ELIMINATION OF THE EFFECT OF CONTAMINATING CO2 IN THE 18O-LABELING OF THE CO2 PRODUCED IN BIOLUMINESCENT REACTIONS

Abstract— Although the mechanism of bioluminescent reactions in various species, such as fireflies, ostracod crustaceans ( Cypridina ), sea pansies ( Renilla ), and the deep-sea shrimp Oplophorus , are thought to involve dioxetanone intermediates, studies reported in the past from different laboratories have included widely different experimental results, most likely due to various factors including the effects of contaminating CO₂. With the improved technique employed in the present study, bioluminescent reactions of the firefly and Cypridina in ¹⁸O₂ gas resulted in an incorporation of over 75% of ¹⁸O into one oxygen of the product CO₂. with a reproducibility within a few per cent. When ¹³CO₂. instead of the product CO₂ of the bioluminescent reaction, was studied in an H₂¹⁸O medium, the exchange of one oxygen of ¹³CO₂ with H₂O was 64%. and the effect of contaminant CO₂ amounted to 1418% of the total CO₂. These results suggest that every molecule of CO₂ formed in the bioluminescent reactions of the firefly and Cypridina had intially contained 1 oxygen atom derived from O₂.     

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.     

PHOTOFRAGMENTATION OF α-OXOCARBOXYLIC ACIDS IN AQUEOUS SOLUTION. AN EPR STUDY. FORMATION OF SEMIDIONE RADICALS BY DECARBOXYLATIVE SUBSTITUTION OF α-OXOCARBOXYLIC ACIDS BY ACYL RADICALS-I: GLYOXYLIC, PYRUVIC, α-OXOBUTYRIC, α-OXOGLUTARIC AND α-OXOISOCAPROlC ACIDS

Abstract— By means of in situ photolysis EPR of aqueous solutions of α-oxocarboxylic acids (RCO-CO₂H) at pH values above 5, semidione radical anions [RC(O^-)=C(O')R] and α-hydroxy-α-carboxy alkyl radicals [RC(OH)CO₂-] were detected. C0₂ was identified as a reaction product. On photolysis of mixtures of α-oxocarboxylic acids (RCOCO₂H and R'COCC₂H), "mixed" semidione radical anions [RC(O^->=C(O)R'] were observed in addition to RC(O^-)=C(O')R, R'C(O^-)=C(O')R', RC(OH)CO₂^- and R'C(OH)CO₂^-. The experimental results are explained in terms of photodecarboxylation (α-clea-vage) of electronically excited RCOCOJ to yield RCO and CO₂. The radicals RC(OH)CO₂^- are formed by reduction of RCOCO₂^- by CO₂^-. The semidione radicals are produced by addition of RCO to RCOCO₂^- followed by decarboxylation of the intermediate adduct. This mechanism was confirmed by generating acyl radicals independently and reacting them with α-oxocarboxylic acids. Selected product studies support the mechanism suggested.     

Aggregation of the Light-Harvesting Complex in Intact Leaves of Tobacco Plants Stressed by CO2 Deficit

Pronounced aggregation of the photosystem II light-harvesting complex (LHC II) was observed in low-lightgrown tobacco plants stressed with a strong CO₂ deficit for 2–3 days. The LHC II aggregates showed a typical band at 697–700 nm (F₆₉₉) in low-temperature emission spectra. Its excitation spectrum corresponded to that of detergent-solubilized LHC II. Formation of F₆₉₉ in stressed plants was not reversed in the dark and leaves did not contain any zeaxanthin showing that neither a light-induced transthylakoid pH gradient nor zeaxanthin was required for LHC II aggregation. The CO₂-stressed plants showed clear signs of photodamage: depression of the potential yield of photosystem II photochemistry (F,/F_M) by 50–70% and a decline in chlorophyll content by 10–15%. Therefore, we propose that the photodamage to the photosynthetic apparatus is the cause of the LHC II aggregation in plants. The F₆₉₉ exhibited a reversible decrease of its intensity upon irradiation of leaves with intensive light. There was no or only slight decrease around 700 nm in unstressed plants. The nonphotochemical quenching of chlorophyll fluorescence showed the opposite relation, being higher before than after the strong CO₂ deficit. This discrepancy was likely related to the different LHC II aggregation state in control and stressed plants.     

PHOTOOXYGENATION OF 3-HYDROXYFLAVONE IN A 12 K O2 MATRIX

Abstract— We present an infrared study of 3-hydroxyflavone in 12 K Ar and O₂ matrices. Although it is extremely photostable in an Ar matrix, a remarkable oxygenation reaction for 3-hydroxyflavone takes place upon photoexcitation in an O₂ matrix. The primary photoproduct is concluded to be a keto-hydroperoxide. On further photolysis the keto-hydroperoxide affords the photostable secondary product 2-(benzoyloxyl)benzoic acid, as well as CO and CO₂ through two independent pathways.     

MULTIPLE ACTION OF FAR-RED LIGHT IN PHOTOPERIODIC INDUCTION and CIRCADIAN RHYTHMICITY

Abstract— It is generally accepted that phytochrome influences the photoperiodic induction of flowering through its interaction with the circadian clock mechanism. We have attempted to separate the effects of phytochrome on the clock mechanism from those that mediate flowering directly by examining a number of responses that are unrelated to flowering but are also regulated by the circadian clock. Gas exchange measurements of both CO₂ and H₂0 vapor were monitored under light conditions (200 μmol m ² s⁻¹) where the addition of far-red energy is required for the maximal promotion of flowering. In addition, photosynthetic capacity and maximal transpiration rates were measured in plants grown under continuous dim (20 μmol m⁻² S⁻') light, with or without supplemental far-red, by exposing them briefly to saturating fluxes (1000 μmol m⁻² s^-l) of light. Net CO₂ fixation was very weakly rhythmic in plants grown under both high and low light and this weak oscillation was completely suppressed by far-red light. Far-red also suppressed the rhythm in transpiration under high light, but the rhythm was immediately reinstated when the far-red light was removed. The phase of this rhythm was also reset with the next peak always occurring15–18 h after the far-red was turned off. When grown under dim light, the transpiration rhythm was not suppressed and the amplitude of the oscillation was more than doubled. Far-red light appears to interact with the rhythm in transpiration in a manner suggesting that the stomatal rhythm may be coupled to the same clock oscillator that regulates the flowering rhythm.     

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis , strainM–2, in the presence and absence of photosynthetic CO₂ fixation. The action spectrum for inorganic carbon accumulation in the presence of CO₂ fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO₂ fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO₂ fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.     

Sensitization of Peroxynitrite Chemiluminescence by the Triplet Carbonyl Sensitizer Coumarin-525. Effect of CO2

Abstract— The flash of spontaneous chemiluminescence (CL) that reflects the formation of electronically excited intermediates during the decay of peroxynitrite (ONOO) to nitrate was investigated. The half-decay time of the CL flash (0.5 ± 0.1 s) was in agreement with the half-life of peroxynitrite obtained in stopped-flow experiments. The spontaneous CL intensity was linearly dependent on peroxynitrite concentration. The yield of spontaneous CL from peroxynitrite decay, 2 × 10^-9 photons/peroxynitrite at pH 9.5, was strongly enhanced by a sensitizer of triplet carbonyl CL, coumarin-525 (C-525). The maximal yield of sensitized CL was calculated to be 3 × 10^-6 photons/ peroxynitrite molecule for infinite concentration of C-525. The dependence of both spontaneous and sensitized CL on pH has a maximum at about pH 9.5. Bubbling with CO₂ or addition of NaHCO₃ considerably enhanced the flash of CL, and it is concluded that the reaction of the peroxynitrite anion with CO₂ is a major pathway leading to the formation of an electronically excited intermediate of peroxynitrite.     

ADENOSINE 5-TRIPHOSPHATE CONTENT AND ENERGY CHARGE DURING PHOTOMORPHOGENESIS OF THE MUSTARD SEEDLING SIN APIS ALBA L.

Abstract— The contents of ATP, ADP and AMP (enzymatic assay) and the rate of respiration (output of CO₂ per h, continuously measured with an infrared CO₂ analyzer) were determined in long-term experiments with intact dark-grown and far-red-light-grown mustard ( Sinapis alba L.) seedlings. While there is a strong effect of far-red light treatment on the respiratory rate (inhibition as well as promotion, depending on the onset of light), an effect on the contents of adenosine nucleotides, and therewith on energy charge, is hardly detectable. At most, there is a tendency that long-term far-red light slightly lowers ATP levels and energy charge. The results suggest that the adenylate system (and therewith the energy charge) in the mustard seedling is controlled by endogenous homoeostasis even under intense phytochrome-mediated photomorphogenesis. It is unlikely that the levels of adenosine nucleotides or the energy charge are links in any causal chain originating from P_fr and leading to phenomena of photomorphogenesis.     

NEAR-UV-INDUCED BREAKS IN PHAGE DNA: SENSITIZATION BY HYDROGEN PEROXIDE (A TRYPTOPHAN PHOTOPRODUCT)

Abstract— Near-UV irradiation of l -tryptophan yields a large number of photoproducts. When this mixture is added to recombinationless ( rec ) mutants of bacteria, the cells are killed. The most toxic component of tryptophan photoproducts has been identified as hydrogen peroxide (H₂O₂). We now report that both tryptophan photoproducts and H₂O₂ sensitize phage DNA to near-UV radiation resulting in enhanced killing as well as enhanced DNA breakages. We conclude that the in situ production of H₂O₂ via tryptophan photolysis may be an important biological event.     

PHOTOLYSIS OF ETHYL CHLOROACETATE IN LIQUID PHASE AT 2537 A°R

Abstract— Results on the photolysis of ethyl chloroacetate, CICH₂COOC₂H₅, at A°≅ 254 nm in liquid phase are presented. GLC and i.r. methods revealed as products: hydrogen chloride and ethyl acetate; in smaller quantities–ethyl succinate and ethyl oxalate; in traces–ethyl malonate, CO₂, CO and CH₃CI; yet other, unidentified products with higher boiling points. Quantum yields are determined for some products. Similar studies were performed in the presence of I₂ as a radical scavenger. In the present case, ethyl iodoacetate I – CH₂COOC₂H₅ was found to arise in the system. The following primary process may be written on the basis of the experimental data:
CICH₂COOC₂H₅← CI + CH₂COOC₂H₅
Attempts to explain the production of these compounds are based mainly on the properties of the CH₂COOC₂H₅ radical.     

THE REACTION OF METHYLENE WITH HYDROGEN

Abstract— An investigation has been made of the reaction between methylene, formed by the photolysis of ketene, and hydrogen. Ethane, ethylene and methane are the major hydrocarbon products, and it has been shown that the formation of these products may be adequately described by the sequence of processes
CH₂CO + hv → CH₂+ CO (1)
CH₂+ H₂→ CH₃+H (2)
2CH₃→ C₂H₆ (3)
CH₃+ H₂+ CH₄+ H (4)
CH₂+ CH₂CO → C₂H₄+ CO (7)
In particular, the relative rates of ethane and methane formation are consistent with the known rate constants for reactions (3) and (4), and it is not therefore necessary to postulate the participation of an 'insertion' process
CH₂+ H₂→ CH₄ (6) to account for the formation of methane.
Decrease of the energy possessed by the methylene, either by increase of the wavelength of ketene photolysis, or by increase of gas pressure, is shown to result in an increase in the reactivity of the methylene towards ketene relative to its reactivity towards hydrogen (i.e. the ratio k₂/k₂ increases).     

THE PHOTOBIOLOGY OF Paphiopedilum STOMATA: OPENING UNDER BLUE BUT NOT RED LIGHT

Abstract— The responses of stomata from Paphiopedilum harrisianum , Orchidaceae, to light and CO₂ were studied in epidermal peels. Stomatal opening under red light was indistinguishable from that in darkness, whereas blue light promoted opening above dark levels. The ineffectiveness of red light in causing stomatal opening was confirmed in the presence of 100 μ M KCN; average apertures in both darkness and red light were 53% of those measured in the absence of the inhibitor, whereas under blue irradiation, the KCN inhibition was only 30%, with average apertures two-fold of those measured under red light or darkness. Fluence rate response curves under blue light were typical of a single photoreceptor; removal of CO₂ increased aperture values without a significant light-CO₂ interaction. The lack of a stomatal red light response contrasts with results obtained in species with chlorophyllous stomata in which red light consistently causes stomatal opening, and suggests that the previously reported red light responses in stomata from intact Paphiopedilum leaves resulted from indirect effects, such as depletion of intercellular CO₂ by mesophyll photosynthesis. In isolation, Paphiopedilum stomata appear to rely on a blue light photosystem for their responses to light and fail to open under red light because of their lack of guard cell chloroplasts.     

SPECIFIC PHOTOREGULATION BY PHYTOCHROME OF EPINASTY and LIGHT-INDUCED ETHYLENE PRODUCTION IN MARCHANTIA POLYMORPHA*

Abstract— A study is made of the influence of phytochrome on light-induced ethylene production and epinasty in green Marchantia polymorpha thalli. Ethylene production of thalli irradiated with terminal far-red is not substantially affected by the presence or absence of CO₂, in contrast with the controls which show a clearcut CO₂ dependency. Photoreversibility and fiuence response data indicate the involvement of the low energy red'far-red reversible type of phytochrome action for both the light-induced ethylene production and the control of epinasty. The far-red effect is further characterized by a difference in escape from reversibility between light-induced ethylene production and the elimination of epinasty.     

Energetics of Photoinduced Electron Transfer in the Indole-O2 Cluster in Gas Phase: Possible Consequences for Photoexcited Tryptophan in Solution

Abstract— A direct process for an activationless electron transfer from photoexcited tryptophan to molecular oxygen is proposed. By photodetachment of mass-selected indole-O_2- clusters in gas phase a neutral indole₊ O_2- charge-separated exciplex state is found at 2.25 0.2 eV above the neutral ground state. By theory also, the existence of an excited charge separated state at 3.05 0.2 eV is postulated. In gas phase both charge-separated cluster states are energetically below the first singlet states ₁L_b and ₁L_a and the lower even below the first triplet state T₁ of indole. In gas-phase clusters these energetics imply a very efficient quenching of photoexcited indole by fast electron transfer to oxygen. We discuss a similar mechanism for tryptophan-O₂ in aqueous environment and find it without activation barrier and presumably extremely fast. In the collisional tryptophan_*-O₂ complex the efficiency and the time scale of the charge transfer process should be mostly solvent independent. In polar solvent a complete charge separation and free superoxide formation are expected. We correlate this model with previous fluorescence and phosphorescence quenching data of excited tryptophan by O₂ and propose electron transfer to be the relevant process.     

ULTRAVIOLET LIGHT-INDUCED INHIBITION OF CELL DIVISION AND DNA SYNTHESIS IN AXENICALLY GROWN REPAIR MUTANTS OF DICTYOSTELIUM DISCOIDEUM

Cell division and DNA synthesis were studied during axenic growth following 254 nm ultraviolet light (UV) irradiation of a repair-proficient parental strain ( rad⁺ , D₁₀ colony formation = 195 J/m²) and two repair mutants ( rad C. D₁₀= 50 J/m²; rad B. D₁₀= 5 J/m²) of Dictyostelium discoideum. Isopycnic CsCI gradients were used to distinguish uptake of labeled precursors into nuclear (n) and mitochondrial (m) DNA, using Netropsin to enhance the density resolution. In all strains, m-DNA synthesis was inhibited to a lesser extent than was n-DNA synthesis. For rad C, which has been shown in other experiments to be slow in incision and dimer removal, the UV-induced lags in division and n-DNA synthesis were longer than for rad⁺. However, rad B showed a more complex response. Although brief division lags were observed for < 10 J/m², little immediate division lag was detected at greater fluences. Instead, a brief period of cell multiplication of up to but not exceeding two-fold occurred, followed by a cessation of division, and then by lysis. Fluences that yielded extensive lags in n-DNA synthesis in rad^- and rad C resulted in little detectable immediate postirradiation lag in n-DNA synthesis in rad B. However, later in the postirradiation period, when DNA synthesis had resumed in rad⁺ and rad C. it gradually declined to near zero in rad B. We conclude: (1) that the more extended lag in division and n-DNA synthesis in rad C is consistent with its slower rate of excision repair, and (2) that rad B contains a defect resulting in less initial blockage of DNA replication by UV lesions.     

CAROTENOID CHROMOPHORE LENGTH AND PROTECTION AGAINST PHOTOSENSITIZATION

Abstract— Carotenoid pigments were extracted and purified from wild-type and mutants 7 and 93a of Sarcina lutea , and tested for their ability to quench ¹O₂. The wild-type pigment (P-438, 9 conjugated double bonds) is as active in quenching ¹O₂ as is β-carotene. On the other hand, the pigment P-422 (8 conjugated double bonds) from mutant 7 is 2 or 3 times less efficient, whereas phytofluene and phytoene from S. lutea are 100 and 1000 times less efficient, respectively, than is β-carotene at quenching ¹O₂. It was also found that the broad EPR signal, induced by light in benzene solutions of chlorophyll a and hydroquinone, and related to chlorophyll oxidation, is efficiently quenched by P-438 and to a much smaller extent also by Sarcina phytoene.     

FLUORESCENCE INDUCTION IN THE RED ALGA PORPHYRIDIUM CRUENTUM

Abstract— The intensity dependence and the spectral changes during the fast (sec) and the slow (min) transient of chlorophyll (Chl) a fluorescence yield, measured at 685 nm, have been analyzed in the red alga Porphyridium cruentum . Both the fast and the slow fluorescence yield changes are affected differently by the inhibitors of electron transport ( e.g ., DCMU) and by the uncouplers of phosphorylation (atebrin and FCCP). Fixation of Porphyridium cells with glutaraldehyde abolishes most of the fluorescence yield changes except for the so-called very fast ( OI ) phase. The same fixed cells, however, reduce DCPIP (a Hill oxidant) but do not evolve O₂ when CO₂ is used as electron acceptor. We interpret these and other results by the hypothesis that fluorescence transients in intact cells are linked to both electron transport and the energy dependent structural changes in the thylakoid membrane.     

A Comparative Kinetic and Mechanistic Study Between Tetrahydrozoline and Naphazoline Toward Photogenerated Reactive Oxygen Species

Kinetic and mechanistic aspects of the vitamin B2 (riboflavin [Rf])-sensitized photo-oxidation of the imidazoline derivates (IDs) naphazoline (NPZ) and tetrahydrozoline (THZ) were investigated in aqueous solution. The process appears as important on biomedical grounds, considering that the vitamin is endogenously present in humans, and IDs are active components of ocular medicaments of topical application. Under aerobic visible light irradiation, a complex picture of competitive interactions between sensitizer, substrates and dissolved oxygen takes place: the singlet and triplet (³Rf*) excited states of Rf are quenched by the IDs: with IDs concentrations ca.  5.0 m m and 0.02 m m Rf, ³Rf* is quenched by IDs, in a competitive fashion with dissolved ground state oxygen. Additionally, the reactive oxygen species: O₂(¹Δ_g), O₂^•−, HO^• and H₂O₂, generated from ³Rf* and Rf ^•−, were detected with the employment of time-resolved methods or specific scavengers. Oxygen uptake experiments indicate that, for NPZ, only H₂O₂ was involved in the photo-oxidation. In the case of THZ, O₂^•−, HO^• and H₂O₂ were detected, whereas only HO^• was unambiguously identified as THZ oxidative agents. Upon direct UV light irradiation NPZ and THZ generate O₂(¹Δ_g.), with quantum yields of 0.2 (literature value, employed as a reference) and 0.08, respectively, in acetonitrile.