Changes In Fluorescence Yield Research Articles

On the origins of 718 nm fluorescence from Porphyridium cruentum at 77 K

Emission spectra and transient behavior of fluorescence in Porphyridium cruentum have been studied in search of the pathway of excitation energy from the phycobilisome to Photosystem I (PS I) of photosynthesis. For activating light at 436 nm, absorbed almost entirely by chlorophyll, fluorescence is dominated by the 718 nm band generally attributed to chlorophyll of PS I. Activating light at 550 nm, absorbed mostly by the phycobilisome, gives rise to the distinctive fluorescence band of PS II chlorophyll at 696 nm but also gives a large component at 718 nm. Analysis depends critically upon the source of emission at 718 nm under 550 nm activation: does it arise from PS I or PS II? Ley and Butler (Ley, A.C. and Butler, W.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3956–3960) have proposed that the 718 nm arises mostly from PS I, to which it is transferred by spillover from PS II. We suggest a different proposition: that under 550 nm activation most of the 718 emission arises from PS II. Analysis shows that this proposition provides an alternative explanation. Using the small change in fluorescence yield observed under 436 nm activation as a monitor of excitation in PS I, we provide evidence that under 550 activation most of the 718 nm fluorescence arises from PS II.

Magnetic field-induced fluorescence changes in chlorophyll-proteins enriched with P-700

Fluorescence yield dependence on external magnetic field (0–600 G) was measured for chlorophyll-protein complexes enriched with Photosystem I. Maximal relative changes of fluorescence yield at room temperature (1.0–2.5%) were dependent on the chlorphyll a: P-700 ratio. Magnetic field-induced changes were observed only in the presence of dithionite. At low temperatures (down to −160°C) the magnetic field-induced effect decreased. The effect is obviously connected with the functions of reaction centers in Photosystem I. An explanation of the effect is proposed based on the hypothesis of radical pairs recombination within the reaction center. For the radical pair ( P-700 +· A −·), an intermediate acceptor, A −·, with a g-value approximately equal to that of P-700 +· is proposed.

Action of salicylaldoxime on electron transport reactions, fluorescence yield, and light-induced field changes in spinach chloroplasts: A new mode of inhibition in photosystem II

Salicylaldoxime (1–10 m m) inhibits chloroplast electron transport reactions by a reversible and an irreversible modification of photosystem II. The irreversible inhibition correlates with removal of the loosely bound pool of manganese associated with the water-splitting mechanism. The reversible inhibition is characterized by (i) a suppression of artificial donor reactions, (ii) a high initial fluorescence yield, and (iii) a decline in the amplitude of the flash-induced electric field across the membrane. After removal of the inhibitor, the initial fluorescence yield declines to near-control levels, but the variable portion of the fluorescence rise remains missing. Addition of an artificial donor restores the variable fluorescence yield and normal electron transport rates to 2,6-dichlorophenolindophenol. Characteristics of the reversible inhibition suggest that salicylaldoxime causes suppression of photochemical charge separation in photosystem II.

The kinetic relationship between the C-550 absorbance change, the reduction of Q( ΔA320) and the variable fluorescence yield change in chloroplasts at room temperature

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolated, envelopefree chloroplasts in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isosbestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differential extinction coefficient Δϵ(540–550) is calculated to be approximately 5 mM −1 · cm −1. The kinetics of the C-550 absorbance change, occurring upon the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical conditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The light-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers. It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Thus, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at room temperature.

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E m 7.6 = + 25 m V and the second with E m 7.6 = -270 m V. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm ( R. Malkin, 1978, FEBS Lett. 87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.

Picosecond time-resolved fluorescence study of chlorophyll organisation and excitation energy distribution in chloroplasts from wild-type barley and a mutant lacking chlorophyll b

Picosecond time-resolved fluorescence spectroscopy has been used to investigate the fluorescence emission from wild-type barley chloroplasts and from chloroplasts of the barley mutant, chlorina f-2, which lacks the light-harvesting chlorophyll a/b-protein complex. Cation-controlled regulation of the distribution of excitation energy was studied in isolated chloroplasts at the F 0 and F m levels. It was found that: 1. (a) The fluorescence decay curves were distinctly non-exponential, even at low excitation intensities (<2 × 10 14 photons · cm −2). 2. (b) The fluorescence decay curves could, however, be described by a dual exponential decay law. The wild-type barley chloroplasts gave a short-lived fluorescence component of approximately 140 ps and a long-lived component of 600 ps ( F 0) or 1300 ps ( F m) in the presence of Mg 2+; in comparison, the mutant barley yielded a short-lived fluorescence component of approx. 50 ps and a long-lived component of 194 ps ( F 0) and 424 ps ( F m). 3. (c) The absence of the light-harvesting chlorophyll a/b-protein complex in the mutant results in a low fluorescence quantum yield which is unaffected by the cation composition of the medium. 4. (d) The fluorescence yield changes seen in steady-state experiments on closing Photosystem II reaction centres ( F m/ F 0) or on the addition of MgCl 2 (+Mg 2+/−Mg 2+) were in overall agreement with those calculated from the time-resolved fluorescence measurements. The results suggest that the short-lived fluorescence component is partly attributable to the chlorophyll a antenna of Photosystem I, and, in part, to those light-harvesting-Photosystem II pigment combinations which are strongly coupled to the Photosystem I antenna chlorophyll. The long-lived fluorescence component can be ascribed to the light-harvesting-Photosystem II pigment combinations not coupled with the antenna of Photosystem I. In the case of the mutant, the two components appear to be the separate emissions from the Photosystem I and Photosystem II antenna chlorophylls.

The interaction of an amphipathic fluorescence probe, 2- p-toluidinonaphthalene-6-sulphonate, with isolated chloroplasts

The amphipathic fluorescence probe, 2- p-toluidinonaphthalene-6-sulphonate has been used to investigate the surface electrical properties of chloroplast thylakoid membranes. The fluorescence yield of 2- p-toluidinonaphthalene-6-sulphonate in aqueous solution increases on addition of hypotonically shocked chloroplast, and the emission maximum shifts towards the blue to 440 nm, although the emission spectrum is somewhat distorted by chloroplast pigment absorption. The intensity of 2- p-toluidinonaphthalene-6-sulphonate fluorescence is further increased on adding salts to the membrane suspension, and changes of >100% are routinely observed. Similar observations have also been made with soya bean phospholipid (azolectin) liposomes. The magnitude of the fluorescence increase is dependent on membrane concentration, being more pronounced at high surface area/suspending volume ratios. The effect of salt addition appears to be that of shielding the fixed negative charges on the membrane surface, thus increasing the fraction of 2- p-toluidinonaphthalene-6-sulphonate molecules at the surface, where the 2- p-toluidinonaphthalene-6-sulphonate has a higher fluorescence yield than in free aqueous solution. This concept is supported by the fact that the effectiveness of salts in increasing 2- p-toluidinonaphthalene-6-sulphonate fluorescence is as predicted by classical electrical double layer theory: governed mainly by the charge carried by the cation with an order of effectiveness C 3+ > C 2+ > C +, and not by the chemical nature of the cation or by the nature of its co-ion. It has been argued that the chlorophyll fluorescence yield, controlled by the cation composition of the suspending medium follows the total diffusible positive charge density at the thylakoid membrane surface (Barber, J., Mills, J. and Love, A. (1977) Febs. Lett. 74, 174–181). Although the cation induced 2- p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yield changes show similar characteristics, there are also distinct differences between the two phenomena particularly when cations are added to chloroplasts initially suspended in a virtually cation-free medium. Therefore it is concluded that although both 2- p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yields are governed by the electrical properties of the thylakoid membrane surface, the mechanism controlling their cation sensitivity is not the same.

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the Pfr/P ratio present at the beginning of the assay, such that far-red was more efficient at high Pfr/P and red at low Pfr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe "Fluram" (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.

LOCAL ELECTRIC FIELD EFFECT ON THE CHLOROPHYLL FLUORESCENCE

Abstract. Polarized absorption and fluorescence of chlorophyll a and bacteriochlorophyll solutions in nematic liquid crystal mixtures have been studied in the presence of an external electric field.The electric field caused a reorientation of the pigment molecules as concluded from changes in the polarized absorption. However, no correlation was found between the change in the polarized absorption and the change in the polarized fluorescence as a function of the field strength. The field influence was much stronger than expected only from the molecular orientation. The fluorescence polarized parallel to the direction of the liquid crystal was found to increase, whereas the similarly polarized absorption decreases. As a whole, the fluorescence yield significantly increased. It is suggested that the additional electric field is reinforced by a local field, most probably due to a protonation of the liquid crystal molecules.Charged solvent molecules are reoriented in an external electric field which changes the local electric field acting on chlorophylls. Similar changes in CHI fluorescence yield due to local electric field can be created in vivo by the shift of charged molecules present in surrounding pigments. Such effects can be at least partially responsible for slow induction of fluorescence phenomena.

Induction patterns of delayed luminescence from isolated chloroplasts. I. Response of delayed luminescence to changes in the prompt fluorescence yield

1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period. 2. Two distinct decay components of delayed luminescence were measured a “fast” component (from ≈1 ms to ≈6 ms) and a “slow” component (at ≈6 ms). 3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change. 4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation. 5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics. 6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity. 7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant. 8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the “lake” model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the “W” hypothesis) were in closer agreement with the results.

Fluorescence changes in isolated broken chloroplasts and the involvement of the electrical double layer

We studied the effects of a variety of cations on chlorophyll fluorescence yield of broken chloroplasts prepared under carefully controlled ionic conditions. In the absence of light-induced electron transport and associated proton pumping, two types of cation-induced chlorophyll fluorescence changes could be distinguished in broken chloroplasts. These are termed "reversible" and "irreversible" fluorescence yield changes. Reversible fluorescence yield changes are characterized by antagonistic effects of monovalent and divalent cations and are prevented by the presence of 5 mM Mg2+ in the suspending media. Reversible-type fluorescence yield changes show little or no dependence on the structure, lipid solubility, or coordination number of the cation, but depend strictly on the net positive charge carried by the ion. It is proposed that these fluorescence changes are brought about through the interaction of monovalent or divalent cations with an electrical double layer at the interface of the outer surface of the thylakoid membrane and the surrounding aqueous solution. The results are interpreted in terms of the Gouy-Chapman theory of the diffuse double layer, indicating that the thylakoid outer surface bears an excess fixed negative charge density of about 2.5 muC/cm2, or approximately 1 negative charge per 640 A2 of membrane surface. Chlorophyll fluorescence quenching in isolated broken chloroplasts suspended in media containing 5 mM MgCl2 is also observed on addition of certain polyvalent cations to the medium. This type of cation-induced fluorescence change appears to be largely irreversible and may occur through specific binding of the cation to the thylakoid as a result of the high electrostatic attraction exerted by the negatively charged membrane surface.

Picosecond time-resolved study of MgCl 2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl 2-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp −At 1 2 , where A was found to be 0.052 ps −1 2 , and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl 2 produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.

9-Aminoacridine Binding to Chloroplast Membranes in Dark. Reversal by Mg2+

Abstract 9-Aminoacridine (9AA) binds to photosynthetic m em branes of unilluminated chloroplasts in low-salt media. The binding was insensitive to the uncouplers of photophosphorylation. The apparent binding constant was 140 μM . The binding isotherm as a function of 9AA concentration was sigmoid, and approximately 3 mol 9AA/mol chlorophyll was bound at saturating concentrations of 9AA. Addition of Mg2+ partially reversed the binding of 9AA in chloroplasts in the dark as observed by a Mg2+-induced increase of 9AA fluorescence as well as by spectrophotom etric measurements of free 9AA. It appeared, however, that use of fluorescence techniques for m easuring free 9AA introduced an error in the estimation of the m agnitude of binding, particularly at low concentration of 9AA ( &lt; 7 5 μM). This is probably due to change in fluorescence yield of membrane-bound 9AA on addition of cations. The nature of the binding of 9AA to the thylakoid membranes and the effects of Mg2+ thereupon suggest that both chemical binding of cations and screening of surface charge of the mem branes should be considered in discussing the mechanism of cation action on chloroplast structure and function. Interpretation of these data with respect to heterogeneity of sites of cation action upon or within chloroplast membranes is discussed.

Does the rate of cooling affect fluorescence properties of chloroplasts at −196 °C?

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to −196 °C rapidly, as very thin samples adsorbed into substrates which were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.

Porphyrins XXXV . Exciton coupling in μ-oxo Scandum dimers

The absorption and emission spectra at room temperature and at 77 K are reported for the monomers and μ-oxo dimers of (OEP)Sc(III) and (TPP)Sc(III). [Here (OEP) is octaethylporphin and (TPP) is tetraphenylporphin.] Exciton coupling effects are strongest in the B(Soret) band of [(OEP)Sc] 2O dimer: (i) The peak is blue shifted by 11 nm; (ii) the Soret band has a long red tail out to 480 mn; (iii) the fluorescence polarization shows a broad negativ band ≈ 440 nm. A vibronic exciton coupling model can roughly interpret the data if there is substantial and variable tilting of the ring planes. Exciton effects are weaker in the B(Soret) band of [(TPP)SC] 2O, presumably because there is less tilting. The effect of dimer formation on the Q band of [(OEP)Scl 2O is to red shift the band ≈ 420 cm −1 and to nearly double the Q(0,0) halfwidth; there is no change in fluorescence yield with dimerization. Presumably for Q bands exciton coupling is weaker than inhomogeneous broadening. Both the phosphorescence yield and triplet lifetime at 77 K drop by case2 3 in the dimer, showing faster radiationless decay.

L-shell x-ray cross sections for Cl-Ar and Ar-Ar collisions

Measurements of total x-ray production cross sections for ${\mathrm{Ar}}^{+}$ -Ar and ${\mathrm{Cl}}^{+}$ -Ar collisions for incident energies between 200 keV and 1.5 MeV are reported. For ${\mathrm{Cl}}^{+}$ -Ar collisions, separate argon and chlorine x-ray production cross sections are extracted. The cross sections exhibit steep increases as bombarding energy is increased. Using existing measurements of Auger cross sections, values for the mean fluorescence yields as a function of bombarding energy are deduced. The increases in the x-ray production cross sections are shown to be due primarily to changes in the mean fluorescence yields. A qualitative explanation for the observations is proposed.

An electrochemical thin-layer cell for spectroscopic studies of photosynthetic electron-transport components.

Abstract An electrochemical thin-layer cell, 0.5 ml in volume and 0.25 mm in pathlength, employing an optically transparent gold minigrid electrode has been constructed and used in redox studies of photosynthetic electron-transport components. Light-induced absorption changes accompanying P700 photo-oxidation in photosystem-I subchloroplasts were measured at a series of electrochemically poised potentials at 88°K. The fluorescence-yield changes in photosystem-II subchloroplasts were measured as the poised potential was varied electrochemically. The utility of the cell was further demonstrated by a redox titration of soluble (spinach) ferredoxin using circular dichroism to monitor changes in the redox state. One important advantage of electrochemical vs chemical titration of photosynthetic electron-transport components is that it is possible to attain much more negative potentials than is thermodynamically possible in chemical titrations. This permits the exhaustive titration of certain components at physiologically reasonable pH values not possible chemically.

Energy transfer in the photochemical apparatus of flashed bean leaves

Fluorescence and energy transfer properties of bean leaves greened by brief, repetitive xenon flashes were studied at −196 °C. The bleaching of P-700 has no influence on the yield of fluorescence at any wavelength of emission. The light-induced fluorescence yield changes which are observed in both the 690 and 730 nm emission bands in the low temperature fluorescence spectra are due to changes in the state of the Photosystem II reaction centers. The fluorescence yield changes in the 730 nm band are attributed to energy transfer from Photosystem II to Photosystem I. Such energy transfer was also confirmed by measurements of the rate of photooxidation of P-700 at −196 °C in leaves in which the Photosystem II reaction centers were either all open or all closed. It is concluded that energy transfer from Photosystem II to Photosystem I occurs in the flashed bean leaves which lack the light-harvesting chlorophyll a b protein.

Cation effects on light-induced chlorophyll a fluorescence in chloroplasts lacking both chlorophyll b and chlorophyll-protein complex II

Chloroplasts from a barley mutant lacking both chlorophyll b and chlorophyll-protein complex II(CPC-II) show cation-induced changes in the fluorescence yield of chlorophyll a. It is concluded that CPC-II is not essential for cation control of the distribution of energy between the two photosystems. The lower extents of the cation effects with the mutant chloroplasts may possibly be related to the smaller degree of membrane appression.

A major site of bicarbonate effect in system II reaction. Evidence from ESR signal II vf, fast fluorescence yield changes and delayed light emission

In order to determine the major site of bicarbonate action in the electron transport complex of Photosystem II, the following experimental techniques were used: electron spin resonance measurements of Signal II vf, measurements of chlorophyll a fluorescence yield rise and decay kinetics, and delayed light emission decay. From data obtained using these experimental techniques the following conclusions were made: (1) absence of bicarbonate causes a reversible inactivation of up to 40% of Photosystem II reaction center activity; (2) there is no significant effect of bicarbonate on electron flow from the charge accumulating S state to Z; (3) there is no significant effect of bicarbonate on electron flow from Z to P-680 +; (4) electron flow from Q − to the intersystem electron transport pool is inhibited by from 4- to 6-fold under bicarbonate depletion conditions.