EFFECT OF TRANSMEMBRANE POTENTIAL ON CHARGE PHOTOSEPARATION IN LIPOSOMES

Abstract— An unexpected transmembrane potential effect on the recombination rate of the pheophytin or bacteriopheophytin anion-radicals (dissolved in membrane) and ascorbic ion-radicals (dissolved in aqueous interior) has been established in liposomes. The influence of transmembrane potential on the recombination rate of Ru³⁺ (dissolved in inner volume) and (C₁₈H₃₇)V⁺ or (C₁₄H₂₉)V⁺ (dissolved in membrane) was observed. The potential was created by a potassium concentration gradient between inner and outer volumes of liposomes in the presence of valinomycin. The effect of the potential was considered on the bases of: (1) it was determined by the diffusional drift of the hydrophobic radicals in a radial direction in the membrane, according to the direction of the electric field; (2) the electric field changed the rate constant of the electron transfer, owing to the effects on the free energy and electronic coupling. Our results show the first explanation to be preferable.     

PREPARATION AND PHOTOPHYSICAL STUDIES OF PORPHYRIN-C60 DYADS

Abstract Porphyrin-C₆₀ dyads in which the two chromophores are linked by a bicyclic bridge have been synthesized using the Diels-Alder reaction. The porphyin singlet lifetimes of both the zinc (Pz_n-C₆₀) and free base (P-C₆₀) dyads, determined by time-resolved fluorescence measurements, are ≦17 ps in toluene. This substantial quenching is due to singlet-singlet energy transfer to C₆₀ The lifetime of Pzn-¹C₆₀ is -5 ps in toluene, whereas the singlet lifetime of an appropriate C₆₀ model compound is 1.2 ns. This quenching is attributed to electron transfer to yield P_zn^bull;+-C₆₀^bull;-. In toluene, P-¹C₆₀ is unquenched; the lack of electron transfer is due to unfavorable thermodynamics. In this solvent, a transient state with an absorption maximum at 700 ran and a lifetime of-10 μs was detected using transient absorption methods. This state was quenched by oxygen, and is assigned to the C₆₀ triplet. In the more polar benzonitrile, P-¹C₆₀ underoes photoinduced electron transfer to give P^•+-C₆₀^bull;-. The electron transfer rate constant is −2 × 10¹¹ s⁻¹.     

ELECTRON AND ENERGY TRANSFER IN ILLUMINATED POROUS VYCOR GLASS

Abstract— Porous Vycor glass samples containing adsorbed molecules were illuminated at 77 K by a mercury lamp jacketed by a filter cutting off wavelengths below 250 nm. Oxygen or carbon dioxide on Vycor produces an asymmetric electron paramagnetic resonance (EPR) signal best described as holes trapped in the glass. Methyl bromide produces an identical EPR signal plus four other lines due to methyl radicals. Evidence is presented that the products result from excitonic energy transfer from the Vycor to the adsorbed materials. Triphenylamine (TPA) adsorbed on Vycor can also be photoionized by similar illumination, and the cation radical TPA⁺ can be stabilized at 77 K if an electron acceptor is also adsorbed. Attachment of the photoejected electron by carbon dioxide forms CO₂^-, and that by methyl bromide leads to methyl radicals. The CH₃ radical yield is dependent on the surface separation between the electron donor (TPA) and the acceptor (CH₃Br). By monitoring the relative quantum yield of the methyl radicals as a function of distance separating the TPA and CH₃Br, it is shown that the photoelectron is capable of migrating on the Vycor glass surface.     

Photosensitized reduction of methyl viologen using eosin-Y in presence of a sacrificial electron donor in water-alcohol mixture

Photoreduction of methyl viologen (MV²⁺) by eosin-Y (EY²⁻) in the presence of triethanolamine (TEOA) has been investigated in water–methanol mixture by means of steady-state photolysis and laser-flash photolysis in the visible/near-infrared regions. The complete conversion to the persistent methyl viologen radical cation (MV^·+) was observed in the presence of lower concentrations of EY²⁻ and excess TEOA. By laser-flash photolysis measurements, electron transfer was confirmed to occur from the triplet state of EY²⁻ [³(EY²⁻)*] to MV²⁺ in the rate constants of ca 2.0 × 10¹⁰ M ⁻¹ s⁻¹. The rates and efficiencies of production of MV^·+ were found to be dependent on solvent compositions and concentrations of MV²⁺ ionic salt and TEOA. The back electron transfer reaction from MV^·+ to EY^·− was retarded in the presence of TEOA, which supports that EY²⁻ is reproduced by accepting an electron from TEOA. In the presence of excess TEOA, the indirect formation of MV^·+ from EY^·3− which was produced by accepting an electron from TEOA, was confirmed. The contributions of both the oxidative and reductive routes of ³(EY²⁻)* for the MV^·+ formation have been confirmed.     

THE PRESENCE OF PHOTOREACTIVE β-CARBOLINE FLUOROPHORES IN WEATHERED WOOL KERATIN

Abstract Solvent-cleaned Merino wool, which was enzymatically digested with protease K, displayed a fluorescence maximum at 500 nm when excited at 430 nm. The yield of this emission was approximately 15 times greater for a fiber tips digest than for a digest of the mid-length region of the same fibers.
Separation of the components in the wool tip digest by silica gel thin-layer chromatography revealed the presence of several fluorescent species. The chromatographic mobility of these species was similar to the behavior observed in a preparation of authentic 1-methyltetrahydro-P-carboline, 1,3-dicarboxylic acid.
Mass spectroscopy of the fluorescent compounds in the wool tip digest displayed molecular ions, [M + H]⁺ with m/z = 273.0890 and 257.0560, and molecular weights that define the formulae C₁₄H₁₂N₂O₄ and C₁₁H₈O₄N₂, respectively, which correspond to β-carboline 1,3-dicarboxylic acids. Mass spectral evidence also indicates the presence of two other β-carbolines.     

MIGRATION OF EXCITATION ENERGY FROM THIONINE TO METHYLENE BLUE IN MICELLES

Abstract— The main absorption bands of thionine (Th⁺) and methylene blue (MB⁺) in aqueous solution lie at 598 nm and 664 nm, respectively. This position permits excitation energy transfer from Th⁺ to MB⁺, but not vice versa. We describe here studies of such transfer between these molecules adsorbed on micelles of sodium lauryl sulfate (SLS), imitating, at least to some extent, the state of pigments in chloroplasts.
The SLS concentration was varied from 3.0 to 11 × 10^-3 M. In the presence of dye, aggregation to micelles, each containing 70–100 detergent molecules, begins at about 3.0 × 10^-3 M SLS. Practically all dye ions are adsorbed on these micelles as soon as their formation begins.
Energy transfer from adsorbed Th⁺ ions to adsorbed MB⁺ ions can be demonstrated by observing the quenching of the fluorescence of thionine and the sensitization of that of methylene blue.
At [Th⁺] = [MB⁺] = 1 × 10^-5 M , the most efficient energy transfer (82 per cent efficiency, as derived from measurements of the quenching of Th⁺ fluorescence, or 90 per cent, as derived from sensitization of MB⁺ fluorescence) is observed at the lowest SLS-concentration (3.0 × 10^-3 M ), when the only micelles present are those formed by aggregation of dye-carrying low molecular complexes of SLS with dye cations. Each micelle carries, under these conditions, 10–14 molecules of the two dyes, and the distance between two closest dye ions is about 16 A. Transfer becomes less efficient as the SLS-concentration increases, causing pigment molecules to distribute themselves among a greater number of micelles.     

DEACTIVATION OF PYRENE BY INDOLE IN MICELLAR SOLUTIONS

Abstract— The deactivation rate of excited pyrene by indole strongly depends on the polarity of the media. In micellar systems (Triton X-100, cetyltrimcthylammonium chloride (CTAC) and sodium dodecylsulfate (SDS) the deactivation efficiency is enhanced due to the high local concentration of indole in the micellar pseudophase. Quantitative interpretation of the data in CTAC and SDS micelles requires to take into account indole exchange between the micelles and the aqueous phase. In SDS micelles, where due to their smaller size the exchange process is more relevant, the exit and entrance rates are (3.0 ± 0.6) x 10⁶ and (1.2 ± 0.3) x 10¹⁰ M ⁻¹s⁻¹ respectively. Intramicellar bimolecular quenching constants are (1.1 ± 0.2) x 10⁸ M⁻¹ s⁻¹ (1.4 ± 0.2) x 10⁸ M ⁻¹ s⁻¹ and (1.5 ± 0.2) x 10⁸ M ⁻¹ s⁻¹ in Triton X-100, SDS and CTAC respectively. These rates are similar to those measured in ethanol rich ethanol-water homogeneous solutions. This is in agreement with the average polarity sensed by both pyrene and indole in the micellar pseudophases.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: SECONDARY OXIDATION OF REDUCED VIOLOGEN ACCEPTORS BY Ru(NH3)63+

Abstract— The kinetics of the oxidation of a homologous series of 4,4'-di(n-alkyl)-bipyridinium (viologen) radicals by Ru(NH₃)₆³⁺ in vesicle suspensions was studied using laser flash photolysis. The viologen radicals were produced photochemically in the bilayer membrane phase of the vesicles by electron transfer from the triplet state of chlorophyll-α. At high concentrations of Ru(NH₃)₆³⁺, the rate of oxidation of the viologen radicals in the aqueous phase was limited by the rate at which the radicals diffused from the membrane to the aqueous phase. The exit rate constant decreased from 2 × 10⁵ s⁻¹ for the methyl viologen radical to 4 × 10³ s⁻¹ for the pentyl viologen radical. Both the exit rate constants and the calculated values for the equilibrium association constants of the viologen radicals were unexpectedly insensitive to the length of their alkyl substituents. This, as well as other data, suggests that the radicals that diffused into the aqueous phase tended to remain associated with the membrane-water interface.     

PHOTOELECTRIC EFFECTS IN BILAYER LIPID MEMBRANE CONTAINING METALLO-PORPHYRINS and DYES

The bilayer lipid membrane (BLM) system containing metallo-porphyrins (M-TPP) and dyes as photosensitizers and electron mediators was studied. Cyclic voltammetry was used to determine photoconductivity and photo-emf of the system. The largest photoconductivity was observed for the Mg-TPP containing BLM with methyl viologen (MV²⁺) and iodine (I₂) present in the aqueous solution. Photoactive dyes, due to their redox ability caused photovoltage up to 30 mV to develop, but no conductance change was observed under illumination in absence of Mg-TPP. The relevance of cyclic voltammetry to the photoconductance and the photo-emf observed in the BLM is discussed.     

REGENERATION OF NAD+ AND NADP+ COFACTORS BY PHOTOSENSITIZED ELECTRON TRANSFER

Abstract The irradiation with visible light of photosensitizer dyes like methylene blue or N-methyl phenazonium methyl sulfate leads to the oxidation of reduced coenzymes such as pyridine nucleotides (NADH or NADPH). This photoredox reaction can be used to regenerate the oxidized form of these coenzymes in enzymatic reactions and total consumption of a substrate with catalytic amounts of enzyme, coenzyme and photosensitizer can be performed. The process has been studied on two common enzymatic reactions: ethanol oxidation by alcohol-NAD ⁺ -oxidoreductase and gluconate-6-phosphate oxidation by 6-phospho-D-gluconate-NADP⁺-2-oxidoreductase. In the first case, a turnover number of 1125 has been obtained for the photoregeneration of NAD ⁺ from NADH.     

DYE-SENSITIZED PHOTOREDUCTION OF METHYL VIOLOGEN

Abstract— The kinetics of the proflavine-sensitized photoreduction of methyl viologen (MV²⁺) to the blue radical cation (MV^.+) are presented. The triplet excited state of proflavine accepts an electron from EDTA to form the singly-reduced species of proflavine (PH^.); this species donates an electron to either the oxidized (MV^.+) or the singly–reduced (MV^.+) species of the bipyridyl. MV²⁺ can be reoxidized by oxygen to MV²⁺ but is decomposed by H₂O₂. The doubly-reduced form can not be reoxidized either by oxygen or by peroxide. Potassium iodide inhibits the photoreduction of MV²⁺ by competing with it as reactant for the singly-reduced form of proflavine (PH^.). The mechanism presented may be analogous to that occurring in the reduction of MV²⁺ by illuminated spinach chloroplasts; its herbicidal action can not be ascribed to the formation of peroxides.     

PHOTOEFFECTS OF PIGMENTED LIPID MEMBRANES IN A MICROPOROUS FILTER

Abstract. The light-induced voltage and current generated by pigmented lipid membranes have been investigated. The membranes, separating two aqueous solutions, were formed in microporous filters with pore sizes ranging from 0.05 to 8μm in diameter and densities of 10⁵ to 6 × 10⁸ pores/cm². The structure and some physicochemical properties of these membranes are described and compared with those of planar BLMs. Photopotentials up to 400 mV could be developed across these membranes having an effective current of 21μA-cm^-2. These and other parameters (redox couples and lifetime) were studied over a period of days. The advantages of this membrane system such as long-term stability and manipulability are discussed.     

AMPHIPHILIC COPOLYMERS AS MEDIA FOR LIGHT-INDUCED ELECTRON TRANSFER-II. ELECTROSTATIC EFFECT ON THE BACK ELECTRON TRANSFER

Abstract— Photosensitized reduction of zwitterionic viologen (SPV) and methyl viologen (MV²⁺) was investigated using an amphiliphilic copolymer having phenanthryl and sulfonate groups (APh) as photosensitizer in aqueous solutions. In the presence of triethanolamine the accumulation of SPV * (photoproduct) was found to be faster than that of MV⁺. This attributed to the electrostatic repulsion between SPV. and anionic segments of APh. Such difference between SPV and MV²⁺ was minimized in the case of the related monomer model. Retardation of the back reaction for the APh-SPV system was also demonstrated by laser photolysis, k _b= 8.7 × 10⁷ M ^-1 s^-1 for the polymer system as compared to k _b= 2.8 × 10⁹ M ^-1 s^-1 for the monomer model system. Strong salt-effects on the yield of the photoreduction and the rate of back reaction confirm the strong electrostatic interaction between the photoproducts and polyanions. This remarkable electrostatic effect of the polyanions was simulated by electrochemical redox reactions by using a graphite electrode coated with APh.     

APPLICATIONS OF CALCIUM-SENSITIVE PHOTOPROTEINS IN EXPERIMENTAL BIOLOGY

Abstract—Calcium-sensitive photoproteins are "precharged bioluminescent proteins that are triggered to emit light by binding Ca²⁺ or certain other inorganic ions. Neither molecular oxygen nor any organic cofactor is required. The first such protein to be described was aequorin, and for various reasons that has been the one most widely studied. Photoproteins have been used as Ca²⁺-indicators both in vitro and in living cells. Their chief advantages for this are (1) ease of signal detection, (2) high sensitivity, (3) relative specificity for Ca²⁺, and (4) lack of toxicity. Difficulties in the experimental use of the photoproteins stem from (1) their one-time reactivity, (2) their large molecular size, (3) their scarcity, (4) the influence of experimental conditions on the sensitivity to Ca²⁺, (5) the nonlinearity of the relation between [Ca²⁺] and light intensity, and (6) the limited speed with which light intensity follows sudden changes in [Ca²⁺]. Photoproteins have now been used as intracellular calcium indicators in more than two dozen types of cells, and experience with the method is rapidly growing. They are also useful in the determination of calcium binding constants for other substances in vitro , and as models for studies of receptor-ligand interactions in general.     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

EFFECT OF TEMPERATURE AND pH ON THE QUENCHING OF TRIPLET LUMIFLAVIN IN THE PRESENCE AND ABSENCE OF FERRI- AND FERROCYANIDE

Abstract— –Flash photolysis at 450 nm over the temperature range 0.8–60°C was used to determine Arrhenius parameters for the first and second order disappearance of triplet lumiflavin (1.66 µ .M ) at a flash energy of 2 kj in deaerated phosphate buffer at varying pH:
³Lf → Lf₀
³Lf +³Lf → Lf₀+ Lf₀
Arrhenius parameters were also determined for the pseudo first-order quenching of triplet lumiflavin by 10 µ M ferri- and ferrocyanide ions,
³Lf + Fe³⁺→ Fe³⁺→ Lf₀+ Fe³⁺ (energy transfer)
³Lf + Fe²⁺→ Lf^-+ Fe³⁺ (electron transfer)
and for disappearance of the semireduced lumiflavin in the presence of ferrocyanide at pH 6.8, by the second-order reaction
Lf^-+Lf ^-→ Lf₀+ Lf⁼.     

pH-DEPENDENCE OF RHODAMINE B SEMIQUINONE DISMUTATION RATE IN AQUEOUS ALCOHOLIC SOLUTION

Abstract— A single transient species is observed in absorption following the flash irradiation of dilute deoxygenated aqueous alcoholic solutions of rhodamine B in hte pH range 5.5–7.0 The second-order decay constant for this semiquinone radical is an approximate linear function of the prevailing hydrogen ion concentration, the transient persisting for a period of from several seconds in neutral solution to several hours at pH = 12.
The slow rate of decay and its pH-dependence are explained in terms of an electron or H-atom transfer between the (unobserved) protonated and (observed) unprotonated semiquinone radical with simultaneous regeneration of hte D^+> and production of the leuco-dye DH:—
D·+ DH·⁺→ D⁺+ DH
Insofar as the reactant concentrations are controlled by the acid dissociation constant K^a of DH^.+ the data are consistent with a value of 5.5±1.0 for pK_a and a pH-independent rate constant K_o of 1.3±0.5×10⁶ 1. Mole^-1 sec^-1 at 20C.     

A Resonance Raman Study Of the C=C Stretch Modes in Bovine and Octopus Visual Pigments with Isotopically Labeled Retinal Chromophores

Abstract— Previous resonance Raman spectroscopic studies of bovine and octopus rhodopsin and bathorhodopsin in the C–C stretch fingerprint region have shown drastically different spectral patterns, which suggest different chromophore-protein interactions. We have extended our resonance Raman studies of bovine and octopus pigments to the C=C stretch region in order to reveal a more detailed picture about the difference in retinal-protein interactions between these two pigments. The C=C stretch motions of the protonated retinal Schiff base are strongly coupled to form highly delocalized ethylenic modes located in the 1500 to 1650 cm⁻¹ spectral region. In order to decouple these vibrations, a series of 11,12-D₂-labeled retinals, with additional 13C labeling at C₈, C₁₀, C₁₁ and C₁₄, respectively, are used to determine the difference of specific C=C stretch modes between bovine and octopus pigments. Our results show that the C₉=C₁₀ and C₁₃=C₁₄ stretch mode are about 20 cm⁻¹ lower in the Raman spectrum of octopus bathorhodopsin than in bovine bathorhodopsin, while the other C=C stretch modes in these two bathorhodopsins are similar. In contrast, only the C₉=C₁₀ stretch mode in octopus rhodopsin is about 10 cm⁻¹ lower than in bovine rhodopsin, while other C=C stretches are similar.     

REACTIVITY OF ACRIDINE DYE TRIPLET STATES IN ELECTRON TRANSFER REACTIONS

Abstract—Rate constants, k _q , for the reaction of cationic and neutral acridine orange and 10-methylacridine orange triplet states (³AOH ⁺, ³AO, ³MAO⁺) with a series of electron donors have been measured. Two different protolytic forms of the semireduced dye radical are produced by acridine orange triplet quenching at various pH^M values in methanolic solution.
It is found that k ₄ decreases with increasing oxidation potential of the reducing agent for all triplet states in a manner which is expected for electron transfer reactions. The different reactivities of the cationic and neutral triplet forms can, therefore, be attributed to the difference in reduction potentials of these species. The difference in reduction potentials is related to the p K ^M values of triplet state, p K _T^M , and semireduced dye radical, p K ^M_S , by thermodynamic consideration. p K _T^M (³AOH⁺/³AO) is determined to be 11.2. From thisp K _S^M (AOH./AO;) is estimated to be 17–18. This is in striking contrast to the protolytic equilibrium of the semireduced dye radicals found to be pK^F= 4.1. We conclude that the last value represents the second protonation equilibrium (AOH⁺₂./AOH). This conclusion is confirmed by spectroscopic data.     

LASER PHOTOLYSIS STUDIES OF QUINONE REDUCTION BY PHEOPHYTIN a IN ALCOHOL SOLUTION

Abstract- Upon laser photolysis of pheophytin-benzoquinone solutions in ethanol, transients due to the pheophytin triplet state (P_t), an exciplex (Pδ⁺ Qδ^-), the pheophytin cation radical (P⁺) and the semiquinone radical (Q^-) can be observed. Kinetic analysis indicates that the evolution of these transients at times longer than one microsecond is due to the decay of the exciplex with the concomitant formation of P⁺ and Q^-, reverse electron transfer to form P and Q, solvent oxidation by P⁺, and Q^- disproportionation. In support of the suggested solvent oxidation reaction, a large deuterium isotope effect is observed upon changing the solvent from methanol to its fully-deuterated counterpart. Comparisons are made between these results and those obtained with chlorophyll as described in the preceding paper.