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1.
The second derivative of absorption, fluorescence and fluorescence excitation spectra of chlorophyll a in concentrated solutions and films was investigated. More than 14 forms of pigment aggregates, which can be divided into two types--with narrow 8-10nm) and wide (25-40nm) low temperature (-196 degrees C) spectra bands, were found. For the most part of the aggregated forms, the position and half width of the bands, as well as the Stokes shift and relative quantum yield were determined. The comparison of the spectral characteristics points to the indentity of the aggregates and corresponding native forms of Chl. a. It is shown that the universal relationship between absorption and fluorescence bands in applicable to the aggregates of the two types and the energy of resonance interaction between monomers in the aggregates is evaluated. 相似文献
2.
Protochlorophyll (PChl) forms were performed in Triton X-100 detergent micelles. The concentration of Triton X-100 was 7·10?4 M (above the critical micellar concentration); the concentration of PChl varied between 1.6·10?5 and 1.8·10?4 M. Absorption, fluorescence and circular dichroism (CD) spectra were registered. The absorption spectra were resolved into Gaussian components by computer analysis. PChl forms with absorption bands at 632–634, 638, 652–654, 663–664, 668 and 676 nm and with fluorescence emission bands at 634–636, 640–644, 652–655, 677–678, 686 and 694–696 nm were observed in micellar solutions of different PChl concentrations. The CD spectra showed a strong dependence on the concentration of PChl: positive CD signals or positive Cotton effects were observed in the vicinity of 650 nm. The intensity of these signals increased in parallel with increasing concentration of PChl. No CD signals were found in the region of the longer wavelength absorption bands. These data show that the PChl exists in many different forms in this system, and the spectroscopic properties of these forms are determined by different molecular interactions viz., interactions of PChl with Triton X-100 or water molecules and/or by the aggregation of PChl. 相似文献
3.
Aqueous solutions of copper-proteins containing type-3 centres (ceruloplasmin, tyrosinase, haemocyanin), excited within their absorption bands at 325-345 nm, show typical luminescence spectra. The emission bands peak at 415-445 nm and their decay time is no longer than 10 ns. A strong analogous fluorescence is obtained also by excitation of concentrated solutions of carboxylic acids and amino acids, which show again absorption bands around 330 nm. Such a fluorescence, although less intense, is also observed in copper(II) carboxylate solutions. In contrast, no fluorescence has been recorded in solutions of acetic anhydride and of polypeptides (valinomycin, gramicidin D), which do not have free carboxyl groups. We tentatively attribute this novel fluorescence in the investigated copper proteins to interactions between carboxyl groups of amino acids at, or near, the active site. 相似文献
4.
1. The absorption spectrum of quinacrine in aqueous solution, in the visible region, changes with the pH of the medium in the pH range from 6.0 to 9.0 with an isosbestic point at 353 nm. This indicates that the monoprotonated (quinacrine - H+) and the diprotonated (quinacrine - 2H+) forms of quinacrine at equilibrium in this pH range have a 1 to 1 stoichiometry. 2. The monoprotonated and the dipronated forms to quinacrine exhibit similar fluorescence emission spectra, but distinctive fluorescence excitation spectra. 3. The relative fluorescence quantum yields of quinacrine in aqueous media of various pH values are estimated. The relative fluorescence quantum yield of quinacrine at pH 9.0 is more than 3 fold of that at pH 6.0. 4. The fluorescence excitation and emission spectra, as well as the relative fluorescence quantum yield of quinacrine associated with non-energized submitochondrial membranes, are similar to those of quinacrine alone. 5. Analyses of the absorption spectra, the fluorescence excitation spectra and the relative fluorescence quantum yield indicate that the energy-linked fluorescence decrease of quinacrine associated with the energized submitochondrial membranes results from the protonation of quinacrine - H+ to form quinacrine - 2H+. 6. Quantitative data are provided indicating that the maximal efficiency of protonation of quinacrine - H+ to form quinacrine - 2H+ depends on the concentration of H+ in the membranes generated through energy coupling, and the concentration of quinacrine - H+ initially present in the reaction medium. Under optimal conditions virtually complete conversion of quinacrine - H+ into quinacrine - 2H+ is observed. 7. The fluorescence intensity of quinacrine, either alone or associated with non-energized submitochondrial membranes, decreases with increasing temperature. When quinacrine is associated with the energized membranes, however, its fluorescence intensity increases slightly with increasing temperature. This unusual fluorescence behavior towards temperature, together with the fact that under optimal conditions virtually all the quinacrine molecules associated with the energized membranes are in the diprotonated form, further substantiate our earlier conclusion that the diprotonated quinacrine molecules are tightly bound to the energized membranes in a fashion which does not permit ready equilibration with the external medium. 相似文献
5.
Absorption maximum positions of three LW Chl forms in pea chloroplasts were estimated using 77 K excitation spectra of fluorescence
detected in their maxima (720, 732 and 746 nm). The 705, 714 and 723 nm components were revealed in the second derivative
plots of the excitation spectra. The same maxima were found in normalized excitation spectra obtained with dividing excitation
spectra by absorption spectrum. It was confirmed that the observed maxima belong to absorption of LW fluorescing Chl forms.
The same maxima were displayed in an action spectrum of P700 oxidation measured at room temperature. It confirms the energy
transfer from LW Chl forms to P700. Close to 50% efficiency of bulk Chl forms in both excitation of LW fluorescence and P700
oxidation was found. Analysis of the shape of normalized excitation spectra suggests that there is no energy exchange among
LW Chl forms. Their location and physiological role are discussed.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
Borisevich NA Dubovsky VL Mikhalchuk AL Raichyonok TF Tikhomirov SA Tolstorozhev GB 《Biopolymers》2004,74(1-2):105-109
Based upon an analysis of the absorption spectra, the fluorescence and fluorescence excitation spectra, and the lifetime of the excited states of aqueous solutions (pH 7.4) of 2,3-dimethoxy-8-azagona-1,3,5(10),13-tetraene-12,17-dione, it is established that two centers of different type exist: one emits long-wave (L-centers) and the other short-wave (S-centers) fluorescence. Irradiation of solutions increases the number of L-centers and greatly decreases that of S-centers. Examination of the picosecond transient absorption spectra and their kinetics revealed that the initial photochemical stage is defined as the ionization of a considered molecule and the subsequent one, as the formation, most probably, of the dehydroderivative of a parent molecule. 相似文献
7.
Absorption, fluorescence and excitation fluorescence spectra of pheophytin a have been measured in aqueous solutions of nonionic (Triton X-100), anionic (sodium lauryl sulphate) and cationic (Cetyl pyridinium chloride) detergents at different concentrations and pH after system relaxation. By measuring the second derivative and differential spectra, it has been shown, that if detergent concentrations are lower than critical micelles concentration, or if the detergent is completely absent, the pigment forms conglomerates containing both dimeric and monomeric forms with an efficient energy transfer between them. If detergent concentrations are higher than critical micelles concentration, pheophytin a localizes in detergent micelle in monomeric form at neutral and acidic pH, and allomerizes at alkaline pH. The spectral characteristics of pheophytin a dimers in the conglomerate and its monomers in micelles poorly (if at all) depend on the sign of the detergent molecule charge. 相似文献
8.
E A Burstein E A Permyakov V A Yashin S A Burkhanov A Finazzi Agro 《Biochimica et biophysica acta》1977,491(1):155-159
The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed. 相似文献
9.
S N Khrapunov A F Protas A V Sivolob A I Dragan G D Berdyshev 《Molekuliarnaia biologiia》1984,18(4):979-987
By optical methods it has been previously shown that the globular "head" of histone H1 forms a hydrophobic cavity containing Tyr72. The latter is screened from the polar water surrounding and its intramolecular mobility is drastically hindered. As a consequence of the alteration in the micromilieu are a long wave shift (lambda max = 279,5 nm) and a more pronounced longwave absorption spectra, higher anisotropy (A = 0,11), augmented quantum yield of fluorescence (approximately 0,2) and a decrease of the Stern-Volmer constant for Hl at fluorescence quenching by acrylamide. It was found that changes in fluorescence intensity of histones are connected with alterations in the quantum yield of fluorescence at lambda exc = = 265 nm, but not at lambda exc = 280 nm. The changes in fluorescence intensity at light excitation 280 nm (F280) and 265 nm (F265) are in good accordance with shift delta E286 in differential absorption spectra. Introduction of parameter Cf = F280/F265 allows to study shifts of excitation spectra instead of shifts in absorption spectra of histones. This method has certain advantages, since it permits investigations with lower protein concentrations and in turbid solutions. The data obtained allow to draw out Tyr72 of histone Hl into a special class of fluorescent-tyrosyls, that differ in properties from those of other tryptophandevoided proteins: RNAse, insulin and core-histones H2A, H2B, H3 and H4. 相似文献
10.
Our aim was to disprove the widespread misconception that Förster resonance energy transfer (FRET) is the only explanation for observing fluorescence from ANS (8-anilino-1-naphthalenesulfonic acid) and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) following excitation at 280 nm in the presence of protein. From ultraviolet (UV) absorption spectra and fluorescence emission spectra of bis-ANS and ANS in buffer and ethanol, direct excitation at 280 nm was found to be the dominant mechanism for the resulting dye fluorescence. Furthermore, Tyr/Trp quenching studies were performed for solutions of N-acetyl-l-tryptophanamide, heat-stressed immunoglobulin G (IgG), and bovine serum albumin (BSA) by monitoring changes in steady state fluorescence spectra and time-resolved fluorescence decays as a function of dye concentration. Stronger quenching of the intrinsic BSA and IgG fluorescence in steady state than in time-resolved fluorescence by bis-ANS and ANS pointed toward static quenching being the dominant mechanism in addition to dynamic quenching and/or FRET. In conclusion, one should consider the role of direct excitation of ANS and bis-ANS at 280 nm to ensure a proper interpretation of fluorescence signals resulting from dye-protein interactions. When ANS or bis-ANS is to be used for protein characterization, we recommend selectively exciting the dyes at the higher absorption wavelength maximum (370 or 385 nm, respectively). 相似文献
11.
Etiolated leaves of three different species, maize, wheat, and pea, as well as a pea mutant (lip1) were used to compare the excitation spectra of protochlorophyllide (Pchlide) in the red region. The species used have different
composition of short-wavelength and long-wavelength Pchlide forms. The relation between different forms was furthermore changed
through incubating the leaves in 5-aminolevulinic acid (ALA), which caused an accumulation of short-wavelength Pchlide forms,
as shown by changes in absorption and fluorescence spectra. This is the first time a comprehensive comparison is made between
excitation spectra from different species covering an emission wavelength range of 675–750 nm using fluorescence equipment
with electronic compensation for the variations in excitation irradiance. The different forms of Pchlide having excitations
peaks at 628, 632, 637, 650, and 672 nm could be best measured at 675, 700, 710, 725, and 750 nm, respectively. Measuring
emission at wavelengths between 675– 710 nm gave an exaggeration of the short-wavelength forms and measuring at longer wavelengths
gave for the pea leaves an exaggeration of the 672 nm peak. In general, an energy transfer from short-wavelength Pchlide forms
to long-wavelength Pchlide forms occurred, but such an energy transfer sometimes seemed to be limited as a result of a discrete
location of the Pchlide spectral forms. The excitation spectra resembling the absorption spectrum most were measured at an
emission wavelength of 740 nm. Measuring the excitation at 710 nm gave higher intensity of the spectra but the short-wavelength
forms were accentuated. 相似文献
12.
Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
13.
Zubova NN Korolenko VA Astafyev AA Petrukhin AN Vinokurov LM Sarkisov OM Savitsky AP 《Biochemistry》2005,44(10):3982-3993
The yellow fluorescent protein from coral (zFP538) forms aggregates in water solutions. According to dynamic light scattering and gel filtration data, the aggregation number is approximately 1000-10000 at pH 8-9 and protein concentration 1 mg/mL. Gel filtration demonstrated that dissociation of the aggregates takes place upon dilution, and the molecular weight of the aggregates decreases with pH. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were used to obtain images of zFP538 in the solid state. It was shown that protein films are comprised of fluorescent ellipsoidal granules with a 50-300 nm major axis and a 30-130 nm minor axis. The dependence of zFP538 fluorescence on protein concentration between 1.2 x 10(-)(9) and 5.5 x 10(-)(7) M can be divided in two linear regions with different slopes indicating the existence of at least two different forms of zFP538. The fluorescence of zFP538 decreases with time upon acidification, and the decrease depends on pH and protein concentration. Between pH 3.5 and pH 5.5, relative residual fluorescence is higher for concentrated zFP538 solutions (about 10(-)(6) M) as compared with diluted ones (10(-)(7) M and below). Aggregation makes zFP538 more stable against fluorescence quenching upon acidification: the decrease in zFP538 fluorescence at protein concentration 1 mg/mL is completely reversible, unlike that observed for less concentrated solutions. This phenomenon may be due to the decrease in the freedom of chromophore mobility in zFP538 aggregates. 相似文献
14.
Dilute aqueous solutions of hematoporphyrin (Hp) and its derivative (Hpd or PF II) have been found to undergo a transformation (aging) on keeping at room temperature leading to (i) shift of the Soret band from 395 nm to 405 nm, (ii) disappearance of visible bands I (610 nm) and IV (503 nm) and (iii) shift of the first emission band from 615 nm to 580 nm. The transformation was concentration dependent. The effects of concentration and temperature on the absorption spectra were much more pronounced in Hp than in Photofrin II (PF II). Variation of pH resulted in changes in the relative intensities of the absorption bands, possibly due to formation of different ionic species at different pH. The rate of transformation was accelerated in the presence of Zn ions (0.01 microM) and considerably increased at higher (50 microM) concentration. The effect of Cu ions was different from the effect of aging. It formed the metal-chelate even when present in very small amounts. The results (absorption and fluorescence analysis) suggest that in dilute solutions (conc. less than or equal to 2 microM) of Hp and PF II, Zn ions present in glass and water as impurity, deform the porphyrin nucleus leading to changes in the conjugated ring symmetry and hence changes in the absorption and fluorescence spectra, while in higher concentrations (greater than 2 microM) it forms the metal chelate as evidenced by their absorption and fluorescence spectra. 相似文献
15.
J E Gill M M Jotz S G Young E J Modest S K Sengupta 《The journal of histochemistry and cytochemistry》1975,23(11):793-799
The optical absorption and fluorescence characteristics of 7-animo-actinomycin D were determined to evaluate its potential as a fluorescent cytochemical probe. At pH 7.0, the absorption maximum and fluorescence excitation maximum are both at 503 nm; the fluorescence emission is at 675 nm. When this compound forms complexes with DNA in solution, the absorption and fluorescence excitation maxima shift to 543 nm and the fluorescence emission shifts to 655 nm. The fluorescence quantum yield is 0.016 for 7-amino-actinomycin D free in solution and 0.01-0.02 for complexes with native DNA. The 7-amino-actinomycin D also exhibits fluorescence shifts characteristic of binding when put into solution with poly(dG-dC) poly(dG-dC), but not with poly(dI-dC) poly(dI-dC). The spectral characteristics are the same at pH 7.0 whether the solvent is 0.01 M PO4 with 0.0001 M EDTA or Earle's salts with 0.025 M N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid. 相似文献
16.
Photoadaptation in marine phytoplankton : changes in spectral absorption and excitation of chlorophyll a fluorescence
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The optical properties of marine phytoplankton were examined by measuring the absorption spectra and fluorescence excitation spectra of chlorophyll a for natural marine particles collected on glass fiber filters. Samples were collected at different depths from stations in temperate waters of the Southern California Bight and in polar waters of the Scotia and Ross Seas. At all stations, phytoplankton fluorescence excitation and absorption spectra changed systematically with depth and vertical stability of the water columns. In samples from deeper waters, both absorption and chlorophyll a fluorescence excitation spectra showed enhancement in the blue-to-green portion of the spectrum (470-560 nm) relative to that at 440 nm. Since similar changes in absorption and excitation were induced by incubating sea water samples at different light intensities, the changes in optical properties can be attributed to photoadaptation of the phytoplankton. The data indicate that in the natural populations studied, shade adaptation caused increases in the concentration of photosynthetic accessory pigments relative to chlorophyll a. These changes in cellular pigment composition were detectable within less than 1 day. Comparisons of absorption spectra with fluorescence excitation spectra indicate an apparent increase in the efficiency of sensitization of chlorophyll a fluorescence in the blue and green spectral regions for low light populations. 相似文献
17.
The polarized absorption, photoacoustic, fluorescence emission, and fluorescence excitation spectra of whole cells of cyanobacteria Synechocystis sp. embedded in a polymer film were measured. The bacteria cells, as it follows from anisotropy of absorption and fluorescence spectra, were even in a non-stretched polyvinyl alcohol film oriented to a certain extent. The measurements were done for such film in order to avoid the deformation of cyanobacteria shapes. Part of the samples was bleached by irradiation with strong polarized radiation with electric vector parallel to the orientation axis of cells. The anisotropy of photoacoustic spectra was higher than that of absorption spectra and it was stronger changed by the irradiation. Polarized fluorescence was excited in four wavelength regions characterised by different contribution to absorption from various bacteria pigments. The shapes of emission spectra were different depending on wavelength of excitation, polarization of radiation, and previous irradiation of the sample. The fluorescence spectra were analysed on Gaussian components belonging to various forms of pigments from photosystems (PS) 1 and 2. The results inform about excitation energy transfer between pools of pigments, differently oriented in the cells. Energy of photons absorbed by phycobilisomes was transferred predominantly to the chlorophyll of PS2, whereas photons absorbed by carotenoids to chlorophylls of PS1. 相似文献
18.
Low-Temperature Fluorescence Excitation Spectra for Long-Wavelength Emission as a Function of Greening in Euglena gracilis and Chlorophyll A Concentration In Vitro: A Mathematical Model to Describe Both Systems
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Seymour Steven Brody 《Biophysical journal》1968,8(2):210-230
A systematic study was made of the spectrum for exciting long-wave-length fluorescence (at 77°K) during the first 100 hr of greening in Euglena gracilis. A band at 705-710 nm is observable after cells have been greening in light for 30 hr. The ratio of the 705-nm to the 675-nm peak increases during greening, reaching a maximum value at 85 hr, then declining. With concentrated solutions of chlorophyll a, fluorescence excitation spectra are similar to those observed in vivo. The ratio of aggregate to monomer bands increases with concentration of chlorophyll, reaching a maximum value in ethanol and in pyridine at about 3 × 10-2 M and 6 × 10-2 M respectively, then declining. Several model systems were analyzed. It is shown that the band observed in solution with maximum at 705-710 nm is not an artifact of the fluorescence apparatus; it does not arise from undissolved chlorophyll; it does not arise from a fluorescent or nonfluorescent impurity; it does not arise solely from light absorption by a dimer or larger aggregate of chlorophyll. Agreement is obtained between the experimental observations and the results of a mathematical model by including terms for the efficiency of energy transfer from monomeric to dimeric chlorophyll, as well as for the formation of dimers by an equilibrium reaction. 相似文献
19.
Adrenolutin 3,5,6-trihydroxy-1-methylindole is an intermediate in the metabolism of adrenaline and in the formation of adrenochromo-melanins. Excitation and emission spectra, quantum yield of the adrenolutin fluorescence in water, D2O, ethanol, methanol, acetone and aqueous phosphate buffer at different pH at 293K temperature are reported. Dependence of the quantum yield of adrenolutin on its concentration are measured. Lifetimes of 0.1 mM adrenolutin in water and ethanol are 32.0 +/- 0.2 ns and 9.2 +/- 0.2 ns respectively. Also fluorescence and phosphorescence spectra of adrenolutin in methanol at 110K are obtained. Degrees of polarization and angles between the dipoles for the three main bands absorption of adrenolutin from measurements at 103K are calculated. Adrenolutin may be classified as one of the most strongly fluorescing metabolites. Broad excitation spectrum and high quantum yields make this compound a potential effective acceptor of excitation energy. 相似文献
20.
Properties of protonated dimeric forms of meso-tetraphenylporphine (TPP) and meso-tetra(p-aminophenyl)porphine (TAPP) bound with copolymer and also complexes produced by associated TAPP bound with copolymer, Mn2+, and Fe3+ are investigated by absorption, luminescence, and Raman spectroscopy. According to absorption spectra of protonated dimers of TPP, three dimeric forms of the porphyrin are observed in the ground state. However, selective excitation of these forms according to the fluorescence spectra reveals only two dimeric forms in the excited state. In contrast, similar selective excitation of TAPP bound with copolymer in aqueous-dioxane solution results in weak changes in the fluorescence spectra, nevertheless, there is strong interaction between porphyrin and macromolecular carboxyl groups in the ground state. In the case of the formation of the complexes between associated TAPP bound with copolymer, Mn2+ and Fe3+, a new band in the near IR region with a maximum at 840 nm is built up in the fluorescence spectrum. However, this near IR emission is completely quenched when new strong vibrational bands at approximately 1800 and 1900 cm-1 are revealed in the resonance Raman spectra of the complexes. The observed effects are explained in terms of direct participation of water molecules involved in the water-porphyrin dimeric complex in the processes of transformation of excitation energy. The involvement of water in this dimeric complex can lead to redistribution of flows of the energy degradation when transition metal ions play a role of the agent which enhances the trapping properties of the porphyrin-metal-ions complexes. 相似文献