PHOTOSENSITIZING EFFECTS OF 8-METHOXYPSORALEN ON THE SKIN OF HAIRLESS MICE—11. TRAIN AND SPECTRAL DIFFERENCES FOR TUMORIGENESIS

Abstract. –The absorption and fluorescence characteristics of subchloroplast particles highly enriched in P700 (1 P700 to 10–15 chlorophyll a molecules) and of two artificial systems are presented here. The fluorescence characteristics measured were excitation and emission spectra, and the degree of polarization of fluorescence. The model systems studied were chlorophyll a and pheophytin a in a polystyrene matrix, and a colloidal mixture of these two pigments with bovine serum albumin. Effects of 0.5% sodium dodecyl sulfate on the optical properties of the P700-enriched particles are also described. The importance of pigment-pigment and pigment-protein interactions in determining the fluorescence properties of these particles are discussed. The possible role of pheophytin in these preparations needs further investigation.     

CHLOROPHYLL-PHOTOSENSITIZED OXIDATION OF HYDROQUINONE AND p-PHENYLENEDIAMINE DERIVATIVES

Abstract— ESR and photovoltaic studies on light-induced one-electron transfer between chlorophyll a and electron donors in the absence of oxygen show (1) the possible conversion of photo-reduced chlorophyll a and p-benzosemiquinone ion radicals to their non-ionic radicals in methanol solutions of low pH, (2) the production of ESR absorption of tetrachloro-p-benzosemi-quinone even in benzene, enhanced by the addition of triethylamine or methanol, and (3) the transfer of one electron from tetramethyl-p-phenylenediamine to either excited chlorophyll a or pheophytin a in methanol at pH above 3.6 but not to pheophytin a at pH below 1 0 where its radical cation appears to accept an electron from excited pheophytin a . Bacteteriochloro-phyll is also shown to be capable of photooxidizing hydroquinones and tetramethyl-p-phenyl-enediamine.
The presence of oxygen enhances chlorophyll a -photosensitized oxidation of hydroquinone and tetrachloro-hydroquinone by one-electron transfer to oxygen and of trimethylhydro-quinone probably by two-electron trnasfer to oxygen. A free radical from excited chlorophyll a-oxygen interaction is formed in these reactions, but rapidly quenched in the case of trimethyl-hydroquinone. This, kind of free radical is not formed in pheophytin a . Tetramethyl- p -phenyl-enediamine readily undergoes chlorophyll a-photosensitized oxidation by oxygen in any pH region.     

CHLOROPHYLLS IN POLYMERS. II. PHEOPHYTIN a IN POLYMERS and THE INFLUENCE OF STRETCHING ON THE STATE OF CHLOROPHYLLS IN ANHYDROUS POLYMER FILMS

Abstract— In the first part of this study the spectral properties of pheophytin a in rigid, unstretched anhydrous polyvinyl alcohol and nitrocellulose films have been studied in order to establish the influence of the central magnesium atom on the state of chlorophylls in polymer systems. The absorption, fluorescence, excitation spectra and fluorescence intensity decays in the polymer films and in the solutions from which they are cast are reported. It is shown that pheophytin a aggregate formation is influenced by the nature of the polymer system. An aggregate of pheophytin a is found in polyvinyl alcohol films over a wide concentration range. On the other hand, pheophytin a exists in the monomeric form in unstretched nitrocellulose films at concentrations below 6 × 10^-6 mol/g.
In the second part of this work, the influence of stretching of the films on the state and distribution of embedded chlorophyll pigments, is described. Here we show that the chlorophyll a molecules are found to undertake a heterogenous distribution in polyvinylalcohol matrices, since stretching partially disrupts the pocket-like structures present in unstretched films. In contrast, chlorophyll a and pheophytin a molecules can be embedded in a monomeric state in nitrocellulose matrices and moreover they remain homogeneously distributed upon stretching. The chlorophyll/nitrocellulose system is concluded to be a useful model system for studies of donor-donor energy transfer processes.     

THE USE OF LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY TO MONITOR THE ALLOMERIZATION REACTIONS OF CHLOROPHYLL a and PHEOPHYTIN a:m IDENTIFICATION OF THE ALLOMERS OF PHEOPHYTIN a

Abstract— This paper reports a new method for monitoring the allomerization reactions of chlorophyll a and pheo-phytin a. Complex mixtures are generated from illuminating pure compounds and monitored using both diode array high-performance liquid chromatography (DAD-HPLC) and liquid chromatography-mass spectrometry (LC-MS). LC-MS allows molecular weight and fragment ion analysis of significant HPLC peaks. Five products of the degradation of chlorophyll a can be simultaneously detected in a mixture, namely the monohydroxy allomer, the methoxylactone allomer, pheophytin a and the two corresponding allomers of the pheophytin. It is demonstrated that more than one pathway must be involved in the in vitro photodegradation of chlorophyll a as shown by the simultaneous existence of several intermediates.     

On the photodecomposition of chlorophyll in vitro. I. Reaction rates

Abstract— Chlorophyll solutions are irreversibly bleached by light in the presence of oxygen. The action spectra parallel the absorption spectra for both chlorophyll a and b. The reaction is of second order with a Q₁₀ of 1.26. The reaction rates for chlorophylls a and b are of the same order of magnitude. Depending upon the light source, the initial rate for chlorophyll a is slightly higher, by a factor of 1.15 to 1.30. The rate for pheophytin is lowe 3 by several orders of magnitude. No pheophytin has been detected in the reaction products of the irradiated chlorophyll solutions in the absence of water.     

A comparative study of dimerization of chlorophylls and pheophytins by fluorescence and odmir

Optical and ODMR data for dimers of pheophytin a and b and chlorophyll a and b are presented. It is proposed that pheophytin a forms a parallel dimer arising from π-π interaction, binding being essentially different from that in the corresponding chlorophyll dimer. The dimer of phcophytin a is less stable (K ≈ 10⁴ ol/mol) than that of chlorophyll a(K ≈ 10⁶ ol/mol).     

Der Einbau von Magnesium in Liganden der Chlorophyll-Reihe mit (2,6-Di-t-butyl-4-methylphenoxy)magnesiumjodid

Introduction of Magnesium into Ligands of the Chlorophyll Series by (2,6-Di-t-butyl-4-methylphenoxy)magnesium Iodide Experimental details are given for the new method of introducing magnesium into porphinoid ligands by (2,6-di-t-butyl-4-methylphenoxy)magnesium iodide (1) , previously published in preliminary form [1]. Besides magnesium octaethylporphyrinate (14) , methyl pyrochlorophyllide a (10) , methyl chlorophyllide a (8) , and methyl bacteriochlorophyllide a (12) , the complexation of pheophytin a (2) to chlorophyll a (3) and of pheophytin b (4) to chlorophyll b (5) are described.     

Chromatographic determination of changes in pigments in spinach (Spinacia oleracea L.) during processing

The content of individual chlorophyll and carotenoid pigments is determined in three spinach varieties (Lorelei, Springfield, and Ballet) after processing. Raw spinach and spinach that is steam-blanched for 3, 9, or 15 min is stored frozen at -24 degrees C for 6 months. In addition, spinach is air-dried at 75 degrees C, packed in atmospheric air or nitrogen, and stored at ambient temperature for 6 months. Processing has a significant effect on the content of individual chlorophyll and carotenoid pigments; however, there are no differences between varieties in their content of total and individual pigments in raw, frozen spinach. Increasing blanching time resulted in decreased contents of chlorophyll a and b and increased contents of chlorophyll a' and b' and pheophytin a and b because of pheophytinization. Changes in the color because of pheophytinization are only detected after 15 min blanching. The carotenoid pigments are more stable than the chlorophyll pigments during blanching. (all-E)-Violaxanthin is significantly reduced, caused by degradation to other xanthophylls, such as neochrome, during blanching. There are no significant differences in the content of chlorophyll a and b of dried spinach and blanched, frozen spinach. Formation of chlorophyll a' and b', pheophytin a and b, and chlorophyll a-1 and b-1 is observed after drying. The content of pheophytin a and b is significantly lower in dried versus blanched frozen samples. In dried spinach that is stored in atmospheric air, the content of beta-carotene [599 mg/kg dry matter (DM)] is significantly lower compared with nitrogen (766 mg/kg DM), and the content of (all-E)-lutein is lower than in blanched frozen spinach. Neochrome is not detected in raw spinach but in steam-blanched and dried spinach. No differences are observed in the content of (all-E)-neoxanthin, (9'Z)-neoxanthin, (all-E)-violaxanthin, (all-E)-lutein epoxide, or neolutein A and B between spinach that is stored frozen after 3 min blanching and dried spinach.     

REVERSIBLE LIGHT-TNDUCED SINGLE ELECTRON TRANSFER REACTIONS BETWEEN CHLOROPHYLL AND HYDROQUINONE IN SOLUTION*

Abstract— EPR and optical studies demonstrate the occurrence of reversible temperature-independent light-induced single electron transfer reactions between chlorophyll (and pheophytin) and hydroquinone in degassed ethanol solutions. Oxygen is shown to quench this process, presumably by interacting with chlorophyll excited states. Pyridine and isoquino-line increase the effectiveness of the hydroquinone as an electron donor to chlorophyll, probably by acting as bases to form hydroquinone anions (or perhaps ion-pairs) which are more easily oxidized. Hydroquinone is found to be more effective in electron transfer than is benzoquinone, suggesting that the chlorophyll excited state is a better oxidizing agent than it is a reducing agent.     

Expanded separation technique for chlorophyll metabolites in Oriental tobacco leaf using non aqueous reversed phase chromatography

An improved separation method for chlorophyll metabolites in Oriental tobacco leaf was developed. While Oriental leaf still gives the green color even after the curing process, little attention has been paid to the detailed composition of the remaining green pigments. This study aimed to identify the green pigments using non aqueous reversed phase chromatography (NARPC). To this end, liquid chromatograph (LC) equipped with a photo diode array detector (DAD) and an atmospheric pressure chemical ionization/mass spectrometer (APCI/MSD) was selected, because it is useful for detecting low polar non-volatile compounds giving green color such as pheophytin a. Identification was based on the wavelength spectrum, mass spectrum and retention time, comparing the analytes in Oriental leaf with the commercially available and synthesized components. Consequently, several chlorophyll metabolites such as hydroxypheophytin a, solanesyl pheophorbide a and solanesyl hydroxypheophorbide a were newly identified, in addition to typical green pigments such as chlorophyll a and pheophytin a. Chlorophyll metabolites bound to solanesol were considered the tobacco specific components. NARPC expanded the number of detectable low polar chlorophyll metabolites in Oriental tobacco leaf.     

PHOTOELECTRON TRANSFER BETWEEN A CHARGED DERIVATIVE OF CHLOROPHYLL AND FERRICYANIDE AT THE LIPID BILAYER-WATER INTERFACE

Abstract— Flash illumination of a lipid bilayer containing a positively charge pigment: chlorophyll b cholyl hydrazone and separating two salt solutions, one of which contained ferricyanide, resulted in a photovoltage of ∼20mV, acceptor side negative. The positive charge on the pigment resulted in several novel effects. (1) The photo-emf is twice that of chlorophyll a and five times that of chlorophyll b at a given concentration. A higher surface concentration of the charged derivative is the likely cause of this effect. (2) The pheophytin of chlorophyll b cholyl hydrazone produces about one-half the photo emf of the magnesium derivative whereas pheophytin a or b produced only one-tenth the signal. This may be a reflection of the changed redox potential of the cation chlorophyll b cholyl hydrazone. (3) A voltage drop of 100 mV across the membrane, the acceptor side negatively biased, causes a 3–4-fold increase in the charge recombination rate. Biasing the acceptor side 100 mV positive has no effect. Chlorophyll a or b do not show this field effect. This asymmetric effect is explained as a movement of the more polar chlorophyll dication towards the water interface, leading to more rapid reaction with donor. Thus the kinetics of the charge reversal are a sensitive and specific probe of the polar interfacial region of the lipid bilayer-water interface.     

The primary electron donor of photosystem II of the cyanobacterium Acaryochloris marina is a chlorophyll d and the water oxidation is driven by a chlorophyll a/chlorophyll d heterodimer

We present a theoretical analysis of the flash-induced absorbance difference spectrum assigned to the formation of the secondary radical pair P(+)QA(-) in photosystem II of the chlorophyll d-containing cyanobacterium Acaryochloris marina. An exciton Hamiltonian determined previously for chlorophyll a-containing photosystem II complexes is modified to take into account the occupancy of certain binding sites by chlorophyll d instead of chlorophyll a. Different assignments of the reaction center pigments to chlorophyll a or d from the literature are investigated in the calculation of the absorbance difference spectrum. A quantitative explanation of the experimental data requires one chlorophyll a molecule per reaction center, located at the site of P(D1). The remaining sites are occupied by chlorophyll d and pheophytin a. By far, the lowest site energy is found for the accessory chlorophyll of the D1 branch, Chl(D1), which represents the sink of excitation energy and therefore the primary electron donor. The cationic state is stabilized at the chlorophyll a, which drives the oxidation of water.     

SPECTROSCOPIC CHARACTERIZATION OF TWO FORMS OF THE D1-D2-CYTOCHROME b559 COMPLEX FROM SUGAR BEET

Two D1-D2-cytochrome b559 complex forms, called RCIIa and RCIIb, with different pigment stoichi-ometry were characterized using absorption and surface-enhanced resonance Raman scattering spectroscopy and spectral gaussian deconvolution. Electronic absorption spectra of the RCIIb at 277 K showed significant differences compared to RCIIa, i.e . a strong decrease in the absorbance due to carotenoid and chlorophyll for the same amount of pheophytin. A reduced carotenoid and chlorophyll content in RCIIb was also observed in the surface-enhanced resonance Raman scattering spectra. Spectral deconvolution elicited three main absorption bands at 680, 672 and 669–670 nm, which were ascribed to P680, pheophytin and accessory chlorophyll, respectively. In addition, a minor component around 667 nm was observed in the RCIIb, most probably due to some reaction center inactivation. Calculation of the relative area under the gaussians together with pigment stoichiometry data suggest that the 680, 672 and 669–670 nm components contain, respectively, two chlorophylls, two pheophytins and four chlorophylls for the RCIIa, and two chlorophylls, two pheophytins and two chlorophylls for the RCIIb.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY-II. PHOTOSENSITIZATION BY CHLOROPHYLL,PHEOPHYTIN AND BACTERIOCHLOROPHYLL OF A ONE-ELECTRON TRANSFER REACTION BETWEEN ETHANOL AND QUINONE*

Abstract— Chlorophyll, pheophytin and bacteriochlorophyll sensitize a one-electron transfer in the presence of quinones in ethanol, to produce a ternary complex of ground state porphyrin analog, alcohol cation radical and semiquinone anion radical as the primary photo-product at low temperature. A similar photo-oxidation of alcohol to produce a binary complex is caused by direct excitation of quin-one in the absence of chlorophyll. The mechanisms of these reactions and their implications for photosynthesis are discussed.     

High-performance liquid chromatographic screening of chlorophyll derivatives produced during fruit storage

Reversed-phase high-performance liquid chromatography with photodiode array and fluorescence detection was applied to the systematic screening of chlorophylls and derivative pigments. The chromatographic procedure proposed made it possible to successfully separate and identify eight chlorophyll derivatives (the a and b forms of chlorophyll, chlorophyllide, pheophytin and pheophorbide) by using a linear gradient of methanol, acetone and ammonium acetate. The method has been routinely applied to study chlorophyll degradation during the postharvest storage of cherimoya (Annona cherimola, Mill.) fruits. The brilliant green colour even at maturity, and its high chlorophyllase and Mg-dechelating activities, indicate that this plant material might be suitable for investigating the as yet not well known chlorophyll breakdown processes.     

Photophysical studies of pheophorbide a and pheophytin a. Phosphorescence and photosensitized singlet oxygen luminescence

The triplet states of pheophorbide a and pheophytin a were studied in several environments by direct measurement of the phosphorescence of the pigments and photosensitized singlet oxygen (1O2) luminescence. The spectra, lifetimes and quantum yields of phosphorescence and the quantum yields of 1O2 generation were determined. These parameters are similar for monomeric molecules of both pigments in all the environments studied. Aggregation of the pigment molecules leads to a strong decrease in the phosphorescence and 1O2 luminescence intensities, which is probably due to a large decrease in the triplet lifetime and triplet quantum yield in the aggregates. The results obtained for pheophorbide a and pheophytin a are compared with those previously reported for chlorophyll alpha. The data suggest that the photodynamic activity of the pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures. Aggregated molecules seem to have a much lower activity.     

ROOM TEMPERATURE TIME-RESOLVED IN μs RANGE DELAYED LUMINESCENCE OF CHLOROPHYLL a AND CHLOROPHYLL-LUTEIN MIXTURE SOLUTIONS

Using time-resolved in μS range luminescence spectroscopy, we observed at 20°C the emission of chlorophyll a, pheophytin a and chlorophyll a-lutein mixture solutions. This delayed emission exhibits several maxima in the650–750 nm region. The positions and kinetics of decay of delayed emission bands depend on chlorophyll concentration, and vary as a result of pheophytinization and addition of lutein. Our results can be explained by supposition that upon excitation, charge transfer species are formed in various pigment complexes. The back electron transfer reactions yield chlorophyll excited singlet states contributing to observed delayed emission. Delay in emission seems to be due also to the trapping of excitation on the triplet states of various forms of pigment and its detrapping with the participation of thermal energy followed by energy transfer to the forms of pigment characterized by different decay times.     

Abnormally high light absorption by pigments adsorbed on metals

An abnormally high extinction coefficient (almost 100 times as high as in solution) has been found for chlorophyll and pheophytin adsorbed on a smooth metal. These and some literature data suggest this phenomenon may be rather general, particularly for substances having a system of conjugated bonds.     

Chemical bonding of chlorophylls and plant aminic axial ligands impact harvesting of visible light and quenching of fluorescence

In the present work, we tested the mode of interaction of all three polyamines (putrescine, spermidine and spermine) with chlorophyll a and b, as well as pheophytin a and b. The results showed that all three polyamines bind to the Mg ion of chlorophyll ring as probed by Raman spectroscopy. The coordination of spermine with Chl b has the most interesting features from all pigments tested. Spermine induces reversible increases and decreases of the fluorescence yield of Chl b at about 661 nm. Interestingly, equilibrium between a high-fluorescence yield conformation and a low yield is feasible by the interaction of chlorophyll b and aminic ligands. Furthermore, absorption data for the diagnostic regions of 518 and 535 nm are provided for all combinations of pigments and ligands. The significance and consistence of these results with respect to photochemical and bioenergetic principles are discussed.     

REDUCTION OF THE EXCITED SINGLET STATE OF CHLOROPHYLL AND PHEOPHYTIN AT THE ORGANIC CRYSTAL/AQUEOUS SOLUTION INTERFACE

Abstract— Reduction of the excited singlet state of chlorophyll a , chlorophyll b , and pheophytin a at the surface of perylene, anthracene, and chrysene single crystals has been measured as hole injection current. The dependence of the quantum yield on the standard free-energy difference of the reaction was in accordance with theoretical expectations without correcting for an interaction energy. The maximum quantum yield of only about 10⁻² holes per absorbed photon is ascribed to a very small effective lifetime of the excited singlet state due to concentration quenching of the excited singlet state in the adsorbed dye layer.