐hexane to 710 nm in acetonitrile). The diboron complexes showed solid‐state fluorescence in the range of 578–706 nm with fluorescence quantum yields of 0.06–0.28. Additionally, the trifluoromethyl derivative exhibited solvent‐inclusion solid‐state fluorescence. The trifluoromethyl derivative formed toluene‐inclusion and ethyl acetate‐inclusion crystals. The toluene‐inclusion crystal (F_max=668 nm, Φ_f=0.16) showed a blueshifted F_max and higher Φ_f value compared to the original trifluoromethyl derivative (F_max=694 nm, Φ_f=0.08) in the solid state. On the other hand, the F_max (709 nm) and Φ_f (0.04) values of the ethyl acetate‐inclusion crystal were redshifted and lower, respectively.     

Laser-induced luminescence from an excited HgNH3 complex

When a Hg/NH₃ mixture is irradiated at 260 nm or longer wavelengths luminescence appears around 340 nm. The luminescence originates from an excited HgNH³ complex formed through a bound—free transition. Both the luminescence excitation spectrum and the dispersed spectrum have been analysed in terms of postulated potentials for the ground and excited-state HgNH₃ complexes.     

Der chemische Transport von FeP2 und FeP4 mit lod

Chemical Transport of FeP₂ and FeP₄ with Iodine Experiments on the chemical transport of FeP₂ and FeP₄ with iodine are discussed, considering the gaseous molecules I₁, I₂, FeI₂, Fe₂I₄, FeI₃, Fe₂I₆, PI₃, P₂I₄, P₄, P₂, and P. Thermodynamic calculations give δH°(298) = 56.322 kcal and ΔS°(298) = 39.5 cal/K for the reaction FeP_2,f + I₂ = FeI₂ + 0.5 P₄ and δG°(923) = 35.8 kcal for the reaction FeP_4,f + I₂ = FeI₂ + P₄.     

Controllable Intramolecular Motions That Generate Fluorescent Signals for a Metal Scorpionate Complex

Simply by changing the pH value, the side chain of complex 1 can be reversibly moved between two positions. Coordination to the metal center through the nitrogen atom of the side chain at moderate pH values is accompanied by a decrease in fluorescence intensity (from I_F=100% to I_F=60%). A further decrease is observed upon deprotonation of the bound water molecule at higher pH (I_F≤2%). Therefore, 1 can be seen as a molecular three-position switch.     

Microwave synthesis and characterization of europium complexes with cinnamic acid and phenanthroline

We report here the synthesis and characterization of a host of Eu(Phen)L₃ with cinnamic acid (C₆H₅CH = CHCOOH, HL) and phenanthroline (Phen), and employing microwave radiation, where the microwave radiation is used just for the uniform heating of the reaction mixture. Its IR absorption spectra, scanning electron microscopy (SEM), and fluorescence spectra were studied. The results show that the particles of Eu(Phen)L₃ phosphors are basically spherical in shape, with good dispersing. The mean particle size is 1–2 μm. The excitation spectrum is a broad band and the main peak is at 320.0 nm. Moreover, excitation peak at 396.0 nm was found in the excitation spectrum. The emission spectrum shows that Eu(Phen)L₃ has narrow emission peaks. The emission peaks are ascribed to Eu³⁺ ions transition from ⁵ D _J (J = 0) to ⁷ F _J (J = 1, 2, 4). However, the strongest main emission peak locates at 614.0 nm, which corresponds to the electric dipole transition of Eu³⁺(⁵ D ₀ → ⁷ F ₂) The article is published in the original.     

KINETIC CHARACTERIZATION OF THE REDOX COMPONENTS IN SOLUBILIZED MEMBRANES FROM PORCINE NEUTROPHILS: REDUCTION WITH DITHIONITE AND PHOTOEXCITED NAD(P)H

Abstract— Cytochrome b₅₅₈ in solubilized membranes prepared from porcine neutrophils was reduced by dithionite with a second-order rate constant of 2.5 times 10⁶ M^-1 s^-1 at pH 7.4 and 20°C accompanied by spectral changes with peaks at 428 nm and 560 nm and isosbestic points at 420 and 441 nm. When an anaerobic mixture of solubilized membranes and NAD(P)H was exposed to a white light, cytochrome b₅₅₈ was reduced biphasically but with almost the same spectral profiles as in the dithionite reduction. Thus, participation of redox component(s) of unknown nature in the photochemical reduction was suggested. The NAD(P)- radical generated by photoexcitation of NAD(P)H with a 355 nm laser pulse under anaerobic conditions also reduced cytochrome b₅₅₈ with a high rate constant of 4.3 times 10⁸M^-1 s^-1 at pH 7.4 and 20°C. The reduction of cytochrome b₅₅₈ accompanied a simultaneous reduction of a component having an absorption band around 420 nm, suggesting participation of an iron-sulfur (Fe-S) cluster. The cytochrome b₅₅₈ reduction was followed by its reoxidation by another component with an apparent second-order rate constant of 6.5 times 10⁵M^-1 s^-1. During the reoxidation, the Fe-S-like component remained in the reduced state, and thus its role other than as electron mediator in neutrophils NADPH oxidase is suggested. Not only the rate constant but also the extent of cytochrome b₅₅₈ reoxidation decreased as the same reaction mixture was exposed to the laser pulse repeatedly. This result clearly indicates that an electron accumulates in this electron-accepting component designated tentatively as the ω component.     

Ion-pair states in the complexes of iodine molecule with rare gases

The luminescence excitation spectra and the luminescence spectra of the I₂ + Rg (Rg = He, Ar, Xe; p _Rg = 2–20 Torr) mixtures measured at room temperature by the method of double optical resonance in the spectral range corresponding to the population of the I₂(f0 _g ⁺, v _f = 8.9) levels and in its vicinity are analyzed in this work. The experimental data and their interpretation, according to which these spectra can be explained by the energy transfer in the intermediate I₂(B0 _u ⁺) and final I₂(f0 _g ⁺) states of the free iodine molecule rather than by the optical population, luminescence, and predissociation of the ion-pair RgI₂(IP) complexes, are discussed. It is shown that these data can be explained only with account taken of the optical population of the RgI₂(IP) complexes. Original Russian Text ? M.E. Akopyan, S.S. Lukashov, S.A. Poretsky, A.M. Pravilov, 2009, published in Zhurnal Fizicheskoi Khimii, 2009, Vol. 83, No. 1, pp. 132–141.     

Laser excited fluorescence of I2

The fluorescence spectrum of iodine was investigated from 200 to 520 nm in the presence and absence of buffer gases following excitation of I₂ with 193 nm photons. The pressure dependence of the fluorescence and tentative transition assignments for one new and several less well-known I₂ emission bands are discussed.     

Rigid cage conversion of a perfluoroalkyl iodide repulsive electronic state into a “bound” electronic state

260–300 nm pulsed “photodissociation” of perfluoroalkyl iodide guests in rare gas and N₂ lattices produces an unstructured structured 850–1050 nm emission. The excitation spectrum is continuous, and the emission maximum and lifetime vary from host to host. It is proposed that this emission occurs from a vibrationally relaxed, stationary “bound” electronic state in which the perfluoroalkyl radical and the I^*(²P_{$\text{1}\text{2}$}) atom are held together by constrictive cage forces.     

PICOSECOND FLUORESCENCE KINETICS and ENERGY TRANSFER IN THE ANTENNA CHLOROPHYLLS OF GREEN ALGAE*

Single-photon timing measurements on flowing samples of Chlorella vulgaris and Chlamydomonas reinhardtii at low excitation intensities at room temperature indicate two main kinetic components of the fluorescence at open reaction centers (F₀) of photosystem II with lifetimes of approx. 130 and 500 ps and relative yields of about 30 and 70%. Closing the reaction centers progressively by preincubation of the algae with increasing concentrations of 3-(3′,4′-dichlorophenyl)-l,l-dimethylurea (DCMU) and hydroxylamine gave rise to a slow component with a lifetime increasing from 1.4 to 2.2 ns (F_max) The yield of the slow component increased to 65-68% of the total fluorescence yield in parallel to a decrease in the yield of the fast component to a value close to zero at the f_max-level. The 130 ps lifetime of the fast component remained unchanged. The middle component showed an increase of its lifetime from 500 to 1100 ps and of its yield by a factor of 1.5. Spacing of the ps laser pulses by 12 μs allowed us to resolve a new long-lived fluorescence component of very small amplitude which is ascribed to a small amount of chlorophyll not connected to functional antennae. The opposite dependence of the yield of the fast and the slow component on the state of the reaction centers at almost constant lifetimes is consistent with a mechanism of energy conversion in largely separately functioning photosystem II units. Yields and lifetimes of these two components are in agreement with the high quantum yield of photosynthesis. The lower lifetime limit of 1.4 ns of the slow component is assigned to the average transfer time of an excited state from a closed to a neighboring open reaction center and the increase in the lifetime to 2.2 ns is evidence for a limited energy transfer between photosystems II. Relative effects of changing the excitation wavelength from 630 to 652 nm on the relative fluorescence yields of the kinetic components were studied at the fluorescence wavelengths 682, 703 and 730 nm. Our data indicate that (i) the middle component has its fluorescence maximum at shorter wavelength than the fast component and (ii) that the antennae chlorophylls giving rise to the middle component are preferentially excited by 652 nm light. It is concluded that the middle component originates from the light-harvesting chlorophyll alb protein complexes and the major portion of the fast component from the chlorophyll a antennae of open photosystem II reaction centers.     

The Photoreceptor for Phototaxis in the Photosynthetic Flagellate Euglena gracilis

The unicellular flagellate Euglena gracilis shows positive phototaxis at low fluence rates (≤10 W m ²) and negative phototaxis at high fluence rates (≥100 W m ²). Currently, retinal or flavins/pterins are discussed as chromo-phores of the photoreceptor. When grown in the presence of 4 mM nicotine, a retinal inhibitor, for several generations, the cells still showed both responses, indicating that retinal is unlikely to be the chromophoric group of the photoreceptor responsible for phototaxis. The native flavin(s) can be substituted by growing the cells in roseo-flavin dissolved in the medium. The absorption spectrum of roseoflavin extends well beyond the action spectrum for phototaxis (up to 600 nm). Excitation at wavelengths >550 nm does not cause phototactic orientation in control cells but causes both positive and negative phototaxis in roseoflavin-grown cells, indicating an uptake and assembly of the chromophore in the photoreceptor complex. The white mutant strain 1224-5/1f, induced by streptomycin treatment, lacks flavins as indicated by fluorescence spectroscopy. The phototaxis-deficient pheno-type cannot be complemented by the addition of external riboflavin. Fluorescence spectra of intact paraxonemal bodies (PAB) indicate that both pterins and flavins are involved in photoperception and that the excitation energy is efficiently funneled from the pterins to the flavins. This energy transfer is disrupted by solubilization of the PAB. In intact PAB flavins are not accessible to reducing or oxidizing substances, indicating that they are located inside the structure, while pterins are accessible, so that their localization can be assumed to be on the surface. The results described above are discussed with regard to the potential involvement of flavins and pterins as well as retinal in photoperception.     

NONRECIPROCAL SYNERGISTIC LETHAL INTERACTION BETWEEN 365-nm and 405-nm RADIATION IN WILD TYPE and uvrA STRAINS OF ESCHERICHIA COLI*

Abstract— Lethality by 405-nm radiation in three repair-proficient and two uvrA strains of Escherichia coli that belong to two isogenic series was greatly enhanced by prior exposures to 365-nm radiation at fluences greater than 1 times 10₆Jm_-2. Fluences at 365 nm that yielded a surviving fraction of 0.10 (>1 times 10₆ Jm_-2) in the 5 strains tested resulted in the following 405-nm fluence enhancement factors (FEF, ratio of the 405-nm F₃₇ in the absence of a prior 365-nm irradiation to that in the presence): strain K.12 AB1157 (wild type), 8.7; strain B/r (wild type), 52; strain WP2 (wild type), 25; strain WP2s (uvrA), 13; strain K.12 AB1886 (uvrA), 15. The maximal 405-nm FEF value obtained after a prior 365-nm irradiation at greater fluences was 83 in the wild-type strain B/r. Enhancement of anoxic 405-nm radiation after a prior aerobic 365-nm exposure was not detectable, suggesting that prior aerobic irradiation at 365-nm increased the effects of damage produced at 405 nm by means of an oxygen-dependent process. Single-strand breaks (or alkali-labile bonds) were produced by 405-nm radiation at 3.0 times 10_-5 breaks per 2.5 times 10₉ daltons per Jm_-2 in the polA strain P3478; pyrimidine dimers were not detected by biological assay (photoreactivation) at 405 nm. Although the introduction of different DNA lesions produced by 365- and 405-nm radiations cannot be ruled out, we propose that the strong synergistic effect of 365-nm irradiation on 405-nm lethality is the consequence of pronounced inhibition by 365-nm radiation of components of the DNA-repair systems that can mend or bypass damage produced by 405-nm radiation.     

Sensitization of Tb3+ luminescence with Ce3+ in LaOBr: Tb3+, Ce3+

Luminescence emission and uv-excitation properties of LaOBr: Tb³⁺, LaOBr: Ce³⁺, and LaOBr: Tb³⁺, Ce³⁺ phosphors were studied. The visible emission spectra of La_0.995Tb_0.005OBr consists of⁵D_3,4 →⁷F_3–6 transitions in the wavelength range of 410–630 nm. The excitation of the Tb³⁺ ion gives a broad 4f → 5d transition band at 254 nm and weaker4f → 4f transition lines above 300 nm. The uv-excitation and emission of La_0.995Ce_0.005OBr at 290, 315, 355 (excitation), and 440 nm (emission) originate from transitions between the 4f-ground state and the four crystal field components of the5d²D excited state. The sensitization of Tb³⁺ luminescence in LaOBr with Ce³⁺ at varying concentrations is described and discussed. With increasing Ce³⁺ concentration the ⁵D₃ →⁷F transitions of Tb³⁺ quench totally and the⁵D₄ →⁷F transitions begin to quench gradually. The excitation spectrum of the⁵D₄ →⁷F₅ transition of Tb³⁺ consists of four bands due to Tb³⁺ and Ce³⁺, of which the three Ce³⁺ bands increase in intensity and the Tb³⁺ band decreases as the Ce³⁺ concentration is increased.     

Spectroscopic properties of Nd ions in nano-perovskite CaTiO3

In this work, we present for the first time the spectroscopic properties of perovskite nanocrystals CaTiO₃: Nd³⁺, measured at room and liquid nitrogen temperature. Samples were prepared by the sol–gel method and annealed at 700 and 1000 °C. The concentration of Nd³⁺ ranged from 0.5% to 5%. Average nanocrystallite size primarily depends on the annealed temperature and is about 25 and 50 nm for 700 and 1000 °C, respectively. The absorption, emission and excitation spectra (monitored at 1078 nm) as well as the decay time profile of the emission from the ⁴F_3/2 energy level of Nd³⁺ are obtained. The increasing amount of Nd³⁺ ions decreases the lifetime of the ⁴F_3/2 level and that changes from 146 μs for 0.5% to 50 μs for 5% of Nd³⁺ concentration. The strongest emission was observed at two regions: 1050–1120 nm and 865–930 nm and was assigned to the ⁴F_3/2→⁴I_11/2 and ⁴F_3/2→⁴I_9/2 transitions, respectively. The spectroscopic quality parameter of neodymium in the CaTiO₃ lattice has been calculated from the emission spectra. The influence of luminescent properties depending on the annealing temperature and concentration of neodymium ions is discussed.     

Fluorimetric assay of serum glutamate oxaloacetate transaminase,glutamate pyruvate transaminase and α-hydroxybutyrate dehydrogenàse by solution an

Fluorimetric methods have been developed for the determination of the activity of the enzymes GOT, GPT and α-HBD with Dade Reagent Tablets, both in solution and via a solid-surface method. In each case the rate of disppearance of NADH fluorescence at 455 nm (λ_ex=365 nm) was measured and equated to the concentration of these enzymes in control serum and blood serum. Pads were prepared for HBD assay with reagent films of the sodium salt of α-hydroxy-butyrate and NAD; the fluorescence of the NADH produced on enzymic reaction was measured, and equated to α-HBD content of serum.Linear responses were found in the ranges 2.2–106 I.U. GOT l^-1. 5–106 I.U. GPT l^-1 and 14–278 I.U.α-HBD l^-1; agreement with standard accepted methods for the assay of these enzymes was excellent.