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.     

PHOTO-EXCITED TRIPLET STATE OF DIMERIC CHLOROPHYLL a IN 3-METHYLPENTANE RIGID MATRIX AT 77 K STUDIED BY FLASH PHOTOLYSIS

Abstract— Flash photolysis studies of dimeric and monomeric chlorophyll a were carried out at 77 K. The triplet-triplet absorption spectrum of the dimeric chlorophyll a in 3-methylpentane at 77 K is interpreted as a sum of the spectra of chlorophyll a in the ground and triplet states. The dimeric chlorophyll a in the triplet state is considered to have the half-excited structure at 77 K without photodisaggregation owing to high viscosity of the solvent.     

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 PHOTOLYSIS STUDIES OF QUINONE REDUCTION BY CHLOROPHYLL a IN ALCOHOL SOLUTION

Upon laser photolysis of chlorophyll-quinone solutions in ethanol, transients due to the chlorophyll triplet state (C_t), the chlorophyll cation radical (C⁺) and the semiquinone radical (Q^-) can be observed. The rise of Q^- parallels the decay of C_t. demonstrating the precursor role of the triplet. The decay of C⁺ is second order, consistent with reverse electron transfer, and has a rate constant which is independent of quinone potential, and an activation energy of 14kJ/mol due mainly to the temperature dependence of solvent viscosity. Triplet quenching and C⁺ yield are found to decrease with decreasing quinone potential.     

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.     

INDICATION OF FAST TRIPLET FORMATION IN BIMOLECULAR QUENCHING OF EXCITED SINGLET STATES STUDIED BY CONVENTIONAL FLASH PHOTOLYSIS

The bimolecular quenching of the first excited singlet state of oxonine by allylthiourea leads to the formation of the triplet state of the dye. This has been proved by comparison with the triplet-triplet absorption spectrum of oxonine obtained by triplet-triplet energy transfer. The conventional flash experiments suggest that the dye triplet state is produced directly rather than by radical recombination.     

FORMATION OF REARRANGEMENT PRODUCT IN PHOTOLYSIS OF α,α-DIMETHYLDEOXYBENZOIN UPON CYCLODEXTRIN COMPLEXATION

Abstract Solid-state irradiation of cyclodextrin complexes of α,α-dimethyldeoxybenzoin results in the formation of a significant amount of rearrangement product, 4-isopropylbenzophenone, in addition to cage products. This behavior is not observed in the photolysis in solution or in micellar media.     

FLASH PHOTOLYSIS STUDIES OF PROTEINS AND INDOLE DERIVATIVES IN SOLUTION

Abstract— Transients obtained upon flash photolysis of a number of proteins in aqueous solution appear to derive from electron ejection from tryptophyl residue side chains. These decay by a second order process. Oxygen is an effective quencher for the protein transients but is less so for the flash-induced signals obtained from simple indole derivatives. Experiments using other quenchers indicate that the signals are not due to an indole triplet state, but that the triplet state may be a precursor of the flash-induced metastabie species. Compounds which bind to the active site of chymotrypsin were found to exert only non-specific effects on the flash-induced signals.     

FLASH PHOTOLYSIS OF 3-IODOTYROSINE IN AQUEOUS SOLUTION AT pH 5

Abstract— The 3-tyrosinyl free radicals (3-Tyr) and iodine atom are formed by flash photolysis of 3-iodotyrosine (3-Tyr-I) in aqueous solutions at pH 5. The presence of iodine atoms in the medium is characterized by the absorption spectrum and the decay kinetics of I formed when KI is added to the system. In the absence of radical scavengers, the 3-Tyr adds to or reacts with the parent molecule to produce a transient species, probably a radical dimer, which has an absorption maximum at 405 nm. The decay of this transient follows second order kinetics whose rate constant increases with decreasing 3-iodotyrosine concentration. Measurements of the dependence of the transient yield on the concentration of added ethanol indicate that the 3-Tyr radical reacts with ethanol by hydrogen abstraction. The rate constants of reaction of the 3-Tyr radical with 3-iodotyrosine and ethanol are deduced from results.     

THE MOLECULAR CHAPERONE FUNCTION OF α-CRYSTALLIN IS IMPAIRED BY UV PHOTOLYSIS

Buffer solutions of the lens protein γ-crystallin and the enzymes aldolase and liver alcohol dehydrogenase became turbid and formed solid precipitate upon exposure to an elevated temperature of 63°C or to UV radiation at 308 nm. When α-crystallin was added to the protein solutions in stoichiometric amounts, heat or UV irradiation did not cause turbidity, or turbidity developed much less rapidly than in the absence of α-crystallin. Hence, normal α-crystallin functioned as a molecular chaperone, providing protection against both UV and heat-induced protein aggregation. When α-crystallin was preirradiated with UV at 308 nm, its ability to function as a chaperone vis-a-vis both UV and heat-induced aggregation was significantly impaired, but only at relatively high UV doses. A major effect of preirradiation of α-crystallin was to cause interpeptide crosslinking among the αA₂ and αB₂ subunits of the α-crystallin macromolecule. In our experiments α-crystallin was exposed to UV doses, which resulted in 0, 50 and 90% crosslinking as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α-Crystallin samples that were 50% and 90% crosslinked gave chaperone protection, which was increasingly impaired relative to unirradiated α-crystallin. The results are consistent with the notion that UV irradiation of α-crystallin results in loss of chaperone binding sites.     

PHOTOFRAGMENTATION OF α-OXOCARBOXYLIC ACIDS IN AQUEOUS SOLUTION. AN EPR STUDY. FORMATION OF SEMIDIONE RADICALS BY DECARBOXYLATIVE SUBSTITUTION OF α-OXOCARBOXYLIC ACIDS BY ACYL RADICALS-I: GLYOXYLIC, PYRUVIC, α-OXOBUTYRIC, α-OXOGLUTARIC AND α-OXOISOCAPROlC ACIDS

Abstract— By means of in situ photolysis EPR of aqueous solutions of α-oxocarboxylic acids (RCO-CO₂H) at pH values above 5, semidione radical anions [RC(O^-)=C(O')R] and α-hydroxy-α-carboxy alkyl radicals [RC(OH)CO₂-] were detected. C0₂ was identified as a reaction product. On photolysis of mixtures of α-oxocarboxylic acids (RCOCO₂H and R'COCC₂H), "mixed" semidione radical anions [RC(O^->=C(O)R'] were observed in addition to RC(O^-)=C(O')R, R'C(O^-)=C(O')R', RC(OH)CO₂^- and R'C(OH)CO₂^-. The experimental results are explained in terms of photodecarboxylation (α-clea-vage) of electronically excited RCOCOJ to yield RCO and CO₂. The radicals RC(OH)CO₂^- are formed by reduction of RCOCO₂^- by CO₂^-. The semidione radicals are produced by addition of RCO to RCOCO₂^- followed by decarboxylation of the intermediate adduct. This mechanism was confirmed by generating acyl radicals independently and reacting them with α-oxocarboxylic acids. Selected product studies support the mechanism suggested.     

FLASH PHOTOLYSIS STUDY OF ALIPHATIC AMINO ACIDS AND PEPTIDES IN AQUEOUS SOLUTION*

Abstract— The intermediates produced in the photolysis of oxygen-free aqueous solutions of a number of aliphatic amino acids and peptides were observed spectrophotometrically using the fast-reaction technique of flash photolysis. Included among the compounds examined are the N-acetyl derivatives of glycine, alanine, sarcosine, glutamic acid and glycylglycine; the esters and amides of these N-acetyl compounds; diketopiperazines; the amino acids glycine, alanine and β-alanine; and finally the oligopeptides di-, tri- and tetraglycine. The direct optical excitation of these compounds was found to lead primarily to a photo-induced decarboxylation reaction:
The transient spectra of the radicals produced have been identified. The quantum yields of these processes were found to be directly proportional to the p K _a of the carboxyl groups of the corresponding ground-state molecules, and hence to the concentration of the non-ionized carboxylic acids. The φ's of these processes for the ionized acids were close to zero. The dependence of φ upon pH is correlated to the absorption spectra of these compounds. The quantum yields of the corresponding esters were lower but independent of pH. No intermediates were observed from excitation of the amine derivatives. Other photolytic reactions are suggested. The photo-decarboxylation of alanine and diglycine were found to be monophotonic, while that of N-acetyl alanine, N-acetyl diglycine, and tetraglycine were found to be biphotonic. A triplet excited state precursor is indicated for the latter group of compounds. These and other results are discussed.     

HYDRATED ELECTRON FORMATION IN NANOSECOND and PICOSECOND LASER FLASH PHOTOLYSIS OF HEMATOPORPHYRIN IN AQUEOUS SOLUTION

Nanosecond (lambda exc = 266, 355 and 532 nm) and picosecond (lambda exc = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X-100. The dependence of the yield of photoproduced hydrated electrons (e-aq) on laser pulse energy was studied over a wide range of energies (0.2 to greater than 1000 mJ cm-2). The results show that e-aq are predominantly formed in a two-photon process at lambda exc = 266 and 355 nm. One-photon quantum yields are higher at lambda exc = 266 nm than at lambda exc = 355 nm. Both one-photon and two-photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self-aggregation. Two-photon e-aq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e-aq could be detected at lambda exc = 532 nm. Nanosecond pulse-induced transient spectra obtained at pH 7.4 are also discussed.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: FLASH PHOTOLYSIS STUDIES OF THE CHLOROPHYLL-QUINONE REACTION IN ALCOHOL SOLVENTS*

Abstract— Flash photolysis of chlorophyll a alone in CBE (cyclohexanol-t-butanol-ethanol) yields a difference spectrum similar to those obtained upon steady illumination of chlorophyll a-quinone mixtures in this solvent. Decay kinetics in CBE and dimethylsulfoxide are faster at the Soret band than at 460–580 nm and red band regions. This difference is not obtained in other solvents (CHCI₃, CCI₄, t-butanol, ethanol), implying that two or more species are obtained in CBE and DMSO. β-Carotene in CBE increases the rate of decay of the flash-induced chlorophyll transients at 430 and 660 nm but only decreases the magnitude of the signal at 470 nm. This implies that the 470 nm absorbance is due to a product formed from the triplet state. This effect is not observed in ethanol. Adding quinone to chlorophyll solutions results in slowly decaying species being generated by flash excitation in CBE. Three components can be distinguished: the first (t_1/2? 0.2 msec) corresponds to the triplet state; the second (t_1/2= 5–10 msec) is quinone concentration and species independent; the third (t_1/2= several seconds) is dependent upon quinone concentration and species (rate is faster for higher concentrations and lower potential quinones). The ESR signal decay rate is approximately equal to the third component flash decay rate when the chlorophyll and quinone concentrations are equal. With excess quinone, the flash decay rate becomes faster, and the ESR decay rate decreases slightly. These slowly-decaying species are not produced when quinone is added to chlorophyll a in ethanol or t-butanol, or to pheophytin in CBE. One observes merely a decrease in signal height with no accompanying increase in decay rate. Mechanisms to account for all of these phenomena are presented which involve an initial chlorophyll triplet-solvent reaction with the subsequent formation of several species of chloro-phyll-quinone radical complexes.     

EXCITATION OF CHLOROPHYLL DURING THE PEROXIDASE-CATALYZED OXIDATION OF ETHYL α-FORMYLPHENYLACETATE

Abstract When micelle-solubilyzed chlorophyll is present during the horseradish peroxidase catalyzed aerobic oxidation of ethyl α-formylphenylacetate its fluorescence is observed. The excitation of chlorophyll may occur via energy transfer from the enzyme-generated triplet ethyl benzoylformate. These results imply that excited states may be generated in the roots of Datura innoxia .     

A LASER FLASH PHOTOLYSIS AND PULSE RADIOLYSIS STUDY OF AMILORIDE IN AQUEOUS AND ALCOHOLIC SOLUTION

Abstract Laser flash photolysis and pulse radiolysis have been carried out on the diuretic drug amiloride. The primary photochemical processes in aqueous solution were found to be photoionisation and triplet formation. Photoionisation was found to occur by a biphotonic process for 265 nm excitation but by a monophotonic process for excitation at 353 nm. The spectral properties of the resulting cation radical of amiloride were determined by pulse radiolysis using one electron oxidation by the radical anion Br₂·^¯ Photoexcitation of amiloride in isopropanol did not result in photoionisation but instead a semireduced radical of amiloride was observed. The spectral properties of the semireduced radical of amiloride were determined using one electron reduction by the CO₂·^¯ radical.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

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.     

CHARACTERIZATION OF A MICROSECOND INTERMEDIATE IN THE LASER FLASH PHOTOLYSIS OF SMALL PHYTOCHROME FROM OAT

Abstract— Irradiation of small phytochrome from oat in its P_r form with 15 ns laser pulses of different wavelengths(605–655 nm) gave rise to a difference absorption with maxima at 400 and 685 nm for the first detectable transient. Bleaching of a 660 nm band was observed, non-recuperable up to 1 ms. The transient absorption has a lifetime of 70±15 μs at 273 K. The transient is tentatively identified as lumi-R and the conformation of its chromophore is postulated to be more extended than that of P_r. A deviation from the exponential decay of the lumi-R absorption at 284 and 300 K and the lack of observable enhancement of the far-red absorption within 1 ms are interpreted in terms of the appearance of still other intermediates on this time scale between lumi-R and P_fr phytochrome.