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.     

INTERMEDIATES IN THE PHOTOLYSIS OF INDOLE: ROOM TEMPERATURE AQUEOUS SOLUTIONS AND LOW TEMPERATURE GLASSES

Abstract— Flash photolysis of indole aqueous solutions produces four transients with absorption maxima, in their order of appearance in time, at 720, 370, 380 and 520 nm. These correlate with low temperature photolysis observations. The results are interpreted in terms of electron-radical cation generation and subsequent production of the indolyl radical.     

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.     

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.     

NANOSECOND LASER PHOTOLYSIS OF PHOBORHODOPSIN: FROM Natronobacterium pharaonis APPEARANCE OF KL AND L INTERMEDIATES IN THE PHOTOCYCLE AT ROOM TEMPERATURE

Abstract— Photochemical and subsequent thermal reactions of pharaonis phoborhodopsin (ppR; absorption maximum, 498 nm) from Natronobacrerium pharaonis were investigated by nanosecond laser photolysis at 20°C. The experimental results clearly showed the presence of two intermediates in the photocycle of ppR besides the K, M and O intermediates detected previously. One was formed immediately after the excitation of ppR with a blue pulse (pulse width, 17 ns; wavelength, 460 nm), and the other was formed by the thermal reaction of this species. The new intermediates' absorption maxima were 512 and 488 nm, their extinction coefficients were 0.85- and 0.68-times smaller than that of ppR, and their lifetimes were 990 ns and 32 μs, respectively. The absorption and kinetic characteristics of these intermediates relative to ppR were similar to those of the KL and L intermediates of bacteriorhodopsin (bR). The formation of KL intermediates from both ppR and bR were observed only at room temperatures. On the other hand, the formation of L intermediate of bR was observed at both of room and low temperature, whereas that from ppR only at room temperature. The unique formation of L intermediate of ppR at room temperature is discussed in relation to high thermal stability of K intermediate of ppR.     

265 NM LASER FLASH PHOTOLYSIS OF SOME ORTHO-SUBSTITUTED ANILIDES AND RELATED N-FORMYLKYNURENINE DERIVATIVES

Abstract— The physical and chemical properties of the triplet state of eight ortho-substituted anilides including N -formylkynurenine (FK), the major trp UV-photooxidation product and a remarkable photodynamic agent, have been investigated using both pulse radiolysis and 265 nm laser flash photolysis techniques. The molar extinction coefficient, the intersystem-crossing quantum yield and the oscillator strength of the T ₁→ T _n absorption band (Λ_max˜ 450 nm) have been determined. It is shown that anilides having n,π* triplets readily react with most solvents whereas those having π ,π* triplets slowly react with alcohols. In both cases, the semi-reduced species are formed. In water, the formation of the semi-reduced. species most probably involves the first excited singlet state. The triplet state properties of the FK derivatives (i.e. ortho-substituted anilides having a side chain bearing charged groups such as carboxylic or amino groups) are strongly modified by the ionization state of the charged side chain. In the case of the FK derivatives possessing an uncharged amino group, quenching of the triplet state occurs via a fast reversible electron transfer reaction from the NH₂ to the triplet anilide.     

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.     

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.     

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.     

THE TRIPLET AND RADICAL SPECIES OF HAEMATOPORPHYRIN AND SOME OF ITS DERIVATIVES

Abstract— Nanosecond laser flash photolysis and pulse radiolysis have been used to generate and characterise the triplet state, and semioxidised and semireduced radicals of haematoporphyrin, and three 0 -acyl compounds derived from it (the monoacetate, the diacetate and the disuccinate).
After 347 nm irradiation in water containing 2% Triton X-100, haematoporphyrin forms the triplet state (φ_T= 0.92) and photoionises monophotonically (φ_I= 0.03). For the O -acyl derivatives, φ_T approaches unity and photoionisation is reduced. In acetone the triplet yield of all four compounds are close to unity. The difference and corrected spectra for the triplet species are presented and decay rates ( k ₁˜10⁴s^-1) and oxygen quenching constants ( k _Q˜1.5times10⁹ M ^-1s^-1) for the triplet state have been measured. The difference and corrected spectra for the semi-reduced species in methanol and semi-oxidised species in aqueous Triton X-100 are presented.
The photophysical characteristics in fluid solution of haematoporphyrin and its 0 -acyl derivatives are rather similar to those previously recorded for other photosensitising porphyrins.     

THERMOLYSIS AND PHOTOLYSIS OF SOME THIOUREA DERIVATIVES (PART I)

Abstract

Thermolysis of N-benzoyl-N′-phenylthiourea (BPTU) on heating in air at 230°C gives NH₃, H₂S, benzaldehyde, benzil, aniline, azobenzene, benzamide, benzanilide, phenylisothiocyanate, phenyl-cyanamide, thiocarbanilide and benzoylisothiocyanate, whereas thermolysis of N-benzoy1-N′-benzylthiourea (BBTU) affords NH₃, H₂S, benzaldehyde, benzil, toluene, bibenzyl, stilbene, benzamide, cyanamide, benzylisothiocyanate, benzoylthiourea and benzoylisothiocyanate. Thermolysis of N-benzoyl-N′-α-naphthylthiourea (BNTU) under the same conditions gives NH₃, H₂S. benzaldehyde, benzil, benzamide, α-naphthylisothiocyanate, α-naphthanilide, α-naphthylcyanamide. benzoylisothiocyanate, α-naphthylamine and N,N′-di-α,α′-naphthylthiourea. Analogous results were also obtained on photolysis of (BFTU), (BBTU) and (BNTU) with the exception of NH₃, and the photodegradation products of phenylthiourea, benzylthiourea and a-naphthylthiourea respectively. The main feature of these pyrolyses is the homolysis of the amide and thioamide bonds providing free radicals that undergo different reactions involving H-abstraction, dimerization, coupling, fragmentation, rearrangement and disproportionation to yield the identified products.     

PRIMARY PROCESSES IN THE LASER FLASH PHOTOLYSIS AND PULSE RADIOLYSIS OF l-EPHEDRINE

Abstract— Transient absorption spectra produced by laser flash-photolysis of an aqueous solution of ephedrine have been measured under a variety of conditions. Ephedrine was found to photoionise via a biphotonic process. The apparent yield of photoionisation increases with lowering of pH, a value of 8.8 being found for the p K _a associated with this change. The cation radical absorption spectrum has been determined using the techniques of both pulse radiolysis and laser flash photolysis. The extinction coefficient of the cation at 295 nm was determined to be 1.37 × 10⁴ dm³ mol^-1 cm^-1 and 1.2 × 10⁴ dm³ mol-¹cm-¹ by the two techniques, respectively, at pH 11. It is also shown that the rate constant for electron abstraction by the azide radical to form the ephedrine cation is controlled by protonation of the amine group in the side chain. The ephedrine anion radical spectrum and its extinction coefficient at 305 nm were also determined. The excited states responsible for photoionisation and photodegradation are discussed.     

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 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.     

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.     

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.     

KINETICS OF RHODOPSIN PHOTOLYSIS INTERMEDIATES IN RETINAL ROD DISK MEMBRANES—I. TEMPERATURE DEPENDENCE OF LUMIRHODOPSIN AND METARHODOPSIN I KINETICS

Abstract— Kinetic measurements have been carried out on rhodopsin photolysis intermediates in retinal rod membrane suspensions on a millisecond time scale over a wide spectral range at 10, 20 and 36°C. To adequately account for the data we find that a three exponential fit is required at most wavelengths and temperatures investigated. The fastest component at 380, 420, 480, 515 and 540 nm is due to the lumirhodopsin → metarhodopsin I transition. The slowest process is not isochromic with the larger amplitude process found on the metarhodopsin I → metarhodopsin II time scale. The properties of the larger amplitude slow component are identical with the classical metarhodopsin I → metarhodopsin II process. Effects of various experimental conditions are discussed. It is shown that scattered light, in particular, can significantly affect the measured kinetics. For example, sonication, low salt and refractive index matching reduce light scattering and increase the contribution of the lumirhodopsin → metarhodopsin I reaction to the absorption transients. Care must also be taken in the analysis because the isosbestic wavelengths in the spectral transients are highly temperature dependent. For example, the lumirhodopsin–metarhodopsin I isosbestic is 490–500 nm at 10°C, 480–490 nm at 20°C and to the blue of 470 nm at 36°C. Activation energies of 77.8, 130.9 and 101.3 kJ/mol were found for the lumirhodopsin → metarhodopsin I, the metarhodopsin I → metarhodopsin II and the slow millisecond processes, respectively. All three processes contribute to the signals at lower temperatures. The amplitude of the slowest component decreases as the temperature is raised, and at physiological temperature its amplitude is essentially negligible compared to the metarhodopsin I → metarhodopsin II reaction. The lumirhodopsin → metarhodopsin I reaction makes a large contribution to the amplitude of the signals at most wavelengths observed from 380–540 nm, especially at physiological temperatures. At physiological temperatures the decay rates of lumirhodopsin and metarhodopsin I are within a factor of three of each other. Thus, lumirhodopsin decay may be much more important for visual transduction than suggested by low temperature studies. In contrast to reports of several other laboratories we have no evidence for kinetic complexity in the metarhodopsin I → metarhodopsin II reaction.     

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.     

THE PHOTOLYSIS RATES OF SOME DI- AND TRIPEPTIDES OF TRYPTOPHAN

We have measured the relative rates of photolysis of free tryptophan (trp), the dipeptides Gly-Trp, Trp-Gly, Leu-Trp, and Trp-Leu, and the tripeptides Gly-Trp-Gly and Leu-Trp-Leu. The photolyses were performed in neutral 0.1 mM aqueous solutions at 25°C using monochromatic 290 nm Xe arc radiation. Tryptophan loss was monitored by absorption, fluorescence and phosphorescence spectroscopy. The rate of tryptophan fluorescence loss was found to be different in the di-and tripeptides than in tryptophan monomer. These rate differences depended on both the identity of the neighboring amino acid (gly or leu) and on the nature of the linkage, e.g., the rate of Gly-Trp photolysis was more than 10 times greater than the rate of Trp-Gly photolysis. Degassing was found to markedly reduce (factor of 8) the photolysis rates of Trp, Trp-Gly, and Trp-Leu, but degassing only slightly reduced (less than a factor of 2) the photolysis rates of the other di-and tri-peptides. Photochemical product structures were not determined, but absorption and fluorescence spectra were obtained and products could be inferred in some cases by comparison with data of previous workers. The products appeared to differ greatly among the various peptides studied; Trp, Trp-Gly, and Trp-Leu gave oxidation products, while Gly-Trp and Leu-Trp apparently gave ring closure products, not requiring oxygen.