INTERACTIONS AMONG PHOTOSYNTHETIC ANTENNA EXCITED STATES

Abstract. The data of Kung and DeVault (1978) showing high-order fluorescence from chromatophores of photosynthetic bacteria are analyzed in relation to other data on first-order fluorescence of photosynthetic systems, particularly that of Monger and Parson (1977). The wavelengths of emission observed (down to 445 nm) require energy equivalent to two lowest singlet-excited states. The dependence on excitation intensity is best explained by any of the following third-order processes: (a) 3 S ₁→3 S ₀; (b) 2 S ₁+ T , → 2 S ₀+ T ₁; (c) S ₁+ 2 T ₁→ 3 S ₀. However, (c) is ruled out because it predicts heavy T ₁-destruction which is not observed. Contribution from the second order process: 2 S ₁→ S ₀ is probable, but even the data of Monger and Parson show that it is insufficient by itself. Two-photon absorption: S ₀+ hv ₁→ S ₁; S ₁+ hv ₁→ S _n; S _n S ₀+ hv ₂ could also account for the high-order fluorescence and its dependence on excitation intensity. [ S ₀, S ₁ S _n are ground, first excited and a higher excited singlet states, respectively, of antenna bacteriochlorophyll, T _t is the lowest triplet state, c/v , is the exciting wavelength (694 or 868 nm) and c/v ₂ the wavelength of the high-order fluorescence (445, 535. or 600 nm), where c = velocity of light.] Maximum values are estimated for some of the rate constants.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

A Water-Soluble Luminescence Oxygen Sensor

We developed a water-soluble luminescent probe for dissolved oxygen. This probe is based on (Ru[dpp(SO₃Na)₂]₃) Cl₂, which is a sulfonated analogue of the well-known oxygen probe (Ru[dpp]₃)Cl₂. The compound dpp is 4,7-diphenyl-1,10-phenanthroline and dpp(SO₃Na)₂ is a disulfonated derivative of the same ligand. In aqueous solution in the absence of oxygen (Ru[dpp(SO₃Na)₂]₃)Cl₂ displays a lifetime of 3.7 μs that decreases to 930 ns on equilibrium with air and 227 ns on equilibrium with 100% oxygen. The Stern–Vohner quenching constant is 11330 M⁻¹. This high oxygen-quenching constant means that the photoluminescence of Ru(dpp[SO₃Na]₂)₃Cl₂ is 10% quenched at an oxygen concentration of 8.8 x 10⁻⁶ M , or equilibration with 5.4 torr of oxygen. The oxygen probe dissolved in water displays minimal interactions with lipid vesicles composed of dipalmityl-L-α-phosphatidyl glycerol but does appear to interact with human serum albumin. The absorption maximum near 480 nm, long lifetime and large Stokes'shift allow this probe to be used with simple instrumentation based on a light-emitting diode light source, allowing low-cost oxygen sensing in aqueous solutions. To the best of our knowledge this is the first practical water-soluble oxygen sensor.     

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.     

ELECTRIC LINEAR DICHROISM OF P700 CHLOROPHYLL U-PROTEIN COMPLEXES

Abstract— The P₇₀₀ chlorophyll a -protein complex (CPI) isolated from green plants was oriented in aqueous solutions using pulsed electric fields of up to 6700 V cm^-1. The electric linear dichroism spectrum is reported in the range of 400–720nm. Positive peaks in the linear dichroism Δ A = A _I - A ₁ (where A_I and A₁ are the absorbance components in which the polarizer orientation is parallel and perpendicular with respect to the electric field. respectively) are observed at 443 and 686 nm. The ΔA signal at 686 nm is discussed in terms of either a specialized chlorophyll form absorbing at 686 nm. or due to an exciton component absorbing at the same wavelength.     

INACTIVATION OF Trypanosoma cruzi TRYPOMASTIGOTE FORMS IN BLOOD COMPONENTS BY PHOTODYNAMIC TREATMENT WITH PHTHALOCYANINES

Abstract— -Three phthalocyanine dyes HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂ (Pc 4), HOSiPc-OSi(CH₃)₂(CH₂)₃N+(CH₃)3I- (Pc 5) and aluminum tetrasulfophthalocyanine hydroxide (AlOHPcS₄) were evaluated for their ability to inactivate the trypomastigote form of Trypanosoma cruzi in fresh frozen plasma (FFP) and red blood cell concentrates (RBCC). The compound Pc 4 was found to be highly effective in killing T. cruzi, Pc 5 less effective and AlOHPcS₄ ineffective. With FFP as the medium, a complete loss of parasite infectivity in vitro (≥5 log₁₀) was found to occur with 2 μ M Pc 4 after irradiation with red light (>600 nm) at a fiuence of 7.5 J/cm², while with RBCC as the medium, a complete loss was found to occur at a fiuence of 15 J/cm². Even without illumination, Pc 4 at 2 μ M also killed about 3.7-4.1 log₁₀ of T. cruzi in FFP during 30 min. Observed differences in T. cruzi killing by the various phthalocyanines may relate to differences in binding; Pc 4 binds to the parasites about twice as much as Pc 5. Ultrastructural analysis of treated parasites suggests that mitochondria are a primary target of this photodynamic treatment. The data indicate that Pc 4 combined with exposure to red light could be used to eliminate bloodborne T. cruzi parasites from blood components intended for transfusion. The inactivation of T. cruzi by Pc 4 in the dark suggests a possible therapeutic application.     

THE EFFECT OF IRRADIANCE ON VIRUS STERILIZATION AND PHOTODYNAMIC DAMAGE IN RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINES

Abstract— Phthalocyanines are being studied as photosensitizers for virus sterilization of red blood cells (RBC). During optimization of the reaction conditions, we observed a marked effect of the irradiance on production of RBC damage. Using a broad-band light source (600–700 nm) between 5 and 80 mW/ cm², there was an inverse relationship between irradiance and rate of photohemolysis. This effect was observed with aluminum sulfonated phthalocyanine (AlPcS_n) and cationic silicon (HOSiPc-OSi[CH₃]₂ [CH₂]₃N⁺[CH₃]₃I^- phthalocyanine (Pc5) photosensitizers. The same effect occurred when the reduction of RBC negative surface charges was used as an endpoint. Under the same treatment conditions, vesicular stomatitis virus inactivation rate was unaffected by changes in the irradiance. Reduction in oxygen availability for the photochemical reaction at high irradiance could explain the effect. However, theoretical estimates suggest that oxygen depletion is minimal under our conditions. In addition, because the rate of photohemolysis at 80 mW/cm² was not increased when irradiations were carried out under an oxygen atmosphere this seems unlikely. Likewise, formation of singlet oxygen dimoles at high irradiances does not appear to be involved because the effect was unchanged when light exposure was in D₂O. While there is no ready explanation for this irradiance effect, it could be used to increase the safety margin of RBC virucidal treatment by employing exposure at high irradiance, thus minimizing the damage to RBC.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

Singlet-Oxygen Generation from Liposomes: A Comparison of 6β-Cholesterol Hydroperoxide Formation with Predictions from a One-Dimensional Model of Singlet-Oxygen Diffusion and Quenching

Abstract— We measured 6β-cholesterol hydroperoxide (6β-CHP), a specific singlet-oxygen (O₂(δg)) product, during irradiation of unilamellar dimyristoyl 1-α-phosphatidylcholine liposomes containing cholesterol and zinc phthalocyanine (ZnPC). The effects of liposome size, the hydrophobic (O₂(¹δ_g)) quencher, β-carotene, and hydrophilic O₂(¹δ_g) quenchers upon the amount of 6β-CHP formed were determined and interpreted in terms of a one dimensional model of ₂(¹δ_g) quenching and diffusion. The model correctly predicted (1) that the amount of 6β-CHP was increased with increasing liposome size, (2) that P-carotene was more effective at reducing 6β-CHP formation in 400 nm diameter liposomes than 100 nm diameter liposomes and (3) that the hydrophilic quencher, water, was also more effective in large liposomes than in small liposomes.
The hydrophobic quencher, β-carotene, was more effective at reducing the formation of 6β-CHP than at reducing the 1270 nm O₂(¹δ_g) emission. This difference was found to be due to the size distribution present in the liposome preparations because the difference between the 6β-CHP data and the 1270 nm emission data was much smaller in liposome preparations with a narrow size distribution. When a significant size distribution was present, the 6β-CHP data were weighted more heavily with large-diameter liposomes, while the 1270 nm emission data were weighted more heavily with small-diameter liposomes.     

THE PHOTOCHEMISTRY OF 14 C-5-BROMO-2''-DEOXYURIDYLYL-(3''→5'')-THYMIDINE DETERMINATION OF QUANTUM YIELDS AS A FUNCTION OF pH

Abstract— Quantum yields for the formation of the major photoproduct of 2-¹⁴C-5-bromo-2'-deoxyuridylyl-(3'→5')-thymidine (BrdUpT) have been determined by irradiation of the 2-¹⁴C-BrU-labeled dinucleotide at pH 2.3, 5.9, 7.05, 8.0 and 10.25 with U.V. light at 280nm. At acidic and neutral pH the quantum yield was 0.0063; the value decreased markedly above pH 8.0 to 0.0025 at pH 10.25. Some evidence of the formation of additional photoproducts at high and low pH was found. Some aspects of the mechanism of the reaction are discussed.
Consideration of p K_a values calculated for singlet and triplet excited states indicates that the decrease in the quantum yield of main photoproduct at high pH is due to dissociation of the excited bromodeoxyuridine moiety. It is suggested that the formation of BrdUpT photoproduct and the debromination of bromouracil-labeled DN A occur via different excited states.     

RECONSTITUTION OF ALLOPHYCOCYANIN FROM Mastigocladus laminosus WITH ISOLATED LINKER POLYPEPTIDE

The core linker polypeptide L_c^8.9 was isolated from Mastigocladus laminosus and purified on a preparative scale. A method for the reconstitution of allophycocyanin (AP)—linker complexes from isolated polypeptides was developed. The complex (α^AP(β^AP)₃ L_c^8.9 was reconstituted and compared to (α^APβ^AP) and (α^APβ^AP)₃ by sucrose density gradient ultracentrifugation, absorption, fluorescence emission and circular dichroism spectroscopy. Differences in the spectra of reconstituted and of directly isolated AP complexes are discussed.     

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.     

Photochemical Reactions and Phototoxicity of Sterols: Novel Self-perpetuating Mechanism for Lipid Photooxidation

Abstract— Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22-ergostatetraen-3bT-ol (9-DHE) and 5,7,9(11)-cholestatrien-3bT-ol (9-DDHC), respectively. Human skin lipids are known to contain 9-DHE. We have also found 9-DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7-dienes (ergosterol or 7-dehydrocholesterol), these photoactive sterols constitute a self-regenerating source of singlet molecular oxygen (₁O₂) during irradiation in vivo or in vitro with UVA bT15-400 nm). The quantum yield for photosensitization of ₁O₂ by 9-DHE was estimated to be 0.09. The ₁O₂ is scavenged by the dienes and the rate constant for ₁O₂ quenching by ergosterol was found to be 1.2 times 10₇ M -₁ s_-1 in methyl t-butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8-endo-peroxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9-trienes and trapped with the spin trap 5,5-dimeth-yl-1-pyrroline W-oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.     

WAVELENGTH DEPENDENCE AND KINETICS OF UV-INDUCED FREE RADICAL FORMATION IN THE HUMAN CORNEA AND LENS

Abstract— The wavelength dependence of free radical formation in the human lens and cornea was obtained in the ultraviolet-visible (UV-VIS) range with electron paramagnetic resonance. The spectral feature of having their maxima close to 290 nm, negligible intensity above 320 nm, and appreciable intensity toward 260 nm is common to both of these tissues.
The kinetics of free radical formation in the lens and cornea was interpreted in terms of a two component exponential function with characteristic rate constants k _l and k ₂, the latter of which has the same value for both the lens and cornea.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY AMINES—I. A FLASH PHOTOLYSIS STUDY OF THE REACTION BETWEEN TRIPLET METHYLENE BLUE AND AMINES

Abstract— Flash photolysis was used to study the reduction of the triplet state of methylene blue by both alkyl- and aryl-amines. The extent of the formation of the semireduced form of the dye yielded rate constants of interaction between the triplet state and the amine ( k ₅). A correlation between log k ₅ and ionization potentials for alkylamines (slope = -1.75 eV^-1) was interpreted as evidence for the formation of a partial charge-transfer intermediate. The rate constants ( k ₅) calculated for aryl-amines approached the rate of diffusion in many cases. A Hammett plot for a series of N, N-dimethyl-anilines yielded a moderately large p value (– 3.28) consistent with the formation of a charge-transfer intermediate. It was concluded that reaction of amines with triplet methylene blue leads to the formation of a partial charge-transfer intermediate which may undergo complete electron transfer to yield radicals, or undergo spin inversion and return to the ground state.     

SOLID PHASE PHOTOREDUCTION OF METHYLVIOLOGEN ON CELLULOSE SENSITIZED BY TRIS(2,2'-BIPYRIDYL)RUTHENIUM COMPLEX

–Methylviologen (MV²⁺) adsorbed on cellulose could be reduced photochemically in the solid phase sensitized by tris(2,2'-bipyridyl)ruthenium(II) complex, [Ru(II)(bpy)₃], using disodium ethylene-diaminetetraacetic acid (Na₂EDTA) as a reducing agent. Formation of the cation radical (MV ⁺.) was confirmed by visible and EPR spectra. The MV⁺. formed on cellulose was remarkably stable against air oxidation and rapidly accumulated even by the irradiation under air. Water adsorbed on the cellulose greatly retarded the photoreaction. Action spectrum showed that the excitation of Ru(II)(byp)₃ is responsible for the photochemical reaction. The quenching of the emission from Ru(II)(bpy)*₃ by MV²⁺. indicated that a primary photochemical reaction occurs between Ru(II)(bpy)*₃ and MV²⁺. The main reaction path is the reduction of MV²⁺ by Ru(II)(bpy)₃, giving MV⁺. and Ru(III)(bpy)₃, followed by the reduction of Ru(III)(bpy)₃ to Ru(II)(bpy)₃ with Na₂EDTA, which in turn is oxidized to give carbon dioxide.     

Generation and Characterization of Psoralen Radical Cations

Abstract— Radical cations of psoralen, 8-methoxypsoralen(8-MOP) and 5-methoxypsoralen have been generated by photosensitized electron transfer in acetonitrile and aqueous buffer/acetonitrile (1:1) and have absorption maxima at 600, 650 and 550 nm, respectively. The radical cations have lifetimes of 5 p.s under these conditions, are unreactive toward oxygen and show behavior typical of ar-ylalkene radical cations in their reactivity toward nucle-ophiles and the precursor psoralens. Direct 355 nm excitation of 8-MOP in aqueous buffer at physiological pH results in monophotonic photoionization to give 8-MOP_*+ with a quantum yield of 0.015.The 8-MOP_*+ reacts with both guanosine and adenosine mononucleotides ( k = 2.5 times 10₉ and 3.4 times 10₇ M_-1 s₁, respectively) via electron transfer to give the purine radical cations, but does not react with pyrimidine mononucleotides. These results suggest that reactions of psoralen radical cations generated by electron transfer or photoionization may be involved in psoralen/UVA therapy.     

SPECTROSCOPY AND PHOTOCHEMISTRY OF 8-SUBSTITUTED 5-DEAZAFLAVINS

Abstract— Absorption, fluorescence and phosphorescence spectra as well as fluorescence and phosphorescence quantum yields of 8-X-5-deazaflavins (X = C1, NO₂, p -NO₂-C₆H₄, N(CH₃)₂, NH₂, p -NH₂-C₆H₄, p -N(CH₃)₂-C₆H₄-N=N) were determined. It was found that all these data are highly influenced by the substituent at position 8 of the 5-deazaisoalloxazine skeleton. Also the photoreduction of 8-X-5-deazaflavins in the presence of electron donors was studied. It was established that the photoreduction leads to the formation of a 5,5'-dimer and/or a 6,7-dihydro compound. Reduction of the C(6)-C(7) bond is promoted by strong electron-donating substituents and bulky electron donors. 5-Deazaftavins with a reducible substituent at position 8 exhibit reduction of the substituent prior to the reduction of the 5-deazaisoalloxazine skeleton.     

MECHANISMS OF PHOTOELIMINATION REACTIONS OF ALKYL BENZOHYDROXAMATES

Abstract— Photolysis at 254 nm of alkyl benzohydroxamates [C, H, CONHOR: R = CH₃ H₂CH₃ CH(CH₃)₂, CH₂C₆H₅ CH(CH₃)C₂H₅ CH(CH₃)- n -C₆H₁₃] in acetonitrile or hydrocarbon solvents gives benzamide. These reactions can be sensitized by benzophenone (at ca. 350 nm) and are quenched by cis-piperylene. Racemization occurred when 2-octyl (+)-benzohydroxamate was irradiated in cyclohexane. These results are consistent with a mechanism involving a triplet biradical. Photolysis of phenyl benzohydroxamate [C₆H₅CONHOC₆H₅] and benzyl N -methylbenzohydroxamate [C₆H₅CON-(CH₃)OCH₂Q₆H₅] cannot be quenched with ris-piperylene and appear to be singlet reactions.     

FREE RADICALS INDUCED BY PHOTOLYSIS OF FROZEN SOLUTIONS OF THYMIDINE-5'-MONOPHOSPHATE: AN ESR STUDY

Abstract— The photolysis (Δ < 220 nm) of thymidine-5'-monophosphate was studied by electron-spin resonance (ESR) in acidic and alkaline phases. In both cases, the H–addition radical at the C₆ position is detected at 77°K. At 225°K, a triplet 1:2:1 is observed, which suggests a H abstraction radical from the CH₃ group. When oxygen is present during irradiation, a peroxide–type radical is observed, which results partly from a reaction like R + O₂→ ROO and partly from an energy transfer from thymidine-5'-monophosphate to oxygen, probably in the ¹π₀ state.