INFLUENCE OF pH AND OXYGEN ON 315 mμ INACTIVATION AND THYMINE DIMERIZATION IN MUTANTS OF BACTERIOPHAGE T4

Abstract— 1. Irradiation with 315 mμ light inactivates phage T4v-x C, and T4v^-x- , and forms thymine dimers in their DNA.
2. Both the rates of inactivation and of thymine dimerization depend upon pH and gaseous environment during irradiation. The U.V. sensitivities are: 1 (pH 7, N₂, 03, 2.2 (pH 3.5, Oz), 3.3 (pH 3–5, N₂; and the corresponding rates of thymine dimerization 1: 2.5: 5.2. The number of thymine dimers per lethal hit observed withT4v-x + are: 5.7 (pH 7, N₂, O₂, 5.4 (pH 3.5, O₂, 10.9 (pH 3.5, N₂); and forT4v-x-: 4.6, 3.4, and 7.1 with the same sequence of conditions.
3. Also the photoreactivable sectors depend upon the environmental conditions at 315 mp inactivation. In T4v-x f this sector amounts to about 50 per cent at pH 7, 18 per cent at pH 3.5, O., and 29 per cent at pH 3.5, N, respectively.
4. The molecular basis of these findings is discussed. It is concluded that, besides thymine dimer, at least one other lethal photoproduct (probably a photoproduct of cytosine) is involved in photoreactivation.     

CONVENIENT AND SIMPLE METHODS FOR THE OBSERVATION OF PHOSPHORESCENCE IN FLUID SOLUTIONS. INTERNAL AND EXTERNAL HEAVY ATOM AND MICELLAR EFFECTS

Abstract— Phosphorescence of organic molecules in fluid solutions may be conveniently and readily observed under certain conditions. If k _p (radiative phosphorescence rate constant) is 10s^-1, then (in the absence of photoreaction) phosphorescence is observable upon N₂ purging. For example, nitrogen purged, acetonitrile solutions of bromo and dibromonaphthalene display readily observable phosphorescence as a result of internal heavy atom enhancement of π_S, and k _p. External heavy atom enhancement of k, (CH₂BrCH₂Br solvent) of aromatic hydrocarbons even allows observation of phosphorescence from these compounds in N₂ purged fluid solutions. Although bromonaphthalenes are not significantly phosphorescent in N₂ purged aqueous solution, phosphorescence is readily observed in N₂ purged detergent (HDTBr, HDTCl, and SDS) solutions above the critical micelle concentration. The general factors which determine whether phosphorescence is "readily" obervable in fluid solution are briefly discussed and the results are interpreted in light of these factors.     

PHOTOVOLTATC EFFICIENCIES OF MICROCRYSTALLINE AND ANHYDROUS CHLOROPHYLL a

Abstract— A thin layer of chlorophyll a (around 2000 Å). a p -type organic semiconductor. was sandwiched between two different metals. aluminum and silver. We used the photovoltaic effect in order to study the efficiency of light conversion by the crystalline and anhydrous forms of Chl a . When the photovoltaic cell is illuminated through the semi-transparent aluminum electrode. an action spectrum similar to the visible absorption spectrum of Chl a is obtained. The anhydrous form. always shows a maximum, in the red, at 672 nm and the crystalline one at 746 nm to 738 nm depending on the amount of water vapor present in the measuring area. The light conversion efficiency has been measured at the maximum absorption in the red for both forms of Chl a . For the anhydrous form, we found values as high as 0.036%, which is very significant even compared to 0.21%, the highest value found for the crystalline form. In both cases. the incident light power was approximately 10 μW on the cell. The light conversion efficiency of both forms of Chl a cells was found to depend on a combine effect of the nature of the gas (O₁. N₂. air. Ar. N₂O. SF ₆ , H₂) and the amount of water vapor present in the measuring area. The best conversion efficiency was obtained with O ₂ , or air saturated with water. This combined effect was very large for the crystalline form whereas the anhydrous form was only slightly affected.     

INTERACTIONS OF ASCORBATE AND CHELATED IRON IN A METHYLVIOLOGEN-MEDIATED MEHLER REACTION

Abstract— –In the light, isolated spinach thylakoids consumed O₂ in the presence of methylviologen, and ascorbate was found to interact with this reaction in various ways. Chelating-resin was used to remove metal impurities from the assay medium. Ascorbate diminished the H₂0₂ pool in resin-untreated solutions, while in resin-treated solutions ascorbate had no effect on H₂O₂ concentrations. A Fenton catalyst (Fe-EDTA) increased O₂ uptake in the presence of ascorbate and decreased the amount of O₂ recovered by catalase. Ascorbate tripled the rate of the methylviologen-mediated Mehler reaction, and the O₂ consumed was liberated to 50% of its original concentration by catalase. Superoxide dismutase reversed the effects of ascorbate on the Mehler reaction rates. These results indicate that ascorbate can stimulate Mehler reactions indirectly by promoting a Fenton-type reaction as well as stimulating Mehler reactions directly by reducing 2O₂^- to 2H₂O₂. The promotion of a Fenton-type reaction by ascorbate appears to be the cause of H₂O₂ depletion in resin-untreated solutions.     

CHEMILUMINESCENCE AND THE FORMATION OF SINGLET OXYGEN IN THE OXIDATION OF CERTAIN POLYPHENOLS AND QUINONES

Abstract— The possibility of ¹O₂ (¹Δ_g) participation in the oxidation of polyphenols and quinones has been investigated in two systems: (1) the system involving autooxidation leading to oxidative polymerization and destruction, and (2) the modified Trautz-Schorigin reaction, i.e. oxidation of polyphenols and HCHO with H₂O₂ in concentrated alkaline solutions. The red band with maximum at 635 nm observed in chemiluminescence of pyrocatechol, adrenaline, pyrogallol, gallic acid, adrenochrome and p -benzoquinone corresponds to the transition 2O₂(¹Δ_g) → 2O₂(³Σ^-_g). Emission bands in the range 475–540 nm arise from the superposition of the 2O₂(¹Δ_g) → 2O₂(³Σ^-_g) transition and radiative deactivation of excited oxidation products. In system (2) chemiluminescence has a broad band from 580 nm beyond 800 nm and much higher intensity than in system (1). Formaldehyde was found to enhance light emission in system (1) by a factor of about 30. The influence of solvents, including D₂O in which ¹O₂ has varying lifetimes, on kinetics of chemiluminescence as well as quenching effect of β-carotene, hydroquinone, cysteine, bilirubin and biliverdin strongly support the involvement of ¹O₂ in the chemiluminescence of both systems.     

PROFLAVINE MEDIATED PHOTOINACTIVATION OFBACTERIOPHAGE φX174 AND ITS ISOLATED DNA: EFFECTS OF AGENTS MODIFYING VARIOUS PHOTOCHEMICAL PATHWAYS

Abstract. Thiols and disulfides protect both φX174 phage and its isolated DNA from the lethal action of proflavine plus light. The protective ability of these compounds appears to be attributed to the -SH or the -S-S- group and the property to interact with the proflavine-phage DNA complex. The phage inactivation efficiency per proflavine bound to DNA is reduced by 50 to 30% upon addition of cysteine or cystamine. Substances that affect the lifetime of singlet oxygen modify the rate of phage photoinactivation in the presence of proflavine; the inactivation rate is decreased by N^-₃ and increased by D₂O. Irradiation under N₂ atmosphere markedly decreases the phage photosensitization by proflavine. Irradiation with monochromatic light of 440 nm is less efficient than irradiation with light of 440 nm plus 360 nm, and the difference is more pronounced in N₂ than in air. These results are discussed in relation to various possible photochemical pathways.     

GROWTH DELAY STUDIES OF THE RESPONSE OF V-79 MULTICELL SPHEROIDS EXPOSED TO DHE and RED LIGHT

Abstract Multicell tumour spheroids (MTS) of V-79 Chinese hamster cells have been used to study the role of a number of treatment and microenvironmental parameters in the modification of tumour response to Photodynamic Therapy (PDT) using visible light in combination with the photosensitizing compound dihematoporphyrin ether (DHE). The kinetics of DHE uptake into MTS, determined by fluorimetry of extracted porphyrins, indicate that after extended incubation (i.e. 24 h) the mean cellular DHE content in larger (˜300 μ.m and 400 u.m) MTS is significantly less than that for smaller (˜200 μm) MTS, consistent with a hypothesis that DHE uptake into the internal regions of spheroids is diffusion-limited. The response of spheroids to PDT, as assessed by the endpoint of growth delay, indicates that the kinetics of spheroid volume alteration and cell loss, as well as the potential for regfrrwth, are markedly dependent on both the drug and light exposure levels used. The oxygen dependence of this response has been investigated after light irradiation of spheroid cultures equilibrated with either 21% O₂ (i.e. air) or 0% 0₂ (i.e. N₂). While treatment in air results in significant growth delay, the growth kinetics of DHE-treated spheroids irradiated under N₂ were essentially unchanged from those of untreated spheroids. These observations clearly demonstrate an important role for oxygen, at the time of irradiation, in determining the response of spheroids to PDT.     

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
     

INVESTIGATION OF DAMAGE TO FOREST BY EPR SPECTROSCOPY in vivo

Spruce needles collected from several trees of the Black Forest were investigaled by EPR spectroscopy. These needles show in the g = 2.00 region a signal II_S(Tyr D ₊) and a light-induced signal I(P700₊) and a Mn₂₊ hyperfine structure which superimposes the other absorptions. Difference spectra, light minus dark, partly eliminate the manganese hyperfine structure, and P700₊ can be observed. By comparison of these EPR signals with those of spinach chloroplast or thylakoid membranes described in the literature, significant deviations were observed, whereas several trees grown in the vicinity of Tubingen exhibit the well known D₊ and P700₊ EPR spectra. After treatment of branches of these 'normal' trees with herbicides like Amitrol and Roundup or chemicals like toluene or trichlormethane the EPR signals obtained are comparable with those observed with needles of the Black Forest.     

COOPERATION OF CHARGES IN PHOTOSYNTHETIC O2 EVOLUTION–I. A LINEAR FOUR STEP MECHANISM

Abstract— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O₂ in weak light and light flashes to analyze the interactions between light induced O₂ precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O₂. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O₂ evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, ( S ) successively accumulates four + charges. The S ⁴⁺ state produces O₂ and returns to the ground state S ₀. 5. Besides S ₀ also the first oxidized state S ⁺ is stable in the dark, the two higher states, S²⁺ and S³⁺ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S *₁← S ₂, has a (first) half time ≤ 200 μsec. The S *₂ state and probably also the S *₀ state are processed somewhat more slowly (˜ 300–400 μsec).     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

HYDROGEN AND OXYGEN PHOTOPRODUCTION BY TITANATE POWDERS

Abstract— Uncoated powders of TiO₂ or SrTiO₃ did not produce H₂ or O₂ on UV irradiation of aqueous suspensions of the powders. TiO₂ powders coated with platinum or rhodium photoproduced H₂ on irradiation (effective wavelengths 334 and 366 nm) and the reaction was stimulated by catalytic quantities of methyl viologen. The turnover numbers for H₂ production relative to TiO₂ were very low suggesting that the powders were not acting catalytically. Hydrogen production was never stoichiometric with respect to TiO₂ and the kinetics of H₂ production were first order, not zero order as would be expected for catalytic photolysis of water. Oxygen was never detected and it appears that H₂ did not arise from water photolysis but rather from oxidation of reduced sites in TiO₂. A rhodium-coated SrTiO₃ powder prepared photochemically produced both H₂ and O₂ on irradiation but the turnover numbers were very low. A Rh-SrTiO₃ powder prepared thermally showed higher turnover numbers for H₂ photoproduction and may be acting catalytically. However, little O₂ was detected with this powder. When the turnover numbers for the different titanate powders were expressed with respect to the number of surface monolayer hydroxyl groups calculated from the surface area of the powders, some turnover numbers greater than one were obtained.     

RATE PARAMETERS FOR THE QUENCHING OF SINGLET OXYGEN BY WATER-SOLUBLE AND LIPID-SOLUBLE SUBSTRATES IN AQUEOUS AND MICELLAR SYSTEMS

Abstract— The laser flash photolysis method has been used to determine the bimolecular rate constants for the reaction between O₂(¹Δ₉) and several lipid-soluble and water-soluble substrates. Values for lipid-soluble substrates have been obtained using aqueous dispersions of surfactants above the critical micelle concentration with 1,3 diphenylisobenzofuran as monitor of singlet oxygen. Under these conditions the hydrophobic substances are solubilized by the micellar phase. For substrates which are water-soluble, 9,10-anthracene dipropionic acid disodium salt was used as singlet oxygen monitor. For several substances, the values obtained are comparable to the values found in homogeneous nonaqueous solutions. In cases where significant differences have been found these have been rationalized according to the individual case. The only major unexpected result concerned β-carotene which, in micellar dispersion, failed to react at all with O₂(¹Δ₉) This may be due to multi-molecular aggregations occurring in the polar medium. The work described herein shows clearly that, under appropriate conditions, singlet oxygen kinetics can be effectively followed in aqueous solutions by time resolved methods. The indiscriminate use of β-carotene as a quencher of O₂(¹Δ₉)in mainly aqueous media is questioned.     

UPCONVERSION OF THE SINGLET OXYGEN 1269 nm EMISSION TO THE VISIBLE

Abstract— Experiments are described that enable the kinetic behavior of singlet oxygen, O₂(^IΔ_g), to be monitored in the time-resolved mode using a photomultiplier to detect deep orange light (γ_max 660 nm). This orange light is a consequence of the upconversion of the natural emission of O₂(^IΔ_g) at 1269 nm.     

ONE-ELECTRON OXIDATION AND REDUCTION OF AZAPROPAZONE AND PHENYLBUTAZONE DERIVATIVES IN AQUEOUS SOLUTION: A PULSE RADIOLYSIS STUDY

Abstract— Using pulse radiolysis techniques, 3 azapropazone and 3 phenylbutazone derivatives all structurally related to the potentially photosensitive anti-inflammatory drug, azapropazone, have been reacted with the free radical oxidants N₃, Br₂^- and (SCN)₂^- as well as with e^-_aq a strong reductant. It is demonstrated that for 5 derivatives, azapropazone (Az), 2-[a-Carboxy-valeryll-3-dimethylamino-7-meth1-1,2-dihydro-1,2,4-benzotriazine (Mi307), phenylbutazone (PB), oxyphenylbutazone (OPB), and ketophenylbutazone (KPB), N₃^- and Br₂^- appear to react via a one-electron removal process. For the other derivative, 8-hydroxy azapropazone (8-OH-Az), Nj and (SCN); oxidise via a one-electron process, while Br₂^- probably fqrms a free radical adduct.
The absolute spectra of the one-electron oxidised and reduced transient species for all six derivatives are thus given in this work and are a basis to the understanding of the action of light on these drugs.     

THE PRODUCTION OF SINGLET MOLECULAR OXYGEN BY ZINC(II) PHTHALOCYANINE IN ETHANOL AND IN UNILAMELLAR VESICLES. CHEMICAL QUENCHING AND PHOSPHORESCENCE STUDIES

Abstract— Zn(II)phthalocyanine (ZnPc) generates O₂(¹Δ_g) with a quantum yield of ca. 0.4 upon photocxcitation at 354 or 600 nm in ethanolic solution as determined by time-resolved phosphorescence studies at 1270 nm and photooxidation experiments using 1,3-diphenylisobenzofuran (DPBF) as substrate. The quantum yield of photooxidation slightly increases upon incorporation of ZnPc into unilamellar liposomes of dipalmitoylphosphatidylcholine. Under our irradiation conditions (600 nm, 18^°C, and short light exposure times), DPBF(5–50 μM) undergoes photooxidation by a pure Type II mechanism; the rate constant for the O₂(¹Δ_g) + DPBF reaction is (1.1 ±0.1) x 10⁹ M^-1 s^_1 in ethanol solution and determined to be about two orders of magnitude smaller when both ZnPc and DPBF are embedded into liposomes.     

PROTONATION AND DEPROTONATION EQUILIBRIA OF LUMICHROME

Abstract— In order to investigate the possibility of the tautomerization of alloxazine to isolloxazine in its ground state, the parameters affecting the redistribution of charges in the lumichrome molecule were studied. The absorption and emission spectra of lumichrome as a function of pH in the range H₀ = - 6 to pH = 12 were recorded. At extreme pH conditions the spectra of lumichrome are similar to those of the isoalloxazine system. At high acid concentration ( H ₀ < - 3.0) the absorption spectrum of lumichrome protonated at N₁₀, is practically identical to that of lumiflavin. The fluorescence quantum yield of the two cations is negligible at room temperature.
At pH = 10.5 lumichrome is deprotonated at positions N₁ or N₃, The two monoanions have different excitation spectra. Except for slight differences in the extinction coefficients, the absorption of the anion deprotonated at N₃ is very similar to the lumichrome spectrum. The absorption spectra of the N₁ monoanion and of the di-anion are similar to the spectrum of lumiflavin except for a blue shift of about 20 nm. Furthermore, the emission spectrum of the N₁ -monoanion is identical to that of the isoalloxazine system. These results indicate that the charge distribution in the lumichrome molecule depends on the protonation and deprotonation of the nitrogen atoms at positions 10 and l. Both processes cause a redistribution of charges so that an isoalloxazine ring system is formed.     

CHEMILUMINESCENCE INDUCED BY OXIDATION OF TRYPTOPHAN BY SINGLET OXYGEN AND BY HYPOCHLOROUS ACID. IMPLICATIONS IN THE LUMINESCENCE EMITTED IN PHAGOCYTOSIS

Abstract— The mechanism of the chemiluminescence emitted during phagocytosis of opsonized zymosan was investigated. The use of an artificial system generating singlet oxygen permitted the distinction between the respective roles of O₂ and HOCl in the light production via the reactions with opsonized zymosan, amino-acids and peptides. The reaction of free hypochlorous acid, produced by the myeloperoxidase H₂ O₂-Cl- system, with free tryptophan appears to be a likely path for the production of chemiluminescence. Implications for the mechanism of in vivo chemiluminescence during phagocytosis are considered.     

BIOLUMINESCENCE FROM THE REACTION OF FMN, H2O2 AND LONG CHAIN ALDEHYDE WITH BACTERIAL LUCIFERASE

Abstract— The bioluminescent oxidation of reduced flavin mononucleotide by bacterial luciferase involves a long-lived flavoenzyme intermediate whose chromophore has been postulated to be the 4a-sub-stituted peroxy anion of reduced flavin. Reaction of long chain aldehyde with this intermediate results in light emission and formation of the corresponding acid. These experiments show that the typical aldehyde-dependent, luciferase-catalyzed bioluminescence can also be obtained starting with FMN and H₂O₂ instead of FMNH₂ and O₂. We postulate that the 4a-peroxy anion intermediate is formed directly by attack of H₂O₂ on FMN. The latter may be bound to luciferase. An enzyme bound intermediate is formed which by kinetic analysis, flavin specificity for luminescence, aldehyde dependence, and bioluminescent emission spectrum appears to be identical with the species generated by reaction of FMNH, and O₂ with luciferase. The quantum yield of the H₂O₂-_- and FMN-initiated biolumlnescence is low but can be enhanced by certain metal ions, which also stimulate a chemiluminescent reaction of oxidized flavin with H₂O₂. The peak of this chemiluminescence. however, appears to be at a shorter wavelength than that (490 nm) of the bioluminescence.     

PHOTOCHEMISTRY OF HEPARIN-ACRIDINE ORANGE COMPLEXES IN SOLUTION: PHOTOCHEMICAL CHANGES OCCURRING IN THE DYE AND POLYMER ON FLUORESCENCE FADING

Abstract— Photolysis of acridine-orange-heparin complexes in N₂- and O₂-saturated solution results in permanent photooxidation of AO with little or no concomitant change in the heparin moiety. The major photoproduct is mono- N -demethylated acridine orange; in O₂-saturated solution an additional minor oxygenated product, most likely the 10-oxide (N-oxide) or the 9-acridanone (acridone) is also formed. The results suggest an intermolecular electron transfer between adjacently adsorbed dye molecules. The heparin moiety plays a significant role in the photochemistry by bringing dye molecules into favorable geometric orientation for biomolecular reaction and by means of specific dye-polymer interactions.