PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

TRIPLET STATES AND cis-trans PHOTOISOMERIZATION PROCESSES IN THE SCHIFF BASES OF RETINAL ISOMERS

Abstract —The triplet states of the n -butyl-amine Schiff bases of 11- cis , 9- cis , 13- cis and all- trans retinal are produced via triplet-triplet energy transfer. Their absorption spectra, peaking around 435 nm, and their decay kinetics are recorded using pulsed-laser photolysis. Direct-excitation (φ^D_ISO) and triplet-sensitized (φ^T_ISO) photoisomerization yields, determined using steady irradiation methods, are found to be: φ^T_ISO (9- cis ) = 0.06, φ^T_ISO (11- cis ) = 045, φ^T_ISO (13- cis ) = 008, φ^T_ISO (all- trans ) = 0.02-0.05, φ^D_ISO (11- cis , = (4 ± 1) × 10^-3, φ^D_ISO (all- trans ) = (2 ± 1) × 10^-3. The possible role of the triplet state in the isomerization of rhodospin is discussed.     

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

Abstract— Intense illumination (60-120 MW/cm²) of an oxygen-free aqueous solution of pyranine (8-hydroxypyrene-l,3,6-tri-sulfonate) by the third harmonic frequency of an Nd-Yag laser (355 nm) drives a two successive-photon oxidative process of the dye. The first photon excites the dye to its first electronic singlet state. The second photon interacts with the excited molecule, ejects an electron to the solution and deactivates the molecule to a ground state of the oxidized dye (φ⁺). The oxidized product, φ⁺, is an intensely colored compound (Λ_max= 445 nm, ε= 43 000 ± 1000 M ⁻¹ cm⁻¹) that reacts with a variety of electron donors like quinols, ascorbate and ferrous compounds. In the absence of added reductant, φ⁺ is stable, having a lifetime of -10 min. In acidic solutions the solvated electrons generated by the photochemical reaction react preferentially with H⁺. In alkaline solution the favored electron acceptor is the ground-state pyranine anion and a radical, φ, of the reduced dye is formed. The reduced product is well distinguished from the oxidized one, having its maximal absorption at 510 nm with e = 25 000 ± 2000 M^-l cm⁻¹. The oxidized radical can be reduced either by φ^- or by other electron donors. The apparent second-order rate constants of these reactions, which vary from 10⁶ up to 10⁹M⁻¹ s⁻¹, are slower than the rates of diffusion-controlled reactions. Thus the redox reactions are limited by an energy barrier for electron transfer within the encounter complex between the reactants.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

Singlet Oxygen Generation by Metallotexaphyrins

Abstract. Metallotexaphyrins have clinical applications as photo-sensitizers of photodynamic therapy (PDT). The singlet oxygen quantum yield (φ>_Δ) was determined for a series of metallotexaphyrin derivatives (Lu [III], Y [III], Cd [II], In [III] and Gd [in]) under conditions where the agents are believed to exist in monomeric form. The results show φ_Δ of metallotexaphyrins vary with the medium and the metal cation. Measurements on the Lu (III) texaphyrin led to φ_Δ= 0.38 in unbuffered 5% Tween 20 and φ_Δ= 0.58 in pH 7.4 phosphate buffer plus 1% Triton X-100 (±10%). The in vitro photodynamic efficiency calculated from φ_Δ is compared to in vivo PDT efficacy in an animal tumor model.     

STEADY-STATE NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN: A STERN-VOLMER QUENCHING EXPERIMENT WITH SODIUM AZIDE

Abstract— A sensitive near-infrared detection system incorporating improvements to existing methodologies has been used to characterize the sodium azide quenching of the steady-state luminescence of singlet molecular oxygen at 1270 nm. Stern-Volmer plots which were linear up to 80% quenching of the ¹O₂ generated by rose bengal and eosin Y yielded a rate constant of 5.8 ± 0.1 times 10⁸ M ⁻¹ s⁻¹ for the quenching of ¹O₂ in water, while the rate constants obtained in deuterium oxide with the same sensitizers were 6.28 times 10⁸ M ⁻¹ s⁻¹ and 6.91 times 10⁸ M ⁻¹ s⁻¹ respectively. A flow system minimized the effects of photobleaching of the rose bengal. With a mercury arc light source, the instrument can be used in photosensitization experiments to detect low levels of ¹O₂ production in aqueous media.     

Depopulation of Highly Excited Singlet States of DNA Model Compounds: Quantum Yields of 193 and 245 nm Photoproducts of Pyrimidine Monomers and Dinucleoside Monophosphates

Abstract— Formation of uracil and orotic acid photodimers, uridine and 5'-UMP photohydrates, TpT photodimers and (6-4)photoproducts, dCpT photohydrates and (6-4)photo-products and UpU, CpC and CpU photohydrates were studied in neutral deoxygenated aqueous solution at room temperature upon irradiation at either 193 or 254 nm. The photoproducts were identified and quantified and the contribution from photoionization to substrate decomposition, using λ_irr= 193 nm, was separated. The ratio of the quantum yields of respective stable products,η=φ¹⁹³/φ²⁵⁴ is indicative of the yield of internal conversion from the second to the first excited singlet state, S₂→ S₁. For the observed photodimers η decreases from 0.94 for uracil to 0.7 for TpT and further to 0.55 for orotic acid. For the (6-4)photoproducts of TpT and dCpT T| = 0.5-0.8 and for the photohydrates in the cases of UpU, CpC, CpU and dCpT TJ ranges from 0.55 to 1.     

EXCITED TRIPLET-SINGLET INTERSYSTEM CROSSING IN ISOALLOXAZINES

Abstract— Higher excited triplet states originating from the lowest triplet state of isoalloxazines by absorption of light with Λ - 600 nm undergo "inverse" intersystem crossing to the singlet manifold [φ( T_n-S_m ) = 8 × 10^-3] followed by rapid internal conversion and "normal" fluorescence S₁-S₀ with Λ_m= 540 nm.     

The Reaction of Triplet 2-Methyl-1,4-Naphthoquinone (Menadione) with DNA and Polynucleotides

Abstract— The photoreaction of 2-methyl-l,4-naphthoquinone (MQ, menadione) with DNA and polynucleotides in argon-saturated aqueous solution (pH 7) was studied. Results from laser flash photolysis experiments indicate that triplet quinone reacts with DNA and polyA but not detectably with polyU by one-electron oxidation of the bases of the nucleic acid with formation of the radical anion of the quinone. Irradiation of argon-saturated solutions containing MQ and DNA or polynucleotides (polyU, polyA, polyG or polyC) with 334 nm light leads to an increase in molecular weight for single-stranded DNA, polyA and to a much less extent for polyU. This finding indicates crosslink formation with quantum yields in the range of 10⁻⁵-10⁻³     

ON THE PHOTOIONIZATION ENERGY THRESHOLD OF TRYPTOPHAN IN AQUEOUS SOLUTIONS

Abstract— To investigate the existence and energy position of a photoionization threshold. tryptophan (Trp) has been photoionized in desecrated neutral aqueous or alcoholic solution under monochromatic light of variable frequency, in presence of N₂O to scavenge the photoelectron.
Present findings and some literature data converge to show the existence of a threshold for the one photon ionization process. This threshold is located at 4.5 ± 0.1 eV and 4.85 ± 0.1 cV for Trp in aqueous and ethanol solutions. respectively, which corresponds to a lowering with respect to the gas phase ionization potential of 3.4 and 3.0 eV.
The photoionization quantum yields for Trp is found about 4 times greater at 250 nm than at Λ_cx= 265 nm, where φe^-_4M=0.080±0.025. In such spectral range. at most one photoelectron out of 4–5 escaping geminate recombination would lead to Trp photodegradation in acrated solutions.
These results also point out that the neutral radical Trp. which has been previously observed for Λ_cx > 275 nm, i.e. below the ionization threshold energy—would not necessarily derive from Trp ⁺ deprotonation or cation-electron dissociative recombination. Similarly, the opening of the indole ring with formylkynurenine (FK) formation which is observed under aerobic conditions and Λ_cx >, 280 nm would not imply an electron attachment on O₂ but reactions such as Trp +³O₂ or Trp*+³O₂ or else
     

PHOTOSENSITIZATION VIA CHARGE TRANSFER OR REVERSIBLE ELECTRON TRANSFER. OXIRANE ISOMERIZATION AND SULFUR DIOXIDE EXTRUSION

Abstract— N, N, N^' N^'-Tetramethylbenzidine (NTMB) photosensitizes the cis-trans isomerization of stilbene oxiranes (SO) and the extrusion of SO₂ from dibenzyl sulfone (DBS). In acetonitrile solution it is found that in the absence of SO or DBS, singlet NTMB undergoes three processes: intersystem crossing to triplet NTMB (φ_ISC= 0.63, k _ISC= 6.3 × 10⁷s^-1), fluorescence (φ_f= 0.30, k _f= 3 × 10⁷s^-1), and formation of a cation by electron ejection (φ_ion= 0.09). Both singlet and triplet sensitization are observed. A charge transfer or reversible electron transfer mechanism is proposed to explain the results.     

PHOTOLYSIS OF AQUEOUS SOLUTIONS OF POTASSIUM CIS-DIAQUOBIS (OXALATO) CHROMATE (III)

Abstract— The photolysis of aqueous solutions of cis -[Cr(C₂O₄)₂(H₂O)₂]- at 254 nm and pH 4 produced CO₂ and H₂ in nearly equal yields. The quantum yield of hydrogen, φ², increased by 9% and the yield of carbon dioxide, φ, by 65% as the pH was lowered from 4 to I. The total gas yield, φ_gas, decreased in the presence of added oxalate or chromium (II) ions and when the light intensity was lowered. The gas yield in D₂O was appreciably higher than in H₂O. The variation of φ_gas with pH (D) and with added oxalate ion was roughly parallel in the two liquid media. The gas yield increased in the series:
A tentative mechanism is suggested to explain the results.     

PHOTOLYSIS OF PHOSPHODIESTER BONDS IN PLASMID DNA BY HIGH INTENSITY UV LASER IRRADIATION

Abstract— The cleavage of phosphodiester bonds in DNA exposed to high intensity UV laser pulses in aerated aqueous solution has been investigated using a krypton fluoride excimer laser (248 nm) and bacterial plasmid DNA. The dependence of strand breakage on fluence and intensity has been studied in detail and shows that the process is non-linear with respect to intensity. The relationship between the quantum yield for strand breakage and intensity shows that the strand breakage reaction involves two-photon excitation of DNA bases. The quantum yield rises with intensity from a lower value of 7 times 10^-5 until a maximum value of 4.5 times 10^-4 is attained at intensities of 10¹¹ W m^-2 and above. This value is approximately fifty-fold higher than the quantum yield for strand breakage induced by exposure to low density UV irradiation (254 nm, 12 W m^-2). DNA sequencing experiments have shown that strand breakage occurs by the specific cleavage of the phosphodiester bond which lies immediately 3' to guanine residues in the DNA, leaving some alkali-labile remnant attached to the terminal phosphate. A mechanism for DNA strand breakage which involves the generation of guanine radical cations is proposed.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

MECHANISM OF THE PHOTOCHEMICAL REACTION OF DIPHENYLAMINES WITH CARBON TETRACHLORIDE

Abstract. The quantum yields of HCI (φ_HC1) formation have been measured for the photolysis of N -methyldiphenylamine (MeDPA), triphenylamine (TPA) and diphenylamine (DPA) in the presence of CCl₄ in polar solvents. The quantum yields of N-methylcarbazole formation (φ_mφca) have also been determined for the system MeDPA-CCl₄. With increasing CCl₄ concentration, φ_HCl increases as φ_MeCA decreases, and φ_HCl reaches maximum values 2.7 at 1 M CCl₄. Using laser photolysis, transient spectra have been recorded for MeDPA in the absence and presence of CCl₄ in polar and non-polar solvents, and for TPA. Transient absorption due to the triplet states and photocyclization products (without CCU), exciplexes, the (C₆H₅)₂ NCHi radical, the MeDPA⁺ cation radical, the (TPA⁺., CCl₄) ion pair, and the TPA⁺ cation radical have been identified. The mechanistic implications of these results are discussed.     

QUANTIFICATION OF THE SOLVENT EFFECTS ON THE TRIPLET QUANTUM YIELD OF PSORALEN BY THE "LINEAR SOLVATION ENERGY RELATIONSHIP"

Triplet formation quantum yields (Φ_τ) of psoralen in a set of 17 pure solvents ranging from n -hexane to water and in dioxane: water mixtures were obtained by nanosecond laser flash photolysis. The triplet yield increases with solvent polarity. The extremum values are 0.009 and 0.545 in n -hexane and water, respectively. Good correlations of the experimental Φ_τ values with empirical "polarity" scales (Dimroth/Reichardt's E_T [30], Kamlet/Taft's solva-tochromic parameters β, and α, and Swains acity/basity A_S/B_S) were obtained: Ln(φ_T^-1 - 1) = 8.86 - 0.143E_T(30) Ln(φ_T^-1 - 1) = 4.40 - 2.34_τ - 1.70α Ln(φ_T^-1 - 1) = 4.65 - 3.72A_s - 1.12B_s The results are discussed in terms of the sensitivity of psoralen triplet quantum yield to solvent polarity and hydrogen-bonding abilities.     

FLUORESCENCE QUANTUM YIELDS OF 124-kDa PHYTOCHROME FROM OAT UPON EXCITATION WITHIN DIFFERENT ABSORPTION BANDS

Abstract— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10⁻³ was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa ) upon excitation in the Soret band at Λ^exc= 380 nm. The small difference between this value and the previously determined quantum yield with Λ^exc= 640 nm, π= (3.5 ± 0.4)10⁻³is attributed to a blue-absorbing emitter responsible for the "anomalous" or "blue" emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10⁻³ is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.     

FLUORESCENCE OF AZINES

Abstract— A comparison of the transient absorption spectra from the photolysis of disulfides in solution suggests that C-S bond breakage is a common primary photolytic process. This process becomes more important as the resulting carbon centered radical is stabilized by increasing alkyl substitution or resonance interaction with an aromatic system. The perthiyl radical product is characterized by λ_max∽380 nm,ε₃₈₀∽1700 M ⁻¹ cm⁻¹ and decays by second order kinetics with k ₂∽3.7×10⁸ M ⁻¹ s⁻¹ in water.
In the presence of O₂, the photolysis of disulfides which produce the thiyl radical give transient absorptions in the 500–600 nm region. Possible identities of these transients are discussed.     

RNA—PROTEIN CROSSLINK AND RNA CHAIN BREAK FORMATION ON UV-IRRADIATION OF TOBACCO MOSAIC VIRUS

Abstract— The ability of UV-irradiation (254 nm) to induce formation of RNA-protein crosslinks in tobacco mosaic virus (TMV) particles have been studied by Cs₂SO₄ density gradient centrifugation, analytical centrifugation, nitrocellulose filter binding and two-dimensional peptide mapping. RNA-protein crosslinks were found to be formed on UV-irradiation of TMV, but the parallel process of UV-induced RNA chain breakage complicated their quantitation. Using speciall devised equations, the quantum yield of RNA-protein crosslink formation was found to be 0.65 × 10⁻⁵ and that of RNA chain break formation 0.95 × 10⁻⁵.     

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.