EFFECT OF AGGREGATION ON THE HEMATOPORPHYRIN-SENSITIZED PRODUCTION OF SINGLET MOLECULAR OXYGEN

Abstract— The hematoporphyrin-sensitized production of singlet molecular oxygen, O₂(¹Δ_g), has been investigated in methanol and in aqueous solution. The quantum yield for formation of O₂(¹Δ_g) (Φ_Δ) has been measured by both steady-state (oxygen consumption) and time-resolved (near-infrared luminescence) methods. In methanol, both techniques indicate that Φ_Δ= 0.76 and the value remains independent of sensitizer concentration over a wide range. This finding is consistent with the dye persisting in a monomelic form in methanol solution. In contrast, Φ_Δ decreases markedly with increasing sensitizer concentration in water due to dimerization of the dye. Analysis of the steady-state data indicates Φ_Δ values of 0.74 and 0.12, respectively, for monomer and dimer. It is further shown that the efficiency whereby quenching of the triplet state by O₂ results in generation of O₂(¹Δ_g) is substantially lower for the dimer than for the corresponding monomer. Because monomer and dimer possess quite different absorption spectral profiles, the efficacy for photodynamic action with hematoporphyrin exhibits a pronounced wavelength dependence.     

SOLVENT EFFECTS ON THE TAUTOMERIZATION OF 5'-DEOXYPYRIDOXAL. A PHOTOPHYSICAL STUDY

Abstract— Absorption and fluorescence spectra of 5'-deoxypyridoxal (DPL) in various pure solvents and mixtures were recorded both at room temperature and over the range10–65°C. The areas under the absorption bands were analyzed to obtain the mole fraction (f_N, f_z) of two tautomers (the zwitterionic, Z, and neutral, N, forms) in the ground state. The following spectral parameters were determined from the fluorescence spectra: Stokes shift (Δ v ), fluorescence quantum yield of the neutral form (Q_N), fluorescence ratio of the neutral to the zwitterionic form (ø_N/ø_Z) and the rate constant of tautomerization ( k ₁) from Z to N in the excited state. Some of these parameters (f_N, Δ v , Q_N, k ₁) were found to depend on the proton donor character of the solvent, whereas others (ø_N/ø_Z) depended on its dipole moment. Thus, the absorption and fluorescence spectra of DPL allow one to obtain information on the polarity and the concentration of –OH groups on its environment.     

ON THE DISTRIBUTION OF EXCITATION ENERGY TO TWO PHOTOREACTIONS OF PHOTOSYNTHESIS

Abstract— From kinetic analysis of the Z-scheme we have derived expressions for fractions of open reaction centers and throughput quantum yield. The fraction of absorbed quanta available to photoreaction II has been treated by the wavelength dependent function, α. However, for purposes of analysis in terms of throughput electron flow it is necessary to introduce a related function, α', for actual processing of excitations by photoreaction II; α' differs from α if the individual quantum yields of the two photoreactions are not equal. 'Included in our findings are the following. As a close approximation quantum yield is maximum at α'= 0.5 and a symmetrical function of α' around the line α' =0.5. As previously noted, lowering of the apparent equilibrium constant between the photoreactions flattens dependence of quantum yield on α' near α' = 0.5 but also lowers the attainable quantum yield. For any given equilibrium constant the effective fractions of open reaction centers are equal at α' = 0.5 and mirror images of each other around the line α' = 0.5. Slow changes in quantum yield following changes in wavelength (the State 1-State 2 phenomenon) are explainable in terms of changes in α' which also have the effect of flattening dependence of quantum yield on wavelength. We have used these changes to estimate the neutral wavelength for α' = 0.5 as 681–682 nm in Chlorella .     

INTRAMOLECULAR EXCIMER FLUORESCENCE: A NEW PROBE OF PHASE TRANSITIONS IN SYNTHETIC PHOSPHOLIPID MEMBRANES

Abstract— The molecule (1,1'-dipyrenyl)-methyl ether [dipyme] shows intramolecular excimer fluorescence in competition with fluorescence from the locally excited pyrene chromophore. This intensity ratio I_c/I_m is sensitive to solvent viscosity. The molecule is soluble in synthetic phospholipid membranes. ¹H NMR studies suggest that the molecule is localized in the hydrocarbon region of the membrane. Fluorescence measurements at various temperatures of dipyme dissolved in these membranes show that I_c/I_m is exceedingly sensitive to fluidity changes accompanying both the pretransitions and the gel to liquid crystalline transitions of the membrane. These studies can be carried out at a mole ratio of probe to lipid 10²-10³ smaller than that necessary to observe bimolecular pyrene excimer formation.     

FLUORESCENCE AND THE LOCATION OF TRYPTOPHAN RESIDUES IN PROTEIN MOLECULES

Abstract— Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (ΔΛ), spectral maximum position (Λ_m), fluorescence quenching by external ionic quenchers, lifetime (b), and quantum yield ( q ) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (Λ_m 330–332 nm, ΔΛ= 48–49nm, q 0.11, τ= 2.1 ns) and two on the surface. One of the latter is completely exposed to water (Λ_m# 350–353 nm, ΔΛ= 59–61 nm, q # 0.2, τ= 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (Λ_m# 340–342 nm, ΔΛ= 53–55 nm, q # 0.3, = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence lifetime for various proteins, show good concordance with independent experimentally determined values.     

ON THE PRIMARY PHOTOPROCESS OF 124-kdalton PHYTOCHROME

The photoreaction between P_τ and the first detectable intermediate, lumi-R, of 124-kdalton oat phytochrome has been investigated at low temperatures. The temperature dependence of the quantum yields of the photoreactions, P_τ to lumi-R and lumi-R to P_τ, has been determined. From measurements over a temperature range from 119 to 155 K, an activation barrier of 3.6 ± 0.5 kJ mol ₁ is found for the photoreaction of P_τ with 661-nm actinic light. A higher value (5.7 ± 0.7 kJ mol ^-1) is found for the photoreaction of lumi-R to P^τ. with 698-nm actinic light. Increased quantum yields are found in deuterated buffer solutions at low temperatures. The activation energies for deuterated phytochrome (3.2 ± 0.7 kJ mol^–1 for P_τ with 661-nm irradiation and 6.2 ± 1.2 kJ mol^-1 for lumi-R at 698-nm irradiation) are identical within the limits of error with those of protonated phytochrome. The lack of a deuterium effect for the activation energies favors the Z,E-isomerization rather than proton transfer or tautomerization for the chromophore photochemistry during P_τ⇄lumi-R conversion.     

SINGLET OXYGEN GENERATION BY PORPHYRINS AND THE KINETICS OF 9,10-DIMETHYLANTHRACENE PHOTOSENSITIZATION IN LIPOSOMES

Abstract— Two new sensitizers are introduced for a potential use in photodynamic therapy: Zn²⁺- and MG²⁺-tetrabenzoporphyrin (ZnTBP and MgTBP). A comparative study of the quantum yields of singlet oxygen generation (Φ_Δ) of hematoporphyrin derivative (HpD), Photofrin II (PF-II), Zn²⁺-phthalocyanine tetrahydroxyl [ZnPC(OH)₄] and the newly introduced sensitizers ZnTBP and MgTBP in liposomes, as well as the kinetics of a photochemical reaction sensitized by them, was made by employing the fluorescent membrane probe 9,10-dimethylanthracene (DMA). We followed the photosensitization of DMA in real time by monitoring its fluorescence decrease at 457 nm and found that DMA's photosensitization is oxygen mediated. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the Φ_Δ values were evaluated. At 10 μ M sensitizer in an aqueous suspension of 2 mg/mL egg phosphatidylcholine (EPC), HpD was found to have the largest value of Φ_Δ (0.215), followed by PF-II (0.191), ZnTBP (0.023), MgTBP (0.019) and ZnPC(OH)₄ (0.005). As a test of the method, Φ_Δ for methylene blue in ethanol was measured and found to be 0.45 as compared to 0.52 reported in the literature. Due to difference in the sensitizers' absorbances at the laser's wavelength, the reaction photosensitized by ZnTBP was the fastest with a time constant of 6.7 min, followed by MgTBP (8.7), PF-II (11.9), HpD (17.1) and ZnPC(OH)₄ (31.2), all at equal sensitizers' concentrations and laser intensities. The binding constants of the sensitizers to EPC liposomes are also reported.     

CHEMILUMINESCENCE IN THE PEROXIDATION OF TANNIC ACID

Abstract— Peroxidation of tannins with alkaline H₂O₂ is accompanied by weak chemiluminescence in the spectral region 480–800 nm. o-Di and tri-hydroxy groups of polyphenols undergo oxidation by a free-radical mechanism and a green intermediate anion-radical with absorption Δ_max= 600 nm is formed. The radical mechanism is supported by the low activation energy 14–20 kJ/mol and the quenching effect of radical scavengers. The reaction of the green intermediate with peroxy anions is the chemiluminescence rate limiting step. In the presence of a-hydroxy-methylperoxide formed from H₂O₂ and formaldehyde, the alkaline peroxidation of tannins is accompanied by strong red luminescence (420–800 nm). The base catalyzed decomposition of peroxides gives only a weak red emission (460–800 nm). Light intensity is enhanced in D₂O by a factor 6.5. Quenchers of O₂(¹Δ_g) and 1,3-di-phenylisobenzofurane diminish light intensity in non-aqueous solutions. The data suggest ¹O₂ participation in the observed chemiluminescence. Thermo-chemical calculations give —ΔH values from 250–1000 kJ/mol for one elementary reaction step which limits the mechanism of chemi-enereization. Chemiexcitation of tannins is relevant to biochemical mechanisms of aerobic degradation of aromatic compounds, energy utilization as well as to defense and resistance processes in plants.     

SPECTRAL PROPERTIES OF THE RHODOPSIN-SYSTEM OF THE CRAYFISH Astacus leptodactylus

Abstract— The absorption spectra of the membrane-bound and of the digitonin-solubilized visual pigment of crayfish Astacus leptodactylus were investigated by conventional spectrophotometry. A method was developed to isolate purified rhabdoms almost entirely free from screening pigments from a single retina. The quantity of isolated and purified rhabdoms from a single retina was sufficient to measure the absorption spectra of the visual pigment.
The absorption spectra of the chromoprotein system (R and M) show that both the membrane-bound and the digitonin-solubilized visual pigment isomers are stable at 0°C and pH 7.0. Rhodopsin and metarhodopsin are photoreversible under these conditions without any light-induced denaturation. The difference spectra for the chromoprotein isomers and those of different photostationary states yield maximal values for ΔE at 570 and 485 nm.
At neutral pH, 0°C, Λ_max of rhodopsin is 530 nm. Irradiation with light of Λ= 630 to 640 nm isomerizes rhodopsin nearly quantitatively to metarhodopsin with Λ_max, of 500 nm. The molar extinction coefficient of metarhodopsin is greater than that of rhodopsin by a factor of ˜ 1.41. each measured at its respective Λ_max Metarhodopsin can be isomerized to rhodopsin by irradiating at Λ > 630 nm. As the absorption spectra of the two chromoprotein isomers overlap, only part of the metarhodopsin can be reversed to rhodopsin. The maximal photoreversion can be achieved by irradiating at 460 nm. The stability of the digitonin-solubilized chromoprotein is remarkably dependent on temperature. Warming the digitonin extract of rhabdoms from 0 to 20 or 30°C caused a shift of the rhodopsin spectrum to shorter wavelengths (Λ_max= 485 nm) accompanied by a decrease of E_Λmax by about 30%.     

PHOTOKINETIC AND PHOTOPHYSICAL MEASUREMENTS OF THE SENSITIZED PHOTOOXIDATION OF THE TRYPTOPHYL RESIDUE IN N-ACETYL TRYPTOPHANAMIDE AND IN HUMAN SERUM ALBUMIN

Abstract— The photosensitized oxidation of 10–100 μ M N -acetyl-L-tryptophanamide (NATA) in neutral aqueous solution and in the presence of various dyes proceeds by a pure O₂(¹Δ_g)-involving mechanism. Incorporation of the tryptophyl (Trp) residue into the polypeptide chain of human serum albumin (HSA) has no influence on the mechanism and efficiency of Trp photooxidation when sensitized either by methylene blue, a non-binding dye, or by rose bengal, a dye that gives non-covalent 1: 1 complexes with HSA. This is due to the location of the Trp residue in close proximity of the protein surface and, in the case of rose bengal, to the coincidence of the photophysical properties (including the quantum yield of O₂(¹Δ_g) generation) for the free and HSA-bound dye. Hematoporphyrin also binds to HSA with 1: 1 stoichiometry, although at a different site from rose bengal. Bound Hp again displays photophysical properties very similar with those of free Hp; however, the efficiency of Trp photo-oxidation in HSA is about 5-fold higher than in NATA owing to a limited rearrangement of the protein structure, induced by Hp binding, which enhances the probability of chemical quenching of O₂(¹Δ_g) by the indole ring.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN IN SOLUTION

Abstract— Bimolecular rate constants for the quenching of singlet oxygen O*₂(¹Δ_g), have been obtained for several transition-metal complexes and for β-carotene. Laser photolysis experiments of aerated solutions, in which triplet anthracene is produced and quenched by oxygen, yielding singlet oxygen which then sensitizes absorption due to triplet carotene, firmly establishes diffusion-controlled energy transfer from singlet oxygen as the quenching mechanism in the case of β-carotene. The efficient quenching of singlet oxygen by two trans-planar Schiff-base Ni(II) complexes, which have low-lying triplet ligand-field states, most probably also occurs as a result of electronic energy transfer, since an analogous Pd(II) complex and ferrocene, which both have lowest-lying triplet states at higher energies than the O*₂(¹Δ_g), state, quench much less effectively.     

ESTIMATION OF ENERGY DISTRIBUTION AND REDISTRIBUTION AMONG TWO PHOTOSYSTEMS USING PARALLEL MEASUREMENTS OF FLUORESCENCE LIFETIMES AND TRANSIENTS AT 77 K

Abstract— A single-sample method for estimating energy distribution and redistribution among the two photosystems using fluorescence lifetimes and transients at 77 K is presented. In this method,α(the fraction of photons absorbed by photosystem I, PSI) is F_1(α)/(F_1(α)+ (τ_{F 1(M)}/τ_{F 2(M)}).F_2(M)) where, F_1(α) is the fluorescence intensity from PSI excited by photons initially absorbed by the latter, τ_{F 1(M)} and τ_{F 2(M)} are the maximum lifetimes of fluorescence from chlorophyll- a in PSI (1) and II (2), and, F_2(M) is the maximum fluorescence intensity from PSII (P level). Analysis of the intensities and lifetimes of wavelength resolved fluorescence of thylakoids (pH 7.0), with and without cations, leads to the following conclusions: The addition of 10 m M Na⁺ to cation-depleted thylakoids (pH 7.0) increases α by ˜ 10%, while the subsequent addition of 10 m M Mg²⁺ leads to three principal concomitant changes (in the order of importance): a 50% decrease in PSII to PSI energy transfer, a 20% increase in other radiation-less losses, and a 10% decrease in α.     

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.     

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.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC TUMOR THERAPY—I. PHOTOPHYSICAL PROPERTIES OF TWO CHLORIN DERIVATIVES

Abstract— Photophysical properties of two chlorin type molecules (CHLI) and (CHLII) were investigated in different solvents. Quantum yields of fluorescence φ_F of S, → T, intersystem crossing φ_T, and of singlet oxygen (¹Δ_g) formation φ_Δ, as well as the Stern-Volmer constants for the quenching of the S, states by oxygen and the bimolecular rate constants of quenching of ¹Δ_g by the chlorins were measured. The values of φ_T and φ_Λ can be given as 0.57 and 0.58 for CHLI and 0.69 and 0.58 for CHLII. The values of the fluorescence quantum yields, the strong absorption of the chlorins in the red (Λ > 630 nm) and the high values of the quantum yields for ¹Δ_g formation recommend the chlorin derivatives as potential markers and photosensitizers for tumor therapy.     

ACTION SPECTRA FOR PHOTOREACTIONS I AND II OF PHOTOSYNTHESIS IN THE BLUE-GREEN ALGA ANACYSTIS NIDULANS

Abstract— Action spectra for photoreactions I and II of photosynthesis were obtained for Anacystis nidulans and three of its variants which had altered chlorophyll/phycocyanin ratios. The spectra are properly scaled to each other. They provide information on contributions of phycocyanin and chlorophyll to initial absorption and final distribution of excitation energy to reaction centers I and II. In normally pigmented cells the light harvesting pigments for photoreaction I include about 40% of the phycocyanin and 84% of the chlorophyll. Both in normal cells and in cells with altered pigmentation excitation energy from phycocyanin is delivered to photoreaction II via a small number of chlorophylls. In response to alterations in chlorophyll/phycocyanin ratio Action I spectra showed large variations whereas Action II spectra were essentially invariant. The result is taken to mean that alteration in chlorophyll components in Anacystis is attended by a special restriction: there are only small changes in amount of chlorophyll accessible to photoreaction II in the face of large changes in amount committed to photoreaction I.     

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.     

MODIFYING FACTORS OF THE CELLULAR CONCENTRATION OF PHOTOLYASE MOLECULES IN Saccharomyces cerevisiae—II. EFFECTS OF PREILLUMINATION WITH LIGHT FLASHES

Abstract The effects of preillumination with photoreactivating light flashes before UV-irradiation on the number of photoreactivable complexes consisting of UV-induced DNA-damages and active photolyase molecules (N_PREact), on the fluence decrements, ΔD_PRE, that are obtained from two UV-survival curves without and with 1 flash photoreactivation and proportional to N_PREact were determined in haploid Saccharomyces cerevisiae cells. ΔD_PRE increased by preillumination from 0.115 Jm^–2 to 0.460 Jm^–2 and from 0.376 Jm^–2 to 0.494 Jm^–2 in cells in logarithmic growth phase and in stationary growth phase, respectively. Δ_PRF in log-cells that were preilluminated before and after resuspension in buffer at 40°C for 60 min was larger than ΔD_PRE in log-cells preilluminated only after resuspension in buffer.     

ELECTRON PARAMAGNETIC RESONANCE OF SOME TRIPHENYLMETHANE DYES IN THE TRIPLET STATE

Abstract— Electron paramagnetic resonance spectra of the triplet states of several triphenylmethane dyes in glassy solutions at 90 K have been measured and their zero-field splitting parameters estimated. Crystal violet and para rosaniline do not possess trigonal symmetry in their triplet states, and the unusually broad absorptions in the Δ M ₈= 1 region of the spectra have been attributed to the presence of different rotational isomers of the dye cations. A number of malachite green derivatives were investigated, but absorption of the triplet states of these dyes was only observed in the low field Δ M ₈= 2 region of the spectrum.     

RESONANCE RAMAN SPECTRA OF THE II-CATION RADICALS OF COPPER, COBALT, AND NICKEL METHYLOCTAETHYLCHLORINS: VIBRATIONAL CHARACTERISTICS OF CHLOROPHYLL MODELS

Abstract— By using excitation at 363.8 nm, resonance Raman (RR) spectra were obtained for Cu(II), Co(II), and Ni(II) complexes of methyloctaethylchlorin (MeOEC) and their ir-cation radicals. Additionally, the Raman spectra of the Cu(II) derivatives of (rarcs-octaethylchlorin (r-OEC) were included for comparison. The alkyl-substituted CuMeOEC exhibits a Raman spectrum that is nearly identical to that of the simpler (-CuOEC in both the neutral and oxidized states. Unlike the latter species, the cation radical of CuMeOEC is immune to oxidative dehydrogenation to porphyrin, and this has facilitated vibrational characterization of the ring-oxidized species. This study aims to compare the vibrational characteristics in the 1450 to 1700 cm^-t region of the metallochlorin ir-cation radicals to those of the corresponding oxidized metalloporphyrins. We focus on two modes in the1500–1520 cm^-1 and1620–1650 cm^-1 region that are analogous to the v₃ and v₁₀ vibrations, respectively, in the metalloporphyrin analogs. These vibrational modes are clearly defined in all species and exhibit a strong core-size dependency in the porphyrin complexes. The core-size study as well as the frequency changes upon oxidation support the conclusion that the v₃-like vibration in the chlorin species features substantial C_bC_b in addition to C_aC_m stretching character. The v,₀-like mode of the chlorin macrocycle, on the other hand, displays characteristics that closely resembles that of the porphyrin analog; consequently, these vibrations are of predominantly C_aC_m stretching character in both cases.