THE COMPLEXITY OF THE FLUORESCENCE OF CHLORELLA PYRENOIDOSA

Abstract— The complexity of the room-temperature emission spectrum of Chlorella was investigated by a matrix analysis method. This approach revealed the presence of two independently fluorescent components in the short-wave region of the spectrum. These components, maximal at about 687 and 695 nm, appeared to correspond to the fluorescence of the bulk pigments of PS II and PS I respectively. The analysis was insensitive to the individual species within the photosystems. As such, other minor fluorescent species, usually observed at low temperatures, which presumably correspond to fluorescence from the trapping centres, did not appreciably complicate the analysis. The absorption spectra of the two photosystems were calculated from the fluorescence data. The results were similar to those that have been obtained by other workers from oxygen evolution and DCMU poisoning data but differed from those obtained by computer analysis of the absorption spectrum. Addition of reduced DCPIP was observed to reverse the increase in fluorescence yield and changes in the spectral distribution of emission taking place on poisoning the algae. The correlation between this and the catalysis of photophos-phorylation in aged or poisoned chloroplasts was noted. This correlation was tentatively interpreted as evidence for a direct interaction between the donor system and the photochemical apparatus associated with PS II, rather than with a member of the electron transport chain as is normally assumed.     

TIME COURSE OF MICROSECOND DELAYED LIGHT EMISSION FROM SCENEDESMUS OBUQUUS AT INTERMITTENT ILLUMINATION

Abstract— For dark adapted cells, the intensity of delayed light at the onset of a light period depends on the length of the preceding dark period. Under our experimental conditions, the exciting laser light seems to reduce the intermediate pool located between the two photosystems. If dark adapted cells are illuminated, mechanisms are established within about 20 s which appear to drain electrons from the intermediate pool.     

AN ANALYSIS OF A TRIPLET EXCITON MODEL FOR THE DELAYED LIGHT IN CHLORELLA*

Abstract— The time dependence of the delayed light in the green alga Chlorella pyrenoidosa has been examined quantitatively in the 1 to 12 msec range after excitation with light pulses (A = 6328 Å) of 100 μsec and 4.5 msec duration. We have confirmed the data of Tollin, Ruby, and Bertsch et al., on the time course of the delayed light in the msec range. New experiments, with 100 μsec flash excitation, on the time dependence of the delayed light emitted by Chlorella treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DMU), hydroxylamine, methyl violgen, and various combinations of these chemicals are presented. Also, data on the dependence of the delayed light intensity on the intensity of the excitation light in the 1.5 and 5.0 msec range are reported. The square law dependence, reported by Jones, in the 140 and 250 msec range is confirmed in the 1.5 and 5.0 msec range at very low light levels. The experimental data on delayed light has been analyzed in terms of a model which incorporates triplet exciton fusion. The following major points result from this analysis: (1) A triplet exciton kinetic model can explain both the time dependence and the excitation intensity dependence of the delayed light emitted by Chlorella. (2) The density of triplet excitons predicted by the model from the observed delayed light intensity is much less than that which can be detected by flash photolysis measurements. Therefore, the failure of such measurements to detect triplet states in vivo does not disprove the model. (3) The possibility of changes in the rate of electron transfer reactions of photosynthesis is included in the kinetic model. The predictions from the model are compared with the effects of chemical additives on the time dependence of the delayed light decay. (4) The proposed triplet exciton model predicts that the delayed light intensity may, under certain specific conditions, be affected by a magnetic field. The negative result of an attempt to observe this effect is reported and discussed. (5) It is concluded that the proposed triplet ‘fusion’ model is a valid alternative to the electron-hole recombination model.     

A PULSED LASER SYSTEM FOR THE STUDY OF MICRO-SECOND DELAYED FLUORESCENCE FROM PLANTS

Abstract—A new laser system is designed for the investigation of S delayed fluorescence. This is intended to overcome some undesirable features present in other current designs, such as low actinic light fluxes and long illumination times, or slow responses owing to long turn-off times of the stimulation source and the presence of fluorescence artifacts.
The system uses a pulsed argon-ion laser and bears little resemblance to either the modified Becquerel phosphoroscope used by Lavorel (1971) or to those systems requiring shutters. Cheapness of design together with the ability to adjust such parameters as pulse duration easily, make this design attractive to most laboratories in which short (10 μs) high intensity (10⁵ W m^-2) pulses with rapid (1.5 μs) turn-off times and high on-to-off contrast ratios (10⁶:1) are required.
Certain design criteria are strictly imposed in order to produce kinetic data that can be meaningfully analysed.     

PROMPT AND DELAYED FLUORESCENCE OF SOME DNA ADSORBATES

Abstract— The absorption and prompt fluorescence spectra of twelve cationic compounds in the free and bound states were surveyed, and four compounds selected for detailed study, viz: proflavine, benzoflavine, acridine orange and thioflavine T. The absorption spectra, prompt fluorescence spectra and delayed fluorescence and phosphorescence spectra of these four compounds in fluid solutions in the free and bound states were measured. The shifts in the promp fluorescence bands of the diaminoacridines on adsorption were found to differ significantly from those of the other compounds, and the behaviour of acridine orange was found to be different from that of the other two diaminoacridines. All three diaminoacridines gave E -type delayed fluorescence in the free and bound states. The ordered structure of the strongly binding adsorption sites in native DNA is associated with a higher intensity of delayed fluorescence than is observed from the diaminoacridines in the free state or adsorbed on denatured DNA. Quenching experiments with proflavine indicated that, in the weakly binding sites, its triplet is easily quenched by unadsorbed quenchers, but in the strongly binding sites its triplet is quenched only with great difficulty.     

FLUORESCENCE INVESTIGATIONS OF ALBUMIN FROM PATIENTS WITH FAMILIAL DYSALBUMINEMIC HYPERTHYROXINEMIA*

Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant syndrome in which clinically euthyroid patients have elevated total thyroxine levels. These high serum thyroxine levels are traceable to altered binding of thyroxine to the patient's albumin. Albumin from FDH patients and normal volunteers have been purified. Reverse-phase and ion-exchange high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the FDH-human serum albumin (HSA) samples show a single band that comigrates with normal HSA. In both protein solutions the intrinsic fluorescence, upon 280 nm excitation, is predominantly due to the single tryptophan residue. The quantum yield of this intrinsic fluorescence in the FDH-HSA solutions is, however, reduced relative to that of HSA. Furthermore, the “average” lifetime value of the tryptophan emission in the FDH-HSA sample is less than that of normal HSA, consistent with its reduced quantum yield. The binding of thyroxine to both albumins effectively quenches the tryptophan emission probably via a nonradiative energy transfer mechanism. Time-resolved data suggest that the albumin from the dysalbuminemic patients is actually an approximately equimolar mixture of normal HSA and FDH-HSA indicative of heterologous expression. Quenching of the intrinsic HSA and FDH-HSA fluorescence by serial additions of thyroxine showed enhanced quenching of FDH-HSA relative to HSA at any T4 to albumin mole ratio, therefore supporting earlier reports of increased thyroxine affinity to FDH-HSA.     

DELAYED FLUORESCENCE AND SOME PROPERTIES OF THE CHLOROPHYLL TRIPLETS

–P-Type delayed fluorescence and sensitised P-Type delayed fluorescence have been observed from solutions of chlorophylls a and b in ethanol. E-Type delayed fluorescence has been observed from solutions in propylene glycol. The measurements in propylene glycol have yielded approximate values for the triplet energies of chlorophylls a and b. Measurements of the emissions from the ethanolic solutions have been used to calculate approximate values of the triplet formation efficiencies in this solvent. The sums of the fluorescence and triplet formation efficiencies in ethanol fall far short of unity and the triplet formation efficiencies therefore need to be confirmed by an independent method before they can be accepted with confidence.     

MICROSPECTROPHOTOMETRIC STUDIES ON THE PIGMENTS IN VIVO OF SINGLE ALGAL CELLS-I. PIGMENTS OF CHLORELLA PYRENOIDOSA

Abstract— The pigments in vivo of single cells of Chlorella pyrenoidosa were studied by the microspectrophotometric technique. An accessory recording the first derivative of absorption was used to obtain fine resolution and enhanced accuracy. The results suggest that there are several long-wavelength components of Chl a in vivo. In addition, there seem to be four short-wave forms of Chl a. It is also likely that Chl b exists in vivo in two different forms. The existence of all these forms was demonstrated at room temperature.     

FLUORESCENCE PROPERTIES OF ETHENOTHIAMINE*

Abstract— The absorption and fluorescence properties of ethenothiamine in neutral and acidic solution are reported, and some are found to be in contradiction to previous findings. The low fluorescence quantum yields of ethenothiamine make it a poor substitute for the vitamin in biological systems.     

THE EFFECT OF INHIBITORS AND UNCOUPLERS OF PHOTOSYNTHETIC PHOSPHORYLATION ON THE DELAYED LIGHT EMISSION OF CHLOROPLASTS*

Abstract— Measurements were made of the 3.7 msec delayed light emission of chloroplasts treated with a variety of agents which affect the rate of electron transport (Hill reaction) or photosynthetic phosphorylation. The presence of the electron acceptors ferricyanide or pyocyanine increased delayed light emission. Inhibitors of electron transport (3-(3,4-dichlorophenyl)-1, -1-dimethylurea or 1,10(ortho)-penanthroline) inhibited delayed light emission. The addition of a phosphate acceptor system inhibited delayed light emission. This inhibition was reversed by inhibitors of the phosphorylation reaction, e.g. Dio-9 or phlorizin. From these results it was concluded that the 3.7 msec delayed light emission probably occurs as a result of back reactions of intermediates in the coupled electron transport and photosynthetic phosphorylation systems.     

THE EFFECT OF DEUTERIUM OXIDE ON THE FLUORESCENCE AND PHOTOTRANSFORMATION OF 124 kDALTON PHYTOCHROME*

Abstract— To probe the nature of primary photoprocess and the mechanism of the phototransformation of undegraded 124 kDa oat phytochrome, solvent deuterium isotope effects on the fluorescence and phototransformation of phytochrome have been investigated. The fluorescence intensity and lifetime of phytochrome (P_r form) are greater in D₂0-buffer than in H₂O-buffer, suggesting a possible involvement of proton transfer in the primary photoprocess of phytochrome. Although the photostationary equilibrium (P_r to P_fr ratio) was not altered by deuterium oxide, in contrast to degraded phytochrome, the rate constants of both transformations, P_r→ P_fr and P_fr→ P_r were enhanced by up to 24%. The P_r to P_fr phototransformation of degraded phytochrome, however, was retarded by about the same percentage in D₂O. These opposite effects of D₂O with degraded and undegraded phytochromes underscore the fact that the P_r form from the former reverts to the P_r form in the dark, apparently catalyzed by deuterated general and/or conjugate acidic group(s). With the degraded phytochrome the deuterium oxide enhancement of the rate of dark reversion was approximately 2-fold (Sarkar and Song, 1981). Both the fluorescence intensity and the rates of phototransformation of phytochrome were enhanced in D₂O with successive photocyclings (P_r→ P_fr→ P_r→ P_fr→ P_r etc.) with alternating red and far-red irradiation. It has been proposed that successive photocycling of phytochrome in D₂O results in proton-deuteron exchange in the partially exposed P_tr chromophore and/or its surrounding amino acid residues.     

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.     

DELAYED FLUORESCENCE AND THE REVERSAL OF PRIMARY PHOTOCHEMISTRY IN RHODOPSEUDOMONAS VIRIDIS

Abstract— Delayed fluorescence from chromatophores of the photosynthetic bacterium Rhodopseudomonas viridis was measured at temperatures below 0°C. A component with a decay half-time of about 7 ms was found. Its intensity was directly proportional to the number of reaction centers in the P985⁺·A^- state. During prolonged illumination it faded as electrons moved forward along the electron transport chain from the primary acceptor, A, (P985⁺·A^-→P985⁺·A), and its decay in the dark paralleled the disappearence of the P985⁺ electron paramagnetic resonance absorption. The data suggest that this component of delayed fluorescence results from a direct reversal of the primary light reaction. While the rate of the P985⁺middot;A^-→P985·A reaction was almost independent of temperature, delayed fluorescence intensity displayed an apparent activation energy of 0°2 eV. It is concluded that the P985⁺·A^-→P985·A reaction proceeds by parallel radiative and nonradiative routes. The direct proportionality between delayed fluorescence and the concentration of P985⁺·A^- pairs seems to preclude an involvement of triplet-triplet annihilation or dependence of delayed fluorescence upon the variable prompt fluorescence yield.     

EFFECT OF SPATIAL NON-UNIFORMITY OF ILLUMINATION IN NON-LINEAR INTENSITY-DEPENDENT PHOTO PHYSICAL EXPERIMENTS. APPLICATION TO SOME FLUORESCENCE YIELD MEASUREMENTS IN PHOTOSYNTHETIC SYSTEMS

The spatial non-uniformity of a light beam must be taken into account in interpreting phenomena which depend on the light intensity in a non-linear manner. Omission of this effect may lead to incorrect values of parameters obtained from a fitting of theoretical curves, which describe the dependence of a given phenomenon on light intensity, to the experimental data. The effect of spatial non-uniformity of excitation light beams is illustrated utilizing non-linear intensity-dependent fluorescence phenomena in photosvnthetic svstems and a Gaussian light beam profile.     

FLUORESCENCE FROM MAJOR AND MINOR BACTERIOCHLOROPHYLL COMPONENTS IN VIVO*

Abstract— In the photosynthetic bacteria Chromatium, Rhoahpirillum rubrum, and Rhodopseudomonus spheroides the fluorescence of bacteriochlorophyll is probably free of contamination by a “fast” component of delayed emission, judging from the characteristics of the delayed light measured 3 msec after excitation. In Rps. spheroides the pigment P870, associated with photochemical reaction centers, is non-fluorescent in its photochemically active state. Fluorescence of P870 can be induced by either of two agencies that suppress its photochemical activity: exposure to Na₂S₂O₄ and (in a dry chromatophore film) dessication. The yield of fluorescence from the major (light harvesting) component of bacteriochlorophyll in vivo is brought to a common maximum value by conditions that suppress the photochemical activity of P870. In addition to dessication and exposure to Na₂S₂O₄ these conditions include saturating illumination and exposure to K₃Fe(CN)₆. Of these four treatments only the last two bleach the long wave absorption band of P870. These experiments support the following assertions: (1) P870 traps singlet excitation energy absorbed by the light harvesting BChl; the trapping function of P870 depends on its ability to initiate and participate in photochemistry. (2) Both dessication and exposure to Na₂S₂O₄ suppress the photochemical activity of P870 by blocking an event that proceeds directly from the excited singlet state in P870. (3) The fluoresecence of BChl in vivo is emitted almost entirely by a major (light harvesting) component.     

QUENCHING OF METHYLCYCLOHEXANE FLUORESCENCE BY METHANOL*

We measured the fluorescence emission spectrum and intensity decays of methylcyclohexane (MCH) when excited by simultaneous absorption of two photons at 298 nm. The steady-state intensities and lifetimes were both decreased by methanol, which was found to be an efficient quencher of MCH fluorescence. Methanol quenching of MCH is clearly dynamic, but the exact mechanism of quenching is unclear. Dynamic quenching of MCH was also observed by water and n-propanol. These results suggest that alkane fluorescence from biopolymers, if observable, will only occur from regions of the macromolecules that are not exposed to water.     

FLUORESCENCE PROPERTIES OF PORPHYRIN-GLOBIN FROM HUMAN HEMOGLOBIN

Fluorescence excitation and emission spectra, decays, and quantum yields are reported for the porphyrin-globin of hemoglobin (Hb^desFe) in aqueous solution of pH 8, at 4°C. A very weak fluorescence was observed in the UV (maximum at 334 nm), due to tryptophan and tyrosine residues, in addition to the strong porphyrin emission in the visible (maxima at 624 and 692 nm) reported previously. The absorption and fluorescence properties of the porphyrins of Hb^desFe were compared to those for free porphyrin in organic solvents and in aqueous solution. The close similarity of the fluorescence decays and quantum yields in Hb^desFe and in solution indicate the absence of stronger, specific porphyrin-protein interactions; however, slight spectral shifts point to the existence of water molecules in the Hb^desFe porphyrin environment. The fluorescence study also demonstrates the existence of efficient Trp-porphyrin energy transfer of Förster type. The extent of transfer is in satisfactory agreement with the value expected from crystallographic data for hemoglobin. The results are discussed and compared to previous fluorescence studies of hemoglobin and apohemoglobin. An improved method for the preparation of Hb^desFe is reported.