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.     

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.     

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.     

CORRELATION BETWEEN DELAYED LIGHT EMISSION AND FLUORESCENCE OF CHLOROPHYLL a IN SYSTEM II PARTICLES DERIVED FROM SPINACH CHLOROPLASTS

Abstract— The induction transient of delayed light of chlorophyll a, excited by repetitive flashes (0.5 ms in duration) and emitted 0.1 - 1.2 ms after the flashes, was measured in system II particles derived from spinach chloroplasts. An uncoupler, gramicidin S, was always added to the particles in order to eliminate the influence of the phosphorylation system on the delayed light and to isolate a direct relationship between the delayed light emission and the primary photochemical reaction, except for the experiments described in the next paragraph. The yield of delayed light emission from the system II particles was found to be about three–times higher than that of chloroplasts on a chlorophyll content basis. System I particles, on the other hand, emitted much weaker delayed light than chloroplasts. Upon intermittent illumination, induction of delayed light in system II particles showed a decrease from the initial rise level to the steady-state level. The initial rise level was the maximum. The fluorescence induction, on the other hand, exhibited an increase from the initial rise level to the maximum steady-state level. The induction of both delayed light emission and fluorescence arrived at their final steady-state levels after the same period of illumination. Induction of delayed light emission was measured under various conditions that changed the oxidation-reduction state of the primary electron acceptor, X, of photoreaction II: by adding an electron acceptor and an inhibitor of electron transport, and by changing the light intensity. The state of A'was monitored by measuring the fluorescence yield. The yield of delayed light emission excited by each flash was found to depend on the amount of oxidized form of X present before the flash. To examine the role of the primary electron donor Y of photoreaction II in delayed light emission, effects of electron donors of photoreaction II such as Mn²⁺, hydroquinone and p-phenylenediamine were investigated. These agents were found to markedly decrease the yield of delayed light emission without altering the pattern of its induction. They had little effect on the induction of fluorescence. These findings are interpreted by a mechanism in which transformation of the reaction center from the form (X^-Y⁺) into (X Y) produces a singlet excitation of chlorophyll a that is the source of millisecond delayed light emission. This reaction is probably non–physiological and must be very slow if compared to the transformation of (X^-Y⁺) into (X^-Y). Since the form (X^-Y⁺) is produced when the excitation is transferred to the reaction center in the form (XY), it is expected in this scheme that the yield of delayed light emission should depend on the amount of the form (X Y) present before the excitation flashes. Electron donors stimulate transformation of the reaction center from (X^-Y⁺) into (X^-Y). Since this reaction competes with the process of delayed light emission, electron donors are expected to suppress delayed light emission.     

HORSE HEART CYTOCHROME c AS AN ELECTRON ACCEPTOR FROM CHLOROPHYLL TRIPLET IN NEGATIVELY CHARGED LIPID BILAYER VESICLES*

Abstract— Cytochrome c has been shown to bind via electrostatic interactions to egg phosphatidylcholine vesicles which contain 5–30 mol percent of negatively-charged surfactant (dihexadecylphosphate) in a low ionic strength medium. Under these conditions the oxidized cytochrome can function as a direct one-electron acceptor from membrane-bound triplet state chlorophyll to produce chlorophyll cation radical and reduced cytochrome. Kinetic experiments using laser flash photolysis have demonstrated that triplet quenching and the yield of electron transfer products increase, and product lifetime decreases, with an increase in the magnitude of the negative charge on the vesicles, and with a decrease in the ionic strength of the medium. Both triplet quenching and product formation rates and yields showed saturation behavior as the cytochrome concentration was increased, and reached limiting values at 20–30 μM cytochrome when the vesicle contained 20 mol percent of the negatively-charged surfactant. This behavior is interpreted in terms of saturation of the vesicle surface binding sites. Under optimum conditions in this system, approximately 20% of the chlorophyll triplet molecules could be converted to electron transfer products which had a halftime for the reverse reaction of approximately 1.5 ms.     

SPINACH PLASTOCYANIN AS AN ELECTRON ACCEPTOR FROM MEMBRANE-BOUND CHLOROPHYLL TRIPLET STATE IN POSITIVELY CHARGED LIPID BILAYER VESICLES*

Spinach plastocyanin is bound to egg phosphatidylcholine vesicles containing 5–25 mole percent dioctadecyldimethylammonium chloride (DODAC) via electrostatic interactions in a 50 mM betaine medium (pH=6.5). This was demonstrated by both gel filtration experiments and kinetic results using laser flash photolysis. Under those conditions, oxidized plastocyanin can function as a direct electron acceptor from membrane-bound triplet chlorophyll to produce chlorophyll cation radical and reduced plastocyanin. The fraction of chlorophyll triplet which is quenched by oxidized plastocyanin increases, and the yield of electron transfer products also increases, with an increase in the magnitude of the positive charge on the vesicles. Product decay and rise halftimes decrease with an increase in the mole percent of DODAC⁺ incorporated into egg phosphatidylcholine vesicles. However, both of these halftimes are independent of oxidized plastocyanin concentration. Even though ～50% of the Chi triplets were quenched, no electron transfer product formation was observed in 5 mM phosphate buffer (pH=7.0). Under similar conditions in betaine, approximately 13% of the Chi triplets could be converted into products.     

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.     

ACTION SPECTRA FOR LIGHT-INDUCED DE-EPOXIDATION AND EPOXIDATION OF XANTHOPHYLLS IN SPINACH LEAF*

Abstract— The action spectra for violaxanthin de-epoxidation and zeaxanthin epoxidation in New Zealand spinach leaf segments, Tetragonia expansa, were determined at equal incident quanta of 2·0 × 10¹⁵ quanta cm^-2 sec^-1. Precise action spectra were not obtained due to variable leaf activity. The de-epoxidation action spectrum had major peaks at approximately 480 and 648 nm. Blue light was slightly more effective than red light and little activity was observed beyond 700 nm. The epoxidation action spectrum showed major peaks at around 440 and 670 nm. Blue light was more effective than red light and light beyond 700 nm showed definite activity. The net result of de-epoxidation and epoxidation is a cyclic scheme, the violaxanthin cycle, which consumes O₂ and photoproducts. The action spectra indicate that the violaxanthin cycle is more active in blue than in red light and therefore could account for O₂ uptake stimulated by blue light. However, the violaxanthin cycle is not the pathway for O₂ uptake by photosynthetic system 1. It was suggested that the violaxanthin cycle may function as a pathway for the consumption of excess photoproducts generated in blue light or the conversion of these photo-products to other forms of energy.     

RESOLUTION ENHANCEMENT AND DECONVOLUTION INTO VIBRONIC BANDS OF THE FLUORESCENCE AND ABSORPTION SPECTRA IN THE RED REGION OF DILUTE SOLUTIONS OF CHLOROPHYLL a IN VARIOUS SOLVENTS

Abstract— The fluorescence spectra of chlorophyll a in less than 10–⁶ mol dm^-3 solutions of benzene, toluene, tetrahydrofurane, EPA and ethanol were determined at both room temperature and at 77 K. Resolution enhancement using Fourier transform methods revealed the presence of vibronic bands with essentially solvent-invariant separation between their origins; this was confirmed by the quantitative deconvolution of the spectra into Gaussian bands. It was concluded that the fluorescence is due to a single spectroscopically distinct species. The absorption spectra in the above solvents include a band absent from the fluorescence spectra which, from its position and intensity, may be identified as the longest wavelength X-polarized electronic transition of chlorophyll a. Differences observed between the shapes of the low-and high-temperature absorption spectra may be attributed primarily to the narrowing of bandwidths with decreasing temperature and to intensity redistribution within the bands; the apparent increase in the integrated intensity on cooling the solutions appears to be due largely to the increase in the volume concentration of the solute as a result of solvent contraction.     

ACTION SPECTRA FOR LETHALITY IN RECOMBINATION-LESS STRAINS OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI*

Abstract— Action spectra for lethality of both stationary and exponentially growing cells of recombinationless (recA) mutants of Salmonella typhimurium and Escherichia coli were obtained. Maximum sensitivity was observed at 260nm which corresponds to the maximum absorbance of DNA. However, a shoulder occurred in the 280–300 nm range that departed significantly from the absorption spectrum of DNA. At wavelengths longer than 320nm, the shapes of inactivation curves departed significantly from those at wavelengths shorter than 320nm and survival curves at wavelengths longer than 320nm had a large shoulder. A small peak or shoulder occurred in the 330–340nm region of the action spectra. The special sensitivity of recA mutants to broad spectrum near-UV radiation may be due to synergistic effects of different wavelengths. Parallels between the inactivation of recA mutants and the induction of a photoproduct of l -tryptophan toxic for recA mutants (now known to be H₂O₂) suggest that H₂O₂ photoproduct from endogenous tryptophan may be involved in the high sensitivity of these strains to broad spectrum near-UV radiation.     

ACTION SPECTRA FOR THE ABSORBANCE CHANGE AT 880 nm AND FOR P870 FLUORESCENCE FROM A PHOTOSYNTHETIC REACTION CENTER*

Abstract— Photosynthetic reaction centers isolated from blue-green mutant strain R-26 of Rhodopseudomonas spheroides were used to study the action spectra for the light-induced absorption changes at 880 nm, and for the P870 fluorescence. These spectra indicated that a pigment, possibly bacteriopheophytin, with an absorption maximum at 757 nm is an intrinsic component of the reaction center and transfers energy to P870 with relatively high efficiency.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: LIGHT-INDUCED ABSORBANCE CHANGES AND ESR SIGNALS FOR VARIOUS PORPHYRIN-QUINONE AND HYDROQUINONE SYSTEMS IN ALCOHOL SOLVENTS*

Abstract— Light-dark optical difference spectra of degassed ethanol or pyridine solutions of chlorophyll and benzoquinone or hydroquinone at temperatures above — 50°C show only the semiquinone absorbance band. Decay of the signals is second order, with a rate constant in agreement with earlier ESR results. Light-induced optical changes due to chlorophyll can be elicited by lowering the temperature of ethanol solutions of chlorophyll and benzoquinone to a region of high viscosity. Hydroquinone is not effective in producing these optical changes. Similar results are achieved at room temperature by using as solvent a degassed mixture of the alcohols: cyclohexanol, tert-butanol, and ethanol (CBE). Difference spectra show bleaching of the chlorophyll bands and increased absorbance in the intermediate wavelength region (460–580 nm). Decay kinetics are first order, while the rise is complicated (probably biphasic). ESR signals have no hyperfine structure and also decay by first order kinetics, at a rate which is faster than that of the optical changes. The ESR signals reach a steady state more rapidly than the optical signals, without biphasic kinetics. These results demonstrate that at least two species are generated. Addition of acid increases the amount of bleaching in CBE, while small amounts of base decrease it. Larger amounts of base cause chlorophyll bleaching to completely disappear and only the semiquinone anion is observed. Activation energies for the chlorophyll a-benzoquinone photoreaction in CBE are 10–14 kcal/mole. Lower potential quinones give lower activation energies. The rate constant for quenching of the triplet state of chlorophyll a by β-carotene in CBE is 7.5±0.5×10⁸ (M set)^-1. β-carotene also quenches photoproduct formation. The bimolecular rate constant for formation of the photoproduct with benzoquinone was calculated to be 7×10⁸ (Msec)^-1. The redox potential of the quinone affects both the magnitude of the chlorophyll absorbance changes and the rate of decay. The higher the potential, the larger the changes and the slower the decay. Other porphyrin systems show similar photoreactions only if they are chelated with a group II metal, such as Mg²⁺, Cd⁺², or Zn⁺². The results are interpreted in terms of the formation, by a triplet-sensitized one-electron transfer from solvent to quinone, of a chlorophyll-semiquinone complex which is stabilized via coordination with the chelated metal.     

ACTION SPECTRA FOR THE PHOTOLYSIS OF 5-IODODEOXYURIDINE IN DNA AND RELATED MODEL SYSTEMS: EVIDENCE FOR SHORT-RANGE ENERGY TRANSFER*

Abstract— A variety of polynucleotides containing 5-iodouracil residues were irradiated in aqueous solution with wavelengths between 240 and 313 nm. From the rate of deiodination the photochemical cross sections (a_B) were determined as a function of the irradiation wavelength (A). The expression was used to relate the observed values of _B to the intrinsic quantum yield, φ_INT, and to the absorption cross sections, and for the iodinated and noniodinated residues, respectively. φ_INT is the probability an excited iodouracil residue will deiodinate, while the parameter b is a measure of the number of noniodinated bases which contribute their excitation energy to the deiodination process. For IdUrd and poly(5-iodouridylic acid), the average values of φ_INT calculated from the experimental _B values were 0.0202 and 0.0188, respectively, for irradiation in air. In native, denatured, and depurinated DNA in which IdUrd was substituted for 10% of the Thd, the average φ_INT values were 0.0069, 0.0088, and 0.0153, respectively, indicating an enhancement in φ_INT upon decreasing the order of the polynucleotide. In contrast, the average values of b bor the same set of compounds were found to be 4 , 2 and 0.4, respectively, indicating a decrease in b with decreasing polynucleotide order, i.e. a loss of base stacking decreases the extent of energy transfer. A value of b= 4 for native DNA is assumed to mean that the extent of energy transfer in native DNA is limited to four base donors per iodouracil residue serving as an energy trap.     

USING TWO DIMENSIONAL NMR TECHNIQUES TO ASSIGN PROTON AND CARBON SPECTRA FOR COBALT(III) COMPLEXES DERIVED FROM LINEAR QUINQUEDENTATE LIGANDS CONTAINING NITROGEN AND SULFUR DONOR ATOMS

Abstract

High resolution NMR spectroscopy was used to study the structure of a cobalt(III) complex derived from the linear quinquedentate ligand, 7-methyl-4, 10-dithia-l, 7, 13-tria-zatridecane, NH₂-CH₂CH₂-S-CH₂CH₂-N(CH₃)-CH₂CH₂-S-CH₂CH₂-NH₂. The flexibility of this ligand allows it theoretically to form four distinct structural isomers with metal ions. The one dimensional proton spectrum exhibited by [Co(NSNSN)Cl]Cl₂, is complicated and does not provide structural insight. A combined utilization of proton, carbon, DEPT, COSY, NOESY and HETCOR spectra was employed to establish geometric structure type, make individual proton assignments, identify proton-proton connectivities that result from scalar coupling, and distinguish interactions occurring as a result of through-space interactions. Information about the five-membered chelate ring orientations was also obtained. Two groups of signals were shown to correlate with each carbon signal, exhibiting a definite nonequivalence between geminal protons. Geminal protons were found to have coupling constants of 14 Hz. In contrast, the vicinal protons were found to have coupling constants of 5-7 Hz.     

ABSORPTION SPECTRA OF DEOXYRIBOSE, RIBOSEPHOSPHATE, ATP AND DNA BY DIRECT TRANSMISSION MEASUREMENTS IN THE VACUUM-UV (150—190 nm) AND FAR-UV (190—260 nm) REGIONS USING SYNCHROTRON RADIATION AS A LIGHT SOURCE

Transmission measurements of 2-deoxy-D-ribose, D-ribose-5-phosphate, ATP and DNA at 5 nm intervals were made with thin films in the wavelength region between 150 nm and 260 nm using synchrotron radiation. ATP and DNA exhibited two peaks in the absorption spectra around 260 nm and 190 nm, and a steep increase below 170 nm, while ribose phosphate and deoxyribose only exhibited the increase below 190 nm with no appreciable absorption above 190 nm. Since adenine does not exhibit the increase of absorption below 180 nm, these results indicate that the absorption of the sugar-phosphate group, rather than adenine, contributed to the increase below 170 nm in the absorption spectra of ATP and DNA.     

AUTOMATIC RECORDING OF ACTION SPECTRA OF PHOTOBIOLOGICAL PROCESSES, SPECTROPHOTOMETRIC ANALYSES, FLUORESCENCE MEASUREMENTS AND RECORDING OF THE FIRST DERIVATIVE OF THE ABSORPTION CURVE IN ONE SIMPLE UNIT

Abstract— A compact, rugged and simply constructed instrument has been designed which measures action spectra of photosynthesis by delivering equal numbers of quanta between 400 and 720 nm to a sample placed upon the cathode of an oxygen sensor. The absorption spectrum is measured delivering equal numbers of quanta over the spectrum to a sample placed directly above a photoreceiver that has been adjusted in wavelength sensitivity to function as a quantum counter. All the details that are recorded by conventional high precision spectro-photometers for samples with relatively broad absorption bands (e.g. chlorophyll, carotenoids, phycobilins and living material such as algal suspensions) are resolved by the instrument. Corrected excitation spectra of fluorescence are obtained with the same instrument. Likewise the first derivative of the absorption curve may be scanned. Such recordings amplify details in the absorption spectrum, and they are particularly useful in analyses of small changes in slope. The functions described above may be operated separately or combined. The instrument has a total weight of about 10 kg, and the dimensions are ca . 60 × 40 × 30 cm. It is constructed for use in field laboratories and on board research vessels, and also for courses in biology where the principles behind photobiological analyses are illustrated. It is possible that a device of this type could be used for investigations of photosynthesis on other planets.     

ABSORPTION MAXIMA OF CATIONS RELATED TO RETINAL AND THEIR IMPLICATION TO MECHANISMS FOR BATHOCHROMIC SHIFT IN VISUAL PIGMENT*

Abstract— The cations resulting from eleven retinal-related polyenes by placing the latter in n-butanol-sulfuric acid were examined spectroscopically in an effort to explain the batho-chromic effect observed in visual pigments. A comparison of the Λ_max values, intensities and 1/2-band widths of the model carbonium ions with those of the visual pigments does not seem to justify the representation of the chromophore in visual pigments as a pure carbonium ion.     

ABSORPTION SPECTRA AND ACID-BASE DISSOCIATION OF THE 4-ALKYL DERIVATIVES OF 7-HYDROXYCOUMARIN IN SELF-ASSEMBLED SURFACTANT SOLUTION: COMMENTS ON THEIR USE AS ELECTROSTATIC SURFACE POTENTIAL PROBES

Abstract— The absorption spectra of the un-ionized and ionized forms of 4-heptadecyl-7-hydroxycoumarin (HHC) in aqueous self-assembled surfactant solution have been investigated. From a comparison with the absorption spectra of 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin (MHC) and HHC in neat organic solvents and organic solvent/water mixtures it is shown that the 7-hydroxycoumarin chromophore of HHC in self-assembled surfactant solution resides, on average, in an interfacial microenvironment which has a lower effective dielectric constant than that of the bulk aqueous solution. The absorption spectrum of the ionized form of HHC in aggregates of self-assembled surfactant molecules with cationic quaternary ammonium headgroups is found to be consistent with there being specific molecular interaction between the anionic chromophore and the quaternary ammonium headgroup. pH titrations performed with MHC in pure water and in four molar aqueous solutions of sodium chloride and tetra-methylammonium chloride indicate that the acid-base dissociation of HHC in charged micelles and vesicles should not be substantially influenced by any interfacial salt-effects, and that the acid-base dissociation of HHC in cationic micelles and vesicles with quaternary ammonium headgroups should not be markedly affected by the specific molecular interaction that exists. Estimates of the electrostatic surface potentials of a number of self-assembled surfactant aggregates are made by utilising the acid-base dissociation of HHC and assuming that the nonionic micelles of n -dodecyl octaoxyethylene glycol monoether (C₁₂E₈) can serve as a reference state of zero surface potential. The validity of this assumption in relation to both micelles and vesicles is discussed in detail.