THE INFLUENCE OF MOLECULAR CONFORMATION ON THE STABILITY OF ULTRAVIOLET STABILIZERS TOWARD DIRECT AND DYE-SENSITIZED PHOTOIRRADIATION: THE CASE OF 2-(2'-HYDROXY-5'-METHYPHENYL)BENZOTRIAZOLE (TIN P)

Abstract— The mechanism for photodegradation of the ultraviolet photostabilizer 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (TIN P) upon direct and dye-sensitized (singlet molecular oxygen [O₂(1Δ_g)]-mediated) irradiation was studied. From the experimental TIN P photodegradation rate data, and low temperature (77 K) fluorescence and phosphorescence quantum yields, one can conclude that the photodegradative process involves phosphorescent states of TIN P. The open conformer of TIN P quenches O₂(¹Δ_g) by physical scavenging with a rate constant (k_q) in dimethylsulfoxide of 2.8 times 10⁶ M ^-1 s^-1. The intramolecular hydrogen-bonded conformer does not appreciably interact with O₂(¹Δ_g). In the presence of a relatively high concentration of OH- (either 5 times 10^-2 M KOH in ethanol or water at pH 13), the ionic form of TIN P (with an ionized phenol group) physically and chemically quenches O₂(¹Δ_g). The reaction rate constant ( k _r) is 1 times 10 ⁸ M ^-1 s^-1, and the ratio k _q/ k _r is approximately three in alkaline aqueous media.     

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.     

THE Py SCALE OF SOLVENT POLARITIES. SOLVENT EFFECTS ON THE VIBRONIC FINE STRUCTURE OF PYRENE FLUORESCENCE and EMPIRICAL CORRELATIONS WITH ET and Y VALUES

Abstract— Pyrene fluorescence spectra have been run in 62 solvents of widely differing solvent polarity. As has been noted previously, the intensity ratio of the first (the 0–0 band) and third bands in vibronic fine structure of these spectra are very sensitive to solvent polarity. These I ₁/ I ₃ values, however, are not sensitive to hydrogen bonding aspects of solvent-solute interactions. Correlations are reported with Winstein's Y values and with Dimrotb's E _T values. On this basis the I ₁/ I ₃ values for pyrene fluorescence are suggested as the basis for a new empirical scale of solvent polarity, called the Py scale, which offers certain conveniences over other scales of solvent polarity.     

Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

EXCITED STATES AND REACTIVITY OF CARCINOGENIC BENZPYRENE; A COMPARISON WITH SKIN-SENSITIZING COUMARINS*

Abstract— Significant spectroscopic difference was found between the ³(π, π*) state of coumarins and the ³L_a state of benzpyrene. No analogy of the partially localized triplet state associated with the coumarin chromophore was revealed in the case of the potent carcinogen, benzo[a]pyrene. Instead, the ³L_a state of benzo[a]pyrene is characterized by more derealization than that of the non–carcinogenic benzo[e]pyrene. Therefore, the predominant photoreaction between benzo[a]pyrene and DNA bases does not seem to involve cycloaddition in contrast to the coumarin-pyrimidine system. Reactivity indices have been calculated, and results are consistent with recent experimental findings. In addition, spectroscopic properties of benzo[a]pyrene and benzo[e]pyrene have been comparatively described in terms of relatively high-resolution spectra, polarization measurements, and molecular orbital calculations.     

FUNCTIONAL ROLE OF CALCIUM IN PHOTORECEPTOR CELLS

Abstract— Ca-uptake by disc membranes prepared from frog rod photoreceptor outer segments was examined. Ca-uptake study revealed two affinity sites which were saturated with 10–⁵ M and 10–³ M of ATP. When disc membranes in 20 m M Tris-HCl buffer (pH 7.5) were stored at -20°C for 6 h, more than 95% of Ca-uptake activity was lost. Ca-uptake activity was, however, preserved if the disc membrane suspension was mixed with 1–10m M ATP and stored at -20°C. Furthermore the reactivation of Ca-uptake was observed if disc membranes, which had lost Ca-uptake ability by storing at 4°C for 3 h, were mixed with 10 m M ATP and then frozen at -20°C for 5 h or 28 h (ATP-induced ATP-dependent Ca-uptake). When the contents of ATP bound to disc membranes were measured during a brief aging at 37°C, the decrement of bound ATP content was correlated well with the decreasing of Ca-uptake activity. Carbonylcyanide m -chlorophenylhydrazone (CCCP), a conductor of protons, inhibited Ca-uptake activity and half maximal inhibition was achieved at 2 × 10–⁸ M. When 10–⁶ M of CCCP was added to the ⁴⁵Ca-accumulated disc membranes, rapid release of ⁴⁵Ca from the disc membranes was observed. These results suggest that ATP may play a role in the Ca-pump regulation in disc membranes and a [H⁺] gradient across disc membrane may be linked to Ca-uptake mechanism.     

THE EFFECT OF MOLECULAR POLARIZATION ON THE ELECTROCHROMISM OF CAROTENOIDS—I. THE INFLUENCE OF A CARBOXYLIC GROUP

Abstract— Electrochromism of oriented all- trans -β-apo-8'-carotenoic acid is studied in thin capacitors. The linear electrochromism is very strong, in contrast to that of symmetrical carotenoids. It is proportional to the first derivative of the absorption spectrum. The quadratic electrochromism can be described as a superposition of fractions proportional to the first and second derivatives of the absorption spectrum. The permanent dipole moment difference between the ground state and the excited state of the carotenoic acid molecule is Δμ= 3.6 × 10^-29 C·m (±20%) (10.7 Debyes). The polarizability difference parallel to the long axis of the molecule is Δα|| = 1.17 × 10^-37 C·m²·V^-1 (±20%) (1050 ų). Furthermore, the relative permittivity of the solid carotenoic ethyl ester is _r= 3.5 ± 0.2.
Δμ is due to the polarizing force of the carboxylic group. This force is equivalent to a mean local electric field of F _t≅3 × 10⁶V/cm. Such a "local field" may also be exerted on a symmetrical carotenoid in the membrane of photosynthesis, e.g. by asymmetrical complex formation with a polarizing molecule. To obtain an effective permanent field of F _p≅ 2 × 10⁶V/cm across the membrane, as postulated in photosynthesis, a local field of F _l≅ 5.5 × 10⁵ V/cm would be sufficient. F _p is shown to be directed from inside to outside of the thylakoid. To realize this, e.g. a positive polar (i.e. electron-attracting) complex partner of the carotenoid, located more to the inside of the thylakoid, can be postulated.     

THE CHROMOPHORES AS ENDOGENOUS SENSITIZERS INVOLVED IN THE PHOTOGENERATION OF SINGLET OXYGEN IN SPINACH THYLAKOIDS

Abstract— The photogeneration of singlet oxygen (¹O₂) from thylakoids and the chromophores involved as endogenous sensitizers were investigated using chloroplasts and thylakoids isolated from spinach. The blue light-induced inhibition kinetics of photosynthetic electron transport and that of CTvCF, ATPase were also studied. The spectral dependence of the generation of ¹O₂ from thylakoid membranes, measured by the imidazole plus RNO method, clearly demonstrated that the Fe-S centers play an important role in ¹O₂ generation, acting as sensitizers in thylakoids. The photoinhibition of the electron transport in isolated chloroplasts was strikingly depressed by a lipid-soluble '0₂ quencher and enhanced by deuterium oxide substitution, indicating that the inhibition processes are mainly mediated by ¹O₂ which is produced via photodynamic activation. The involvement of chloroplast cytochromes in the production of ¹O₂ was deduced from the action spectrum for the photodynamic inhibition of the electron carrier chain. The results obtained from the kinetic studies appear consistent with the involvement of some components such as the Fe-S centers and cytochrome chromophores of the carrier chain in the generation of ¹O₂.     

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.     

PHOTOSENSITIZING EFFECTS OF HEMATOPORPHYRIN DERIVATIVE IMMOBILIZED ON SEPHAROSE

Abstract— The cytotoxicity that ensues following photosensitization by hematoporphyrin derivative (Hpd) is attributed to production of singlet oxygen. Many of the cellular end points reported to be affected are localized to membranes, hydrophobic environments conducive to partitioning of hydrophobic porphyrins in Hpd. In order to test the hypothesis that efficacy of Hpd-induced photosensitization is enhanced by its ability to freely enter cells or subcellular organelles, we immobilized Hpd on a sepharose support. This immobilized reagent was found to produce ¹O₂ when photoradiated, in yields similar to those observed for Hpd in solution, as evidenced by the bleaching of p -nitrosodimethylaniline in the presence of imidazole. The immobilized Hpd was capable of photosensitizing, i.e. inhibit, cytochrome c oxidase activity in intact mitochondrial membranes and in aqueous solution. However, enzymes located on the interior of mitochondrial membranes (F₀F₁ ATP synthase and succinate dehydrogenase), in the mitochondrial matrix (malate dehydrogenase), or on the inside of the plasma membrane, (Na++ K+)- ATPase, were unaffected by immobilized Hpd plus photoradiation compared to free Hpd. The results suggest that photosensitization by Hpd most likely arises from entry of the photosensitizer into the biological membrane, although proteins on the exterior membrane surface may be susceptible to damage by ¹O₂ produced in proximity to their location.     

ACTION SPECTRA FOR THE GENERATION OF SINGLET OXYGEN FROM MITOCHONDRIAL MEMBRANES FROM SOYBEAN (Glycine max) HYPOCOTYLS

Abstract— The action spectrum for the generation of singlet oxygen (¹O₂) from mitochondrial membranes under aerobic conditions was measured at wavelengths between 360 and 600 nm, using sub-mitochondrial particles (SMP) prepared from soybean hypocotyls. The spectrum, showing a peak at about 420 nm, remarkably resembles the absorption spectra of the Fe-S centers of nonheme iron proteins. Disruption of the Fe-S centers by treating SMP with mersalyl acid resulted in a substantial decrease in the efficiency of ¹O₂ generation, leaving an action spectrum whose pattern is significantly similar to the absorption spectrum of flavins, at least in the region of near UV and blue light wavelengths. Estimating the contribution of the Fe-S centers to the generation of ¹O₂ from SMP, we suggest that the Fe-S centers act as very important endogenous photosensitizers in plant cells, in so far as the type II mechanism is concerned. Possible involvement of mitochondrial flavoproteins in the generation of ¹O₂ is also discussed.     

EXCITED STATES OF ANTHOCYANINS: THE CHALCONE ISOMERS OF MALVIDIN 3,5-DIGLUCOSIDE

Abstract The excited state properties of the chalcone isomers of malvidin 3,5-diglucoside (malvin) in acidic aqueous solution (0 < pH < 4) were investigated using steady-state and time-resolved fluorescence spectroscopy. The two chalcone isomers of malvin were first isolated by high-performance liquid chromatography and then characterized by UV/visible absorption and fluorescence spectroscopy. The results were supported by molecular orbital calculations. The rate constants for photodeprotonation ( k ₁= 1 × 10⁹ s^−l) and protonation ( k ₋₁= 1.3 × 10¹⁰ L mol⁻¹ s^−l) were determined, both from the multiexponential fluorescence decays and the fluorescence intensities measured at the emission wavelengths of the neutral and ionized chalcone forms. The results here obtained are relevant for the understanding of the photoreactivity of anthocyanins in acidic medium.     

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.     

LUMINESCENCE STUDIES OF QUINIZARIN AND DAUNORUBICIN

Abstract— The absorption and emission spectra of quinizarin (1,4-dihydroxy-anthraquinone) have been investigated in hydrocarbon and alcoholic solvents. Fluorescence spectra in 3 different Shpolskii matrices were recorded at 14 K. Vibrational analyses of these spectra revealed the presence of 3, 8, and 9 sites in octane, heptane, and hexane matrices, respectively. The fluorescence lifetime was found to be 6.5 ns in hexane and EPA. Fluorescence photoselection measurements in EPA (77 K) showed that the first 4 electronic transitions of quinizarin are polarized parallel, parallel, perpendicular, and parallel to the long molecular axis and can be assigned, in order of increasing energy, to ¹B₂, ¹B₂, ¹A₁ and ¹B₂ (ππ*)→¹A₁(C_2v,) transitions, respectively. The fluorescent transition is assigned as ¹B₁ (ππ*)→¹A¹. The absence of phosphorescence is attributed to the intramolecular hydrogen bonding present which displaces the parent anthraquinone n →* states above the ππ* states, thereby rendering the intersystem crossing (S₁-T₁) radiationless pathway inefficient.
Photoselection measurements on daunorubicin, a substituted quinizarin and known anticancer drug, revealed an absorption band polarization pattern identical to that of quinizarin. These results are in part at variance with assumptions used in previous work on the intercalation specificity of daunorubicin with DNA.     

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.     

Molecular Requirement of Chlorosomal Chlorophylls. Self-Organization of a Chlorophyll Derivative Possessing a Hydroxyl Group at Ring II

Aggregation of zinc 7¹-hydroxyl-13²-demethoxycarbonyl-pheophytin a (Zn-7¹-OH-Chl) was examined in relation to the structure and function of the self-aggregates of 3¹-OH-type chlorophylls (Chi) in chlorosomes of green photosynthetic bacteria. The Zn-7¹-OH-Chl aggregates yielded a Q_y absorption band at 712 nm with a 1.2-fold larger width (full width at half maximum, 500 cm⁻¹) than the monomer's (420 cm⁻¹). Infrared and NMR spectroscopies revealed that each molecule in the aggregate links together with simultaneous coordination (C7¹-OH…Zn) and hydrogen bonding (C7¹-OH … O=C13¹). A nonlinear alignment of the constituent molecules in the oligomeric structure was assumed. Despite the similar molecular linkages, linearly aligned Q_y, moments in the Zn-3¹-OH-Chl aggregate gave a chlorosome-like broader, more redshifted Q_y band (740 nm; 670 cm⁻¹, 2.1-fold larger than the monomer's). Because it is advantageous for efficient light harvesting and energy transfer to have several Q_y, spectral components, spread over a wide spectral range, that can act as the energy gradient, it is concluded that not only the intermolecular linkages but the linear locations of OH, C=0 and Mg in the molecule are crucial for photosynthetic antenna of the self-assembled chiorosomal Chl.     

EPR STUDIES ON SINGLET OXYGEN PRODUCTION BY PORPHYRINS

Abstract— EPR studies of the porphyrin-sensitized photooxidation of 2, 2, 6,6-tetramethyl-piperidine to the nitroxide demonstrate that all the porphyrins examined are able to generate ¹O₂, although the efficiency of the photoprocess is dependent on the nature of the side chains. Incorporation of metal ions into the porphyrin molecule depresses or even inhibits the formation of ¹O₂. Comparison of these results with previously obtained kinetic data points out that the efficiency of porphyrins as photosensitizes is controlled by the lifetime of their lowest triplet state.     

UVA self-photosensitized oxygenation of beta-ionone

The steady-state UVA (350 nm) photolysis of ( E )-β-ionone ( 1 ) in aerated toluene solutions was studied by ¹H NMR spectroscopy. The formation of the 1,2,4-trioxane ( 2 ) and 5,8-endoperoxide ( 5 ) derivatives in the ratio of 4:1 was observed. Time-resolved laser induced experiments at 355 nm, such as laser-flash photolysis, photoacoustic and singlet oxygen ¹O₂ phosphorescence detection, confirmed the formation of the excited triplet state of 1 with a quantum yield Φ _T = 0.50 as the precursor for the generation of singlet oxygen ¹O₂ ( Φ _Δ = 0.16) and the isomeric α-pyran derivative ( 3 ), which was a reaction intermediate detected by NMR. In turn, the reaction of ¹O₂ with 1 and 3 occurred with rate constants of 1.0 × 10⁶ and 2.5 × 10⁸  m ⁻¹s⁻¹ to yield the oxygenated products 5 and 2 , respectively, indicating the relevance of the fixed s-cis configuration in the α-pyran ring in the concerted [2+4] cycloaddition of ¹O₂.     

QUENCHING OF CHEMIEXCITED TRIPLET ACETONE BY BIOLOGICALLY IMPORTANT COMPOUNDS IN AQUEOUS MEDIUM

Abstract— Thermolysis of tetramethyl-l,2-dioxetane is a convenient source of triplet acetone, which can be monitored in aerated solutions by the sensitized fluorescence of 9,10-dibromoanthracene. We have investigated the quenching of chemiexcited triplet acetone in air-equilibrated aqueous solutions containing the 9,10-dibromoanthracene-2-sulfonate ion by five classes of compounds: indoles, tyrosine derivatives, quinones, riboflavin, and xanthene dyes. Quenching rates for indoles, tyrosine and its 3,5-dihalogenoderivatives, and xanthene dyes (k_q= 10⁸-10⁹ M^-1 s^-1) are considerably smaller than the diffusion controlled rate, whereas those for quenchers with high electroaffinities, such as quinones (IP = 10–11 eV), approach the diffusion controlled rate (k_q= 10¹⁰ M^-1 s^-1). Energy transfer for riboflavin probably occurs by a triplet-singlet Förster type process.
A comparison of the present data with previous studies of quenching of enzymically generated triplet acetone (isobutanal/O₂/horseradish peroxidase) by the same classes of quenchers (except riboflavin) reveals that, independent of the nature of the quencher and the deactivation mechanism, the Stern-Volmer quenching constants ( k_q t⁰) are systematically about one order of magnitude higher in the enzymatic system. The difference is attributed to a longer lifetime of triplet acetone in the latter case, "protected" in an enzyme cavity against collisions with dissolved oxygen.     

CHEMILUMINESCENT REACTIONS OF SO

Abstract— The rapid bimolecular reaction SO + O₃= SO₂+O₂+ 106 kcal/mole
yields electronically excited SO₂ in the ³B ₁ and ¹B₂ states with some vibrational excitation, as well as SO₂ in its electronic ground state. It is shown that k₁ = 1.5 x 10¹² exp (-2100/ RT ) cm² mole^-1 s-¹ and that the formation of electronically excited SO₂ involves higher activation energies.