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.     

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.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

FLASH PHOTOLYSIS OF ROD PARTICLE SUSPENSIONS

Abstract. Flash photolytic experiments with suspensions of rod particles isolated from cattle eyes reveal that at low temperatures a transient is formed following irradiation which preceeds lumirhodopsin in the bleaching process. Kinetic studies, in the range -75°C to -50°C, suggest that the transient is composed of three intermediate species, having similar absorption spectra but changing to lumirhodopsin at different rates. The energies of activation of the decay reactions appear to be identical and equal 12.5°3 kcal/mole. Spectrophotometric experiments have demonstrated that the low temperature transient is stable at liquid nitrogen temperatures and suggest that it can be reversibly converted to a mixture of rhodopsin and isorhodopsin at -195°C.
A detailed comparison of light initiated reactions of rhodopsin in rod particle suspensions and in solutions reveals striking similarity in the two systems.     

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.     

LASER FLASH PHOTOLYSIS OF RHODOPSIN AT ROOM TEMPERATURE

Abstract. Nanosecond flash photolysis of rhodopsin with 530 or 353 nm light produces an initial transient absorption spectrum with peaks at ˜57O and ˜420nm, and a subsequent transient species with a maximum absorption at 480 nm. These results are interpreted as the initial formation of prelumi-rhodopsin (570 nm) followed by its conversion to lumirhodopsin (470 nm). The peak at 420 nm in the first transient may be due to either hypsorhodopsin or isorhodopsin.     

LASER FLASH PHOTOLYSIS AND PHOTOINACTIVATION OF SUBTILISIN BPN'

Abstract— Laser flash photolysis of subtilisin BPN'at 265 nm has shown that photoionization of tryptophanyl (Trp) and tyrosinyl (Tyr) residues are the principal initial photochemical reactions. The initial products are the corresponding oxidized radicals. Trp and Tyr, and hydrated electrons (e_aq) which react with the enzyme at: k (e_aq+ subt. BPN') = 2.1 × 10¹⁰ M⁻¹ s⁻¹. The photoionization quantum yield was 0.032 ± 0.005 at 265 nm, which was enhanced 3.5-fold by simultaneous excitation at 265 and 530 nm. The photoionization yields were unchanged by 3 M bromide ion and 8 M urea. which did affect the enzyme fluorescence excited at 265 and 295 nm. A similar lack of correlation between the effects of perturbants on the photionization yields and fluorescence yields was found for subtilisin Carlsherg. The results indicate that the monophotonic and biphotonic ionization of the Trp residues does not involve the thermally-equilibrated. lowest excited singlet state and that singlet energy transfer from Tyr to Trp does not contribute to Trp photoionization. The photoinactivation quantum yield was 0.014 for 265 nm laser excitation. which was not changed by simultaneous 530 nm excitation. The corresponding quantum yield was 0.009 for low intensity 254 nm radiation, indicative of a biphotonic contribution to photoinactivation. The results are explained by postulating that photolysis of Trp-113 leads to disruption of hydrogen bonding to Asn-117 and a shift in the primary chain sequence associated with the aromatic substrate binding sites. The photoionization quantum yields in subtilisin BPN'and subtilisin Carlsberg agree with a model based on the assumption that exposed Trp and Tyr residues contribute independently at intrinsic photoionization efficiencies characteristic of the chromophores.     

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 FLASH PHOTOLYSIS STUDIES OF DNA-COMPLEXED ETHIDIUM BROMIDE

Abstract— Laser flash photolysis studies of DNA-complexed ethidium bromide were undertaken. We have observed a singlet-singlet (S₁-S_n) absorption process for DNA-complexed ethidium bromide. The observed lowest singlet excited state lifetime was 21 ± 2 ns. The molar difference extinction coefficient was measured to be 2.4 ± 0.4 × 10³M^-1 cm^-1 at 370 nm. The assignment of this transition was confirmed by time resolved fluorescence measurements.     

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.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

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.     

MECHANISM OF FORMATION OF FORMALDEHYDE IN THE FLASH PHOTOLYSIS OF METHYL FORMATE

Abstract —From flash photolyses of methyl formate (HCOOCH₃), d -methyl formate (DCOOCH₃), methyl formate- d ₃ (HCOOCD₃) and fully ***deuterated methyl formate (DCOOCD₃) in the vapour phase at room temperature, the relative efficiencies of the formyl and methoxyl radicals in producing formaldehyde have been determined.     

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.     

LASER FLASH PHOTOLYSIS OF INDOLE IN THE PRESENCE OF AMINO ACIDS

The laser flash photolysis of indole at 265 nm in the presence of glycine, proline and hydroxy proline was studied. The relative yields of c _aq, triplet state, and indole cation radical were determined in the absence and in the presence of the amino acids. The yields were determined as a function of laser intensity and the values at very low intensity were compared with the fluorescence quenching results. It was concluded that in these conditions the photoionization of indole occurs via the fluorescent state. From the curves of triplet yield vs laser intensity, the triplet quantum yield extrapolated at low laser intensity was obtained, φr = 0.55 φ 0.05, relative to the literature value of 0.15 for φ_eag. This gives φ_F+φ_eaq= 1.0 ± 0.1 at room temperature. When proline and hydroxy proline were used as singlet quenchers, the yield of In was greater than the yield of c_aq. This was considered as evidence that a fraction of the quenching processes leads to complete electron transfer from indole to the amino acids.     

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.     

CIDNP DETECTED FLASH PHOTOLYSIS OF cis–syn 1,3 DIMETHYLTHYMINE DIMER

Abstract— Theanthraquinone–2-sulfonate photo-sensitized splitting of cis-syn 1,3-dimethylthymine dimer gives rise to large CIDNP effects in the reaction product 1,3-dimethylthymine. The polarization originates from a radical ion pair formed by electron transfer from the dimer to the triplet state sensitizer. In a deoxygenated solution the sign of the polarization of theC–6 proton is reversed compared to the predicted one on account of the CIDNP sign rules. In an aerated solution the correct sign is observed. This can be accounted for by assuming reduction of the lifetime of the radical pair in the presence of oxygen. The time-resolved photo-CIDNP technique was used to study the time dependence of the 1,3-dimethylthymine signal. To account for this time-dependence a cation radical disproportionation reaction is proposed.