INTERFACING FOR LASER FLASH PHOTOLYSIS EXPERIMENTS: A NEW APPROACH

Abstract— A computer-controlled nanosecond laser flash photolysis system incorporating a CAMAC interface system is described. The resulting system provides many benefits, particularly with regard to ease of operation and flexibility. The use of readily available, off-the-shelf components results in rapid installation, high-level language programability, expandability, and moderate cost. Transient absorption spectra of the benzophenone triplet, ketyl radical and ketyl radical anion in aqueous acetonitrile, demonstrate the utility of the apparatus described.     

AN ULTRAVIOLET PULSE GENERATOR IN THE SUBMICROSECOND RANGE FOR FLASH PHOTOLYSIS STUDIES

Los métodos experimentales que se emplean en la actualidad par identificar el electrón hidratado y otras especies transientes no pueden extenderse a escala de tiempos más cortos (10^-7–10^-8 seg), que se requiere para la observación de moléculas excitadas de corta vida, que son precursoras de estas especies transientes, sin sustancial desarrollo. Para alcanzar estos tiempos, los pulsos de excitation sean de electrones, rayos X o luz, así como la luz pulsada para el análisis espectro-fotométrico debe acortarse en varios órdenes de magnitud. Hemos diseñado un generador de luz ultravioleta y visible que emite pulsos menores que 10^-7 seg y potencia de miles de megavatios. El principio básico consiste en aumentar la tensión de chispa hasta próximo a 100 kV y decrecer la capacidad del circuito de descarga como √V, para mantener la energía de la descarga. Suponemos que estos no son los límites finales de intensidad y tiempo. Para obtener pulsos de luz netos, con tiempo de decrecimiento corto y sin cola se han mejorado considerablemente el circuito elétrico y el montaje del generador. Los primeros resultados en el estudio de transientes de corta vida muestran que esta fuente de luz mejorada permite extender el estudio de transientes en la región de 10^-7 seg.     

FREE RADICAL PRODUCTION BY CHLORPROMAZINE SULFOXIDE, AN ESR SPIN-TRAPPING AND FLASH PHOTOLYSIS STUDY

Abstract— Using the spin-trapping technique we have investigated the photolysis of chlorpromazine sulfoxide and promazine sulfoxide. Photolysis of these sulfoxides in aqueous solution resulted in a species which is capable of oxidizing ascorbate, cysteine, glutathione, NADH, and azide by one electron, in addition to extracting hydrogen atoms from ethyl alcohol and dimethyl sulfoxide. These oxidations were not dependent on the presence of dissolved oxygen. The oxidizing species is proposed to be the hydroxyl free radical arising from the homolytic cleavage of the S-O bond of the sulfoxide. Flash photolysis of the chlorpromazine and promazine sulfoxides demonstrated the formation of cation radicals consistent with the loss of the hydroxyl radical from the sulfoxides. In addition we present a simple direct method for the quantitative synthesis of promazine and chlorpromazine sulfoxides from the parent promazine derivatives.     

REPAIR SYNTHESIS IN HUMAN LYMPHOCYTES PROVOKED BY PHOTOLYSIS OF ETHIDIUM AZIDE

Abstract— The azide analog of ethidium was mixed with human lymphocytes and when photolyzed with visible light provoked repair synthesis as shown by incorporation of tritiated thymidine in the presence of hydroxyurea. The use of photolyzed drug, or incubation of drug-cell mixtures in the dark was without effect. These experiments should prove useful in targeting drug action sites and in studying the details of DNA repair.     

FLASH PHOTOLYSIS OF RIBONUCLEASE A

Abstract— Flash photolysis spectra show that ultraviolet irradiation of RNase (Λ > 250 nm) at pH 11.5 generates the hydrated electron and a long-lived transient with absorption maxima at 390 nm and 410 nm, attributed to the phenoxyl type radical from tyrosyl residues. Comparison of the initial yields with flash photolysis spectra obtained from aqueous tyrosine and mixtures of the chromophoric amino acids indicates that 3–4 tyrosyl residues are photoionized in the primary act. This process is almost completely quenched at pH 1–9, even though the p -alanylphenoxyl radical is obtained with tyrosine over this pH range and the accompanying electron is observed at pH 7. The negative result is not altered by denaturation of RNase with 8 M urea or heating to 70°C, suggesting that a primary chain interaction is responsible for the suppression of tyrosyl residue photolysis. This mechanism is supported by flash photolysis spectra of small peptides, showing that the initial radical yield from tyrosylglycylglycine is strongly quenched compared to tyrosine when the phenolic group is protonated. Comparion of this work with published results on fluorescence and inactivation quantum yields indicates that photochemical electron ejection from RNase in alkaline solutions takes place in the dissociable residues and does not contribute to loss of enzymic activity.     

PHOTOSENSITIZATION OF PYRIMIDINES BY 2-METHYLNAPHTHOQUINONE IN WATER: A LASER FLASH PHOTOLYSIS STUDY

2-Methylnaphthoquinone (MQ) has been excited in water with a 20 ns laser flash at 353 nm and the resultant transient species have been observed optically. Triplet-state MQ (³MQ) decays on a sub-microsecond time scale. It has been characterized in terms of its absorption spectrum and quantum yield. Rate constants have been measured for the decay of ³MQ in infinitely dilute solution, for self-quenching by ground-state MQ, and for reactions of ³MQ with oxygen, thymine, uracil, 6-methyluracil, and orotic acid. The interaction of ³MQ with pyrimidines involves charge transfer to give the pyrimidine cation radical and the MQ : anion radical. These reactions are discussed in relation to the mechanism of pyrimidine photooxidation sensitized by MQ.     

A FLASH PHOTOLYSIS STUDY OF 1-METHYLINDOLE

Abstract— The conventional flash photolysis of 1-methylindole in aqueous media was studied at Λ_excitation≥290 nm. The transients observed 20 μs after excitation consisted mainly of the radical cation (R⁺). the hydrated electron (e^-_aq) and the triplet state (T). Electron counting experiments indicate that photoionization is the only source of R⁺ with e^-_aq/R⁺= 1.07±0.09 in neutral media. Quenching of the R⁺ yield with H⁺ indicates that the fluorescent state is the precursor to 80% of the photoionization events with the remainder probably arising from a prefluorescent state. The triplet decays with a lifetime of 29 μs in deaerated neutral media. This decay is unchanged by N₂O saturation, but T reacts with acrylamide with k ≥2.8 × 10⁹ M ^-1. In 2 M Br^-, R⁺ and T yields are increased by factors of 2–3. Consideration of fluorescence quenching and T enhancement by Br-permits an estimate of φ_Isc between 0.33 and 0.49. The increased R⁺yield at high Br-concentrations cannot be accounted for by induced photoionization or triplet state reactions.     

TEH PHOTO-OXIDATION OF SIMPLE N-ALKYL ANILINES STUDIED BY FLASH PHOTOLYSIS

Abstract— The flash-initiated photo-oxidation of six simple N-alkyl tertiary, secondary and primary anilines was studied in aqueous solution by kinetic spectroscopy. Two transient species were observed with the tertiary anilines, but only one "transient" was observed with the secondary and primary anilines. Where possible, the kinetics of formation and decay of these transients was studied in detail. The formation of highly coloured products was observed on flashing the anilines in air-saturated solution and the variation of their colour with the pH of the solution was noted. Preliminary observations on the nature of possible blue products from the tertiary anilines were made. The results for thwe tertiary anilines are interpreted in terms of the mechanism previously for the photo-oxidation of HEMA.     

LASER FLASH PHOTOLYSIS OF EOSIN AND ITS COMPLEX WITH LYSOZYME

Abstract— The transient absorption spectra of aqueous solutions of eosin and of the lysozyme-eosin complex have been examined after excitation with a Q -switched frequency-doubled (347 nm) ruby laser pulse. Eosin itself gives three broad, intense short-lived absorption bands with maxima at wavelengths of 410, 460 and 580 nm, which other workers have identified with the semi-reduced and semi-oxidized radicals and the triplet state of the dye, respectively. In the complex with lysozyme, the yield and lifetime of the eosin triplet are greatly reduced in comparison with the free dye. It is suggested that excited eosin, when bound to lysozyme, decays mainly from the singlet state by pathways such as charge transfer which are not available to the free molecule.     

TRIPLET EXCITATION TRANSFER INVOLVING β-IONONE. A KINETIC STUDY BY LASER FLASH PHOTOLYSIS

Abstract— Employing nanosecond laser flash photolysis, β-ionone (BI) has been examined as an acceptor and as a donor of triplet excitation. In the limit of diffusion-control as well as below it, the rate constants for the quenching of a series of sensitizer triplets by BI are2–3 times smaller than those by 2,4-hexadienal (HD), although the triplet energies (spectroscopic) of the two carbonyl-containing dienes are estimated to be the same (∼55 kcal mol^-1). We attribute the difference to a steric effect arising from ground-state geometric distortion and heavy alkyl-substitution in BI. In spite of possible exothermic energy transfer, BI triplet is nearly nonquenchable by azulene and ferrocene; this is explainable by torsional relaxation to an equilibrium geometry at which the vertical energy gap is smaller than 40 kcal mol^-1. The singlet oxygen yield from the interaction of BI triplet with oxygen in benzene is estimated to be 0.5, suggesting that spin-exchange and energy-transfer may be involved to the same extent in the oxygen quenching process.     

THE PHOTOREDUCTION OF FLAVINS BY AMINO ACIDS AND EDTA. A CONTINUOUS AND FLASH PHOTOLYSIS STUDY

Abstract— Primary and secondary photochemical processes in oxygen-free aqueous solution have been characterised for FMN alone and in the presence of EDTA and four amino acids using nanosecond and microsecond flash photolysis and continuous photolysis techniques. The relative contributions of oneelectron and two-electron (group or hydride transfer) reactions to the deactivation of the triplet has been determined by comparing the radical concentration (560 nm) with the bleaching of the ground state (446 nm). It was concluded that one-electron reactions (hydrogen atom or electron abstraction) are the major mode of reactivity of the flavin triplet state with all the suhstrates studied.
The nature of the reactions of the flavin semiquinone radical have been studied quantitatively by microsecond flash photolysis. These secondary reactions consist of either a 'back reaction' between the flavin and substrate radicals (tryptophan or glycyl-tyrosine) or the transfer of a second electron (or hydrogen atom) from the substrate radical to the flavin radical (EDTA, methionine and possibly cysteine) to form reduced flavin and oxidised substrate. From a comparison of the quantum yields of formation of reduced flavin using 'flash' and continuous irradiation, an additional pathway for the decay of the flavin radical is suggested to occur at low light intensities in the presence of glycyl-tyrosine or histidine.     

DETERMINATION OF THE pk VALUES OF THE LUMIFLAVIN TRIPLET STATE BY FLASH PHOTOLYSIS

Abstract— The triplet-triplet absorption spectra in aqueous solution of the acid (³LfH₂⁺), the neutral (³LfH) and the basic (³Lf^-) forms of lumifiavin (6,7,9-trimethylisoalloxazine) were measured by flash photolysis. The p K _a values of the corresponding protolytic equilibria of the lumifiavin triplet were found to be 4.4₅±0.1 and 9.8±0.15.     

PRIMARY PHOTOCHEMICAL PROCESSES OF CATIONIC ACRIDINE ORANGE IN AQUEOUS SOLUTION STUDIED BY FLASH PHOTOLYSIS

Abstract— A transformation system in Escherichia coli was employed to verify the extent of the lesions caused by ultraviolet (UV) and ionizing radiations. DNA inactivated at 280 nm could be reactivated to some extent by exposing the transforming DNA solutions at 240 nm. This reactivation has been tested using more than one strain of E. coli as recipient. Transforming DNA inactivated by ionizing radiations (⁹⁰Sr beta rays and ⁶⁰Co gamma rays) was not reactivable. Low doses of beta rays, however, reactivated the DNA inactivated by 280 nm UV to a slight but significant extent.     

MECHANISM OF THE PHOTOHYDRATION OF PYRIMIDINES: A FLASH PHOTOLYSIS STUDY*

Abstract— Based on comparisons between the microsecond flash photolysis of uracil and substituted uracils and the radiation chemistry of the corresponding 5,6-dihydro-2,4-dioxopyrimidines, it is proposed that the mechanism of photohydration of pyrimidine bases involves the formation of a pyrimidine carbocation. The effects of substitution in the pyrimidine ring and of pH are consistent with a proton transfer from water to the excited singlet state at theC–5 position of the pyrimidine ring. The resultant carbocation is thought to undergo solvolysis to form the photohydrate or eliminate a proton at N-l producing an intermediate isomeric form of the pyrimidine (isopyrimidine) which re-arranges to the parent pyrimidine by a first-order process.     

THE PHOTOPHYSICAL AND PHOTOCHEMICAL PROCESSES OF TRYPTOPHAN IN INTERACTION WITH POLYNUCLEOTIDES: LASER FLASH PHOTOLYSIS STUDY

Abstract— The influence of nucleotides or polynucleotides on the photophysics and the photochemistry of tryptophan (Trp) derivatives has been investigated in aqueous solutions using the 265 nm laser flash photolysis technique. In solutions containing mixtures of N -acetyltryptophanamide and uridine monophosphate (UMP) or mercurated dUMP, the Trp triplet and the hydrated electron (e_aq) are quenched at almost diffusion controlled rates by the nucleotides leading to uracil reduction. Lysyl-tryptophyl-α-lysine (Lys-Trp-Lys) forms stable complexes in solution with normal or mercurated poly(uridylic acid) [poly(U)]. In the Poly(rU)-Lys-Trp-Lys complex the Trp triplet state is completely quenched, whereas the Trp triplet formation quantum yield is enhanced in complexes with mercurated poly(U). In this last case, the 'heavy atom effect' is characterized by a shortening of the Trp triplet lifetime in agreement with low temperature experiments. Our results also show that photoionization of Trp does occur in the complexed state with both polymers. The e_aq lifetime is however longer with the complexed than with the free peptide.     

A STUDY OF THE PHOTOCHEMICAL PROPERTIES OF SOME CINNAMATE SUNSCREENS BY STEADY STATE AND LASER FLASH PHOTOLYSIS

The E ⇄ Z photoisomerization of 4'-methoxycinnamates, used as sunscreens in cosmetics, has been studied by steady state and laser flash photolysis, in aqueous and organic solutions. Photoisomerization quantum yields are found to be fairly high (˜0.5-1), although no intermediate is detected upon laser flash photolysis. Cinnamates are not photodynamic sensitizers but are able to quench the 8-methoxy-psoralen and 5-methoxypsoralen triplets which produces E → Z isomerization.     

A FLASH PHOTOLYSIS STUDY OF INTERMEDIATES IN PHOTOCHEMICAL REACTIONS OF CHLOROPHYLL

Abstract— The photochemical reactions of chlorophyll intermediates in vitro have been studied by the flash photolysis method. The flash excitation of pigment solutions has been shown to involve the population of a chlorophyll triplet state where the oxidation-reduction processes occur. The mechanism and kinetics of pigment triplet decay have been investigated from 20°to — 50°C and the ability of chlorophyll molecules to carry out triplet-triplet energy transfer has been established. The latter phenomenon has been used to show up the role of chlorophyll triplets in the reversible photooxidation reaction with P -quinone. There have been studied initial products of pigment photoreduction with ascorbic acid and phenylhydrazine. Experimental data of the mechanism of the initial oxidation and reduction in chlorophyll photosensitized reactions have been analysed. There have been also obtained the differential spectra of chlorophyll triplets and radicals. A calculation has been made of rate constants for a few elementary reactions.     

PHOTOCHEMICAL LABELING OF YEAST ALCOHOL DEHYDROGENASE WITH AN AZIDE ANALOG OF NAD +

Abstract— 3-Azidopyridine adenine dinucleotide, an azide analog of NAD⁺, has been synthesized as a potentially general photochemical labeling reagent for the active sites of NAD-dependent dehydrogenases. The analog is a competitive inhibitor of NAD reduction by yeast alcohol dehydrogenase (YADH). Upon photolysis of the ³H-analog in the presence of YADH, 7% of the label becomes covalently bound. The results are discussed in terms of desired properties of a photochemical labeling reagent.