LOW-TEMPERATURE FLUORESCENCE SPECTRA OF CHLOROPHYLL a SOLUTIONS

Abstract Fluorescence and fluorescence polarization spectra of chlorophyll a dissolved in ethanol, n -propanol, EM (ethanol-methanol, 4:1) and EPE (ether, n -pentane, ethanol, 5:5:2) were measured at 77 K. An emission band ('shoulder') between the two usual fluorescence bands appears in such spectra of dilute solutions (concentration ˜ 10^-5 M ) of chlorophyll a (except in EPE). The position, intensity and half-width of this band were calculated using a computer. The correlation between electronic transitions of chlorophyll a and these emission bands is discussed.     

ABSORPTION AND FLUORESCENCE SPECTRA OF CHLOROPHYLL-a IN POLAR SOLVENTS AS A FUNCTION OF TEMPERATURE

Abstract— Absorption and fluorescence spectra of chlorphyll-a in aqueous alcohol were determined as a function of temperature between 173° and 293°K. From a comparison of these spectra with ones obtained in pure methanol, it is suggested that changes in molecular association occur within aggregates of chlorophyll-a in aqueous-alcoholic solution induced by variation of the dielectric constant. The latter varies over a wide range in aqueous alcohol as a function of temperature.     

水杨酸及其酯类化合物在不同溶剂中吸收光谱和荧光光谱的研究

本工作测定了水杨酸和水杨酸甲酯在不同溶剂中的吸收光谱和荧光光谱,并按在不同温度下测得的结果计算出它们在不同溶剂中平衡过程的函变△H.数据表明,在质子溶剂中,上述化合物有较小的△H值,即形成的分子内氢键只有较低的稳定性.根据这些结果可对该类化合物在不同溶剂中形成内氢键的能力作出恰当的评价.     

THE CORRELATION BETWEEN THE ABSORPTION AND THE FLUORESCENCE ENERGY SPECTRA,AND THE QUANTUM YIELD OF CHLOROPHYLL a IN DIFFERENT SOLVENTS†

Abstract— The applicability of Stepanov's relation to solutions of chlorophyll a in 17 solvents was determined. The value of T* (the local temperature of the excited molecules calculated by the relation) was higher than T (the ambient temperature) in 7 solvents and lower in one. The relative quantum yield determined by comparing the fluorescence spectrum with the thermal emission spectrum was fairly constant (? 1.0) up to 690 nm in dioxane, methylene chloride and dimethylformamide. In other solvents its value at 690 nm varied between 0.2 and 067. It is suggested that existence of a solute-solute or solute-solvent interaction in some solvents could possibly cause such a drop in the quantum yield.     

RESOLUTION OF THE FLUORESCENCE EXCITATION SPECTRUM OF INDOLE INTO THE 1La AND 1Lb EXCITATION BANDS*

Abstract— The fluorescence excitation spectrum and the excitation polarization spectrum of indole in propylene glycol were measured at — 58°C, after selecting by optical filters the emission originating from the ¹L_a electronic level. From the analysis of these spectra, the excitation spectrum was resolved into the ¹L_a and ¹L_a excitation bands. A similar resolution of the excitation spectrum of tryptophan is given. This method can also be applied to the resolution of the emission spectrum in cases of dual emission.     

CHLOROPHYLL a FLUORESCENCE YIELD IN LIVE CELLS AND SOLUTIONS

Abstract— Fluorescence yields of chlorophyll a in ether and methanol solutions ( F_s ), on the one hand, and in Chlorella pyrenoidosa and Anacystis nidulans cells (F_c), on the other hand, were determined by excitation at 600 mμ (instead of at 436 mμ, as was done in earlier research in our, and other, laboratories). The ratio of the yields Fs/F_c , was found to be 5.9 for Chlorella (compared to an ether solution of chlorophyll a ), and of 4.5 (as compared to a methanol solution—not too different from the corresponding ratios of fluorescence lifetimes, τ_s /τ_c which were determined earlier as 3.1 and 4, respectively. The much higher values of the yield ratio, previously reported for Chlorella (about 13, compared to chlorophyll a in ether solution), may have been due to disregard of light absorption in carotenoids in live cells; and perhaps also to quenching of chlorophyll excitation by carotenoids. The latter can occur (as suggested in an earlier publication) when chlorophyll is excited to its second singlet excited state.
For Anacystis , the yield ratios were now found to be 5.1 and 3.8, when compared to ether and methanol solutions respectively; while the previously determined lifetime ratios were 3.7 and 4.9, respectively. It remains to be seen whether the remaining differences between the lifetime ratios and the yield ratios are real and significant.     

RESOLUTION OF OVERLAPPING BANDS IN THE NEAR-UV ABSORPTION SPECTRUM OF INDOLE DERIVATIVES

Abstract— The second derivative spectra of tryptophan in water and in ethylene glycol at 22°C have been integrated in order to obtain the corresponding primitive functions. The integration was carried out by making use of Tchebychev polynomials. The results show that the integrated primitive functions do not correspond to the original absorption spectra of tryptophan in various solvents, but they reflect only the contributions of the ¹L_b bands of the indolic chromophore. The identification of the electronic component, which generates the second derivative spectrum, was based on the solvent insensitivity of the derivative peaks. The comparison between the absorption spectra reported in this paper and those calculated for the ¹L_b←¹A electronic transition of indole confirmed the assumption that the derivation process eliminates the broad, although more intense, contributions coming from the ¹L_a←¹A electronic transition.     

IN VIVO FLUORESCENCE SPECTROSCOPY OF CHLOROPHYLL IN VARIOUS UNRIPE AND RIPE FRUIT

—Low temperature (77 K) fluorescence emission spectra of slices obtained from the peel and various layers of the pericarp were recorded for fruits which remain green or undergo color break during ripening.
Fluorescence emission peaks characteristic of the photosystem II antennae (λ_F 686 nm) and reaction center (λ_F 696 nm), as well as of the photosystem I antenna (λ_F 730-740 nm), were present in the peel and all parts of the green pericarp of ripe kiwi, avocado and cantaloupe, as well as in ripe tomato and tangerine after color break. The pattern of the fluorescence emission spectra of all samples except that of the kiwi fruit was similar to that obtained from green photosynthetic tissue of leaves, indicating a normal organization of the chlorophyll-containing complexes of thylakoidal membranes. This pattern is characterized by a significantly higher emission at 730-740 nm relative to that of the 696 and 686 nm peaks. In contradistinction, the fluorescence emission at 686 and 696 nm was higher than that at 730 nm in the kiwi fruit, indicating a reduction in the size of the photosystem I antenna chlorophyll. In the innermost yellowish layers of the kiwi pericarp, a further loss of this antenna occurred, as well as disorganization of the photosystem II complex. The above conclusions are suggested also by measurements of variable fluorescence kinetics.
The results presented here indicate that fluorescence spectroscopy might be used as a tool for the study of chlorophyll organization during the growth and ripening periods of fruit.     

THE EFFECT OF pH ON THE ABSORPTION AND FLUORESCENCE SPECTRA OF SANGUINARINE

Abstract— The influence of pH in the range 1.0 to 13.0 on the structure of sanguinarine has been investigated by spectrophotometric and spectrofluorimetric measurements. The data on absorption maxima, molar extinction coefficient, fluorescence emission maxima, relative fluorescence intensity and fluorescence quantum yield of the sanguinarine under various pH are presented. It is suggested that the pH dependent absorbance and fluorescence property of sanguinarine is due to the formation of the carbinolamine by hydroxylation at C-6 at alkaline pH.     

ACIDITY DEPENDENCE OF THE ABSORPTION AND FLUORESCENCE SPECTRA OF ISOFLAVONE AND 7-HYDROXYISOFLAVONE

-The pH and H₀ dependence of the absorption and fluorescence spectra of isoflavone and 7-hydroxyisoflavone are reported. Isoflavone is fluorescent in acidic solution only, whereas 7-hydroxyisoflavone is fluorescent in all acidity ranges under investigation. Ground and first excited singlet state p K _a's have been determined spectrophotometrically and fluorimetrically, respectively. Excited state protolytic equilibration processes via a second order reaction (proton gain) are found to be too slow to compete efficiently with fluorescence. This is deduced from the close agreement between the p K _a's of the conjugate acids obtained by absorption and fluorescence titrations. On the other hand, photodissociation of 7-hydroxyisoflavone proceeds faster than its fluorescence decays. The experimental p K _a(S₁) is in fair agreement with the calculated one. 7-Hydroxyisofiavone forms a phototautomer (or exciplex) in the pH 2 to H₀-1 acidity range, which is characterized by its long wavelength emission. Quantum efficiencies are given for isoflavone and 7-hydroxyisoflavone in aqueous solutions of various acidities. Deuteration effects thereon are discussed.     

SOLVENT EFFECTS ON THE ELECTRONIC ABSORPTION AND FLUORESCENCE SPECTRA OF INDOLE-4-CARBOXYLIC ACID: PROTOTROPIC EQUILIBRIA IN AQUEOUS SOLUTIONS

Abstract— The absorption and fluorescence spectra of indole-4-carboxylic acid in various solvents have indicated that the -COOH group is more planar with respect to the indole ring in the first excited singlet state (S₁) than in the ground (S₀) state. Relatively large Stokes' shifts indicate that polarisability and dipole moment of the molecule are increased predominantly upon excitation. Prototropic reactions in the S₀ and S₁ states are the same. The -COO^- and -COOH+₂ groups are not coplanar in the S₀, but coplanar in the S₁ state. pH-dependent fluorescence spectra have revealed that both protonation and deprotonation of the -COOH group increase the basicity of the molecule upon excitation.     

FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDY OF THE STRUCTURE OF CHLOROPHYLL a and CHLOROPHYLL b IN HIGHLY DILUTE WATER-SATURATED CARBON TETRACHLORIDE SOLUTIONS

Abstract— Fourier-transform (FT) infrared (IR) absorption spectra have been measured for chlorophyll a (Chi a ), chlorophyll b (Chi b ), pheophytin a (Pheo a ), and pheophytin b (Pheo b ) in highly dilute (10^-5-10^-6 M ) water-saturated carbon tetrachloride solutions. Frequencies of IR bands due to C=O stretching modes of the 9-keto group indicate that Chi a assumes largely a dimeric structure in the concentrated (10^-2-10^-3 M ) water-saturated carbon tetrachloride solutions but it remains mostly a monomer with one or two coordinated water molecules in dilute (10^-5-10^-6 M ) solutions. Although it seems that Chi b also assumes predominantly dimeric form in concentrated solutions and monomelic form in dilute solutions, the relative intensity change of two C=O stretching bands ascribed to the free and coordinated 3-aldehyde groups with decreasing concentration suggests that the aldehyde group is also involved in formation of the dimer. The relative intensity of two C=O stretching bands due to the free and coordinated aldehyde groups changes significantly for Pheo b in water-saturated carbon tetrachloride solutions. This observation suggests that some of Pheo b also assume dimeric form via the aldehyde group in concentrated solutions.     

FLUORESCENCE CHARACTERISTICS OF INDOLES IN NON-POLAR SOLVENTS: LIFETIMES, QUANTUM YIELDS AND POLARIZATION SPECTRA

Abstract— Fluorescence lifetimes, quantum yields and polarization spectra were measured for indole, 3-methylindole and 2,3-dimethylindole in non-polar solvents. The results indicate simultaneous emission from thermally equilibrated ¹L_a and ¹L_b levels, with ¹L_a→¹ A dominating the 2,3-dimethylindole emission, and ¹L_b→¹ A dominating the indole emission. These results are consistent with previous assignments of the 0-0 transitions in absorption for these compounds. Radiative rates are: ¹L_a→¹ A , 2·0 × 10⁸ S^-1 and ¹L_b→¹ A . 0·62 → 10⁸ S^-1. In addition, the temperature dependence of the excitation and emission spectra are presented, which show that aggregation occurs with these indoles in hydrocarbons below approximately - 110°C. Possible applications to tryptophyl emission in the hydrophobic interiors of proteins are briefly discussed.     

OPTICAL ABSORPTION AND ELECTRON SPIN RESONANCE SPECTRA OF CATION RADICALS OF DIMERIC CHLOROPHYLL a IN LOW-TEMPERATURE MATRICES

Abstract— A chlorophyll (Chl) a solution in 3-methylpentane at 77 K exhibits an absorption spectrum with a distinct peak at 706 nm in the red-band region. The formation of the 706 nm absorbing species (S706) was reversible with respect to temperature change; no chemical change was observed. γ-Irradiation of the rigid 3-methylpentane solution at 77 K yields an absorption spectrum which can be ascribed to S706⁺ and S706⁻. When carbon tetrachloride, an electron scavenger, was added to the solution, the absorption of S706⁺ survived, which has peaks at 850 and 956 nm. It is assumed that the S706 is hydrogen-bonded dimeric Chi a , which may be regarded as a model of P700 in photosynthesis. Cation radicals of monomeric Chi a were formed in a γ-irradiated sec -butyl chloride solution at 77 K, and an absorption spectrum with peaks at 730 and 813 nm was recorded. ESR spectra of the cation radicals of S706 and monomeric Chi a are of a similar shape but their linewidths are 7.5 and 11.0 Gauss, respectively. The linewidth narrowing observed for S706⁺ is clear evidence for the assumption that S706 is dimeric Chi a. Comparison was made of the absorption spectrum of S706⁺ with the light-induced spectrum of P700 reported earlier.     

MULTIPLE EXCITATIONS AND THE YIELD OF CHLOROPHYLL a FLUORESCENCE IN PHOTOSYNTHETIC SYSTEMS

Abstract. Analysis of the effect of multiple excitations on chlorophyll a fluorescence yields in the green alga Chlorella reveals several distinct reactions. The first excitation in dark-z-adapted units produces photochemistry and the high yield state with a rise time of 35 ns. It is ascribed to a change in coupling between the antenna pigments and the photochemical trap. The second hit produces with the same quantum yield a quenched state which changes to the high yield state with a rise time of 4 μ s. This is ascribed to the formation and the decay of a particular carotenoid triplet state near the funnel or antenna-trap junction. Further hits produce enhanced quenching assigned to mobile triplets with lifetimes in the order of 100 ns. The fluorescence yield decreases monotonically with increasing excitations during the 7 ns pulse. This effect can be adequately ascribed to annihilation of excitations with lifetimes longer than the trapping time, or by a unique model of a multi-trapped unit. The latter model is favored by arguments based both on the absence of a local maximum in the graph of fluorescence yield vs excitation energy and on the fact that the high yield state shows a different behaviour on multiple excitation, fit by a single-trapped unit. This analysis is related to that used in experiments with ps flashes and is applied to the qualitatively different bacterial system.     

PHOTOSYNTHETIC ACTIVITY AND CHLOROPHYLL ABSORPTION SPECTRA OF FRACTIONS FROM THE ALGA,DUNALIELLA*

Abstract— Dunaliella chloroplasts were fractionated according to C. Arntzen et al, Biochim. Biophys. Acta 256 , 85–107, 1972. The initial French-press treatment and differential centrifugation produced Fraction 1 (Fr 1) enriched in photosystem I activity and a heavier Fraction 2 (Fr 2). When Fr 2 was treated with digitonin followed by either gradient or differential centrifugation, two more fractions were recovered: Fr 1 g with a photosystem 1 activity similar to that of Fr 1, and Fr 2 g with very low photosystem II activity. Photosystem II activity was considerably lower in these Dunaliella chloroplasts and fractions than in spinach particles measured under the same conditions, but the relative activities between the fractions were similar to those for spinach. Fr 2 always had greater photosystem II activity than Fr 1, but the digitonin fractions were low and similar in photosystem II activity. Photosystem II activity was measured as the reduction of 2, 6–dichlorophenol indophenol (DCIP) with H₂O, diphenylcarbazide (DPC) or Mn²⁺ as electron donor. The results indicated that exogenous manganous ion competed with H₂O as an electron donor to photosystem II in broken chloroplasts initially, but after 10–15 s of illumination, the Mn³⁺ formed began to reoxidize DCIP and a cyclic reaction ensued. DPC and Mn²⁺ appeared to react at different sites. Computer-assisted curve analysis of the absorption spectrum of each fraction revealed four major component curves representing the absorbing forms of chlorophyll a at 663, 670, 679 and 684 nm seen in numerous other in vivo chlorophyll spectra (C. S. French et al., Plant Physiol. 49 , 421–429, 1972). However, Fr 2g had approx. 20 percent more of Ca663 and Ca670 and 10% more absorption by chl b than Fr 1 which correlated with the difference in photosystem II activity. On the long wavelength side, Fr 2 g had no Ca694 and almost no photosystem I activity. The results are not sufficient to answer the question of whether the photosystem I particle obtained from the original homogenate is significantly similar to or different from the corresponding fraction obtained from Fr 2 with digitonin.     

ROLE OF HYDROXYL-BEARING AMINO ACIDS IN DIFFERENTIALLY TUNING THE ABSORPTION SPECTRA OF THE HUMAN RED AND GREEN CONE PIGMENTS

The human red and green cone pigments differ at either 15 or 16 amino acids, depending upon which polymorphic variants are compared. Seven of these amino acid differences involve the introduction or removal of a hydroxyl group. One of these differences, a substitution of alanine for senne at position 80, was found previously to produce a 5 nm blue shift. To determine the role of the remaining six hydroxyl group differences in tuning the absorption spectra of the human red and green pigments, we have studied six site-directed mutants in which single amino acids from the green pigment have been substituted for the corresponding residues in the red pigment. Blue shifts of 7 and 14 nm were observed upon substitution of phenylalanine for tyrosine at position 277 and alanine for threonine at position 285, respectively. Single substitutions at positions 65, 230, 233, and 309 produced spectral shifts of 1 nm or less. These data are in good agreement with a model based upon sequence comparisons among primate pigments and with the properties of site-directed mutants of bovine rhodopsin. Nonadditive effects observed in comparing the absorption spectra of red-green hybrid pigments remain to be explained.     

IN VIVO CHLOROPHYLL a FLUORESCENCE TRANSIENTS AND THE CIRCADIAN RHYTHM OF PHOTOSYNTHESIS IN GONYAULAX POLYEDRA

Abstract— The intensity of chlorophyll a fluorescence during the early part of fluorescence induction at O , initial fluorescence, and P, peak fluorescence, was higher during the day phase of the circadian cycle than during the night phase in continuous light (LL) conditions and was positively correlated with the rate of oxygen evolution. The circadian rhythm in fluorescence in LL persisted in the presence of 10μM 3–(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which blocks electron flow from photo-system (PS) II in photosynthesis. The rhythmic changes in fluorescence intensity are consistent with a lower rate constant for radiationless transitions during the day phase than during the night phase of the circadian rhythmicity. The circadian changes in the intensity of fluorescence were abolished at 77K, which may indicate the importance of structural changes in membranes in circadian oscillations.     

THE EFFECT OF RED AND FAR RED LIGHT ON CAROTENOID AND CHLOROPHYLL FORMATION IN PEA SEEDLINGS

Abstract— Treatment of etiolated pea seedlings with a short exposure to red light caused a stimulation of growth (size and dry wt production) and carotenoid synthesis during the following 48 hr compared with seedlings kept entirely in darkness.The effect is nullified by a following dose of far red light and thus the phenomenon is probably phytochrome-controlled.
Similar treatment with red light one hour before continuous illumination with white light tended to reduce the lag period for chlorophyll synthesis.Again a following dose of far red light reversed this response.     

SPECTRA AND EXTINCTION COEFFICIENTS OF NEAR-INFRARED ABSORPTION BANDS IN MEMBRANES OF Rhodobacter sphaeroides MUTANTS LACKING LIGHT-HARVESTING AND REACTION CENTER COMPLEXES

Abstract— Membranes from Rhodobacter (formerly Rhodopseudomonas) sphaeroides mutant strains that lack one or more of the bacteriochlorophyll a (BChl)-protein complexes were used to obtain spectra and molar extinction coefficients of the near-IR absorption bands. The strains examined were NF57 which lacks the B875 light-harvesting and reaction center complexes, and M21 which lacks the B800–850light-harvesting complex. The extinction coefficients obtained for the B800, B850 and B875 bands were 226 ± 10, 170 ± 5, and 118 ± 5 m M ^-1cm^-1, respectively, in reasonable agreement with values reported for detergent-isolated complexes (Clayton, R. K. and B. J. Clayton, Proc. Natl. Acad. Sci. USA 78 ,5583–5587, 1981). The results also demonstrated that detergent solubilization altered the spectra, causing a band broadening on the blue side of the B875 and B800 peaks. The data obtained from the analysis of the mutant strains were used for deconvolution of the BChl species in membranes of the wild-type. A short BASIC computer program for performing this deconvolution is included.