INTERMEDIATES IN THE ROOM TEMPERATURE FLASH PHOTOLYSIS AND LOW TEMPERATURE PHOTOLYSIS OF PURINE SOLUTIONS

The flash photolysis of purine in acetonitrile and in water at different pH was studied. The transients produced on flash excitation of degassed aqueous solutions have been identified as the triplet excited state, the hydrated electron, a purine radical cation and radical anion on the basis of quenching experiments and comparison to transients observed in low temperature photolysis.     

PHOTOCHEMICAL HYDROXYLATION OF 7,8-DIMETHYLALLOXAZINE (LUMICHROME)-A FLASH PHOTOLYSIS STUDY

Abstract— Flash photolysis experiments on the hydroxylation of lumichrome (L) in aqueous 0.5 M H₂SO₄ solution in the presence of O₂ or Ni²⁺ as triplet quenchers and quantum yield measurements confirm the assignment of the photoreactive species to the protonated form of the excited singlet state. A mechamism concerning the photochemical step is proposed, accounting for the formation of protonated 9-hydroxy-5,10-dihydrolumichrome (LOH₃⁺). This primary stable photoproduct was characterized by spectral and kinetic data. The dark reactions originating from LOH₃⁺ were investigated, and data regarding the successive steps are presented. The reaction LOH₃⁺ L→ LO + LH₃⁺ is demonstrated to be a two-electron reduction. The rate constant for the reaction of LH₂⁺ with O₂ is much larger than that for the oxidation of LH₃⁺ by oxygen.     

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 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.     

INTERMEDIATES IN THE ROOM TEMPERATURE FLASH PHOTOLYSIS OF ADENINE AND SOME OF ITS DERIVATIVES

The transient absorption spectra of aqueous solutions of adenine, 2′-deoxyadenosine, 2′-deoxyadenosine-5′-phosphate and 2′-deoxyadenylyl-(3′-5′)-2′-deoxyadenosine have been determinated at different pH values using conventional flash photolysis. Reactives intermediates produced in the flash photolysis of these adenine derivatives present similar absorption regions: two higher intensity bands in the UV and 560–720 nm wavelength region and a third weaker band at 420–560 nm. On the basis of the effects produced by triplet quenchers and/or electron scavengers the bands have been assigned to hydrated electrons, radical cations, radical anions and/or neutral radicals resulting from neutralization reactions of the charged radicals. The results indicate that the bases photoionize via a triplet state under these conditions.     

A FLASH PHOTOLYSIS STUDY OF 5-METHOXYINDOLE

Abstract— The microsecond flash photolysis of 5-methoxyindole in aqueous solutions has been studied at γ_exc≥ 290 nm. Transients identified in this time realm in neutral solutions are: e_aq^-, the 5-methoxyindole radical cation (γ_max≅ 440 nm), the neutral transient with γ_max≅ 530 nm) and an unidentified oxygen sensitive transient with γ_max≅ 435 nm. Radical cations and e^-_aq are shown to be produced in equal amounts consistent with a photoionization process as the only source of both transients. H⁺ quenching of fluorescence and radical cation production gives equivalent Stern-Volmer constants indicating that photoionization occurs from the fluorescent state. The unidentified oxygen sensitive transient exhibits a pK _a of2–2.5 and is quenched at lower pH values indicating that it also has a fluorescent state precursor.     

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.     

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.     

A FLASH PHOTOLYSIS STUDY OF THE TRIPLET STATE OF ACRIDINE ORANGE IN BASIC SOLVENTS

Abstract— Photochemical reactions of acridine orange (AO) in basic aqueous and ethanolic solutions were studied using the flash photolysis technique. The absorption spectrum of AO was determined in detail (230–900 nm) and extinction coefficients were obtained. The decay of the triplet state is the result of a first order process, a triplet-triplet annihilation process and a quenching by the dye in the ground state. The main part of the triplet decays to the ground state; however the observation of semi-reduced AO shows that the decay is partly due to chemical reactions.
An efficient reversible reaction is observed on flashing aqueous solutions of AO containing tetramethyl- p -phenylenediamine: semi-reduced AO is formed in high yield by reaction between the triplet dye and the diamine. In addition, irreversible reactions of AO occur; these are shown to be due to the triplet state by the method of triplet energy transfer.     

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.     

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.     

FLASH PHOTOLYSIS OF BOVINE SERUM ALBUMIN: IDENTIFICATION AND DECAY KINETICS OF TRANSIENT INTERMEDIATES

Abstract. Using the method of flash photolysis, the triplet of the single indole side chain of human serum albumin was detected at room temperature. In a nitrogen saturated solution, this species was found to decay exponentially for over a factor of ten with a lifetime τ 0.5 ms. Analogous experiments, reported here, with bovine serum albumin yield a non-exponential decay which may be decomposed into two components. The yield of the longer lived triplet, with an average τ of ∼6 ms, is significantly enhanced by addition of a 20 fold excess of sodium dodecyl sulfate or 1 M Br^-. The yield of the shorter lived triplet, τ 0.4 ms, is unaffected by these treatments as was previously observed for the single indole in HSA. Thus, the short lived triplet may be assigned to the indole in BSA which is homologous to the one in HSA. The longer lived triplet may be assigned to the remaining indole of BSA. On the bases of wavelength dependence studies, two additional transients may be identified; the electron adduct of the disulfide bond, λ; 420 with a τ 30 ms, and the neutral indole radical,λ; 520 nm with τ ls. These results suggest that the triplet, because of its long τ, will be a valuable intrinsic reporter group for the study of the structure and dynamics of proteins in solution at room temperature.     

PHOTOPRODUCTION OF IONS FROM CHLOROPHYLL α IN SOLUTION STUDIED BY FLASH PHOTOLYSIS and PHOTOCONDUCTIVITY

Abstract— Ionic species were detected electrically on flash excitation of chlorophyll α following rapid production of the triplet state of the pigment. The action spectrum of photocurrent agreed well with the absorption bands of the pigment in the visible region; the threshold for the formation of ionic species in acetonitrile was 1.8 eV. Oxygen gas introduced into the solution completely prevented the appearance of the triplet and ionic species. The latter are suggested to result from electron-transfer between the triplet pigments. Decay processes of the ionic species varied markedly with changes in the solvent polarity. Direct photo-ionization of the pigment could be observed by UV excitation in some non-polar solvents.     

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.     

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.     

LASER PHOTOLYSIS STUDY OF TRIPLET EOSIN AND THIONINE REACTIONS IN PHOTOSENSITIZED OXIDATIONS

Abstract —Nanosecond laser photolysis was used to determine the rate constants of quenching (including chemical reactions) of the triplet states of eosin and thionine, at pH 7, by imidazole, l-histidine, l-methionine, l-phenylalanine, l-tryptophan, 3-indole-acetic acid, cytidine, guanosine, α-chymotrypsin, lysozyme, allylthiourea, thiourea, p -nitrosodimethylaniline, I^- N₃^- and Fe(CN)₆^4-. The quenching rate constants for thionine were found to be up to three orders of magnitude higher than those for eosin. The mechanism of photosensitized oxygenations (free radical vs singlet oxygen) is discussed on the basis of the present results.     

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.     

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.     

A LASER FLASH PHOTOLYSIS STUDY OF THE NATURE OF FLAVIN MONONUCLEOTIDE TRIPLET STATES AND THE REACTIONS OF THE NEUTRAL FORM WITH AMINO ACIDS

Abstract— Primary photochemical processes in aqueous solution have been characterised for FMN. The influence of pH on these processes is attributed to protonation of the neutral triplet and not to the presence of a dimeric species as postulated earlier. Second order rate constants for reaction between the neutral triplet and some naturally occurring amino acids are reported.