Anthroyl stearate as a fluorescent probe of chloroplast membranes.

1. A reversible light-induced enhancement of the fluorescence of a "hydrophobic fluorophore", 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1'-ethylene-2,2'-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response. 2. This effect is decreased under phosphorylating conditions (presence of ADP, Pi, Mg2+), and completely inhibited by the uncoupler of phosphorylation NH4Cl(5-10mM), as well as the ionophores nigericin and gramicidin-D (both at 5 - 10(-8)M). The MgCl2 concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a "high energy intermediate". The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the "high energy state" of the thylakoid membrane rather than a terminal phosphorylation step. 3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fllorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H+ uptake per se because (a) the pH dependence of H+ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H+ uptake, reduces anthroyl stearate fluorescence. Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate of Diquat catalyzed electron flow and is the first "non-amine" fluorescence probe utilized for this purpose in chloroplasts.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

Effect of filipin on Rat-liver and yeast mitochondria

1. Under the appropriate conditions intact yeast and mammalian mitochondria exhibit a heretofore unobserved sensitivity to the polyene antibiotic, filipin. The activity of the “filipin complex” (Filipins I, II, III and IV) is shown to be primarily due to the component designated Filipin II.

2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited -ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate.

3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg²⁺, K⁺, Ca²⁺ or 2,4-dinitrophenol, but is reversed by cytochrome c.

4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes.     

Bongkrekic acid sensitivity of respiration-deficient mutants and of petite-negative species of yeasts

1. Growth on glucose of cytoplasmic respiration-deficient (ρ⁻) mutants isolated from five strains of Saccharomyces cerevisiae and one strain of Saccharomyces carlsbergensis were arrested by the inhibitor of mitochondrial adenine nucleotide translocation, bongkrekic acid. This indicates that the mitochondrial adenine nucleotide translocation system is preserved and necessary for growth in a number of independent ρ⁻ mutants.

2. Growth of three “petite-negative” yeast species was arrested by a combined inhibition of respiration by antimycin A and of adenine nucleotide translocation by bongkrekic acid. Thus, the arrest of growth upon inhibition of adenine nucleotide translocation in non-respiring cells is not specific for ρ⁻ mutants and may be a general characteristic of eucaryotic cells.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

A low-temperature-sensitive intermediate state between S2 and S3 in photosynthetic water oxidation deduced by means of thermoluminescence measurements

The temperature dependence of S-state transitions in Photosystem II was measured by means of thermoluminescence using two different protocols for low-temperature flash excitation: protocol A, “last flash at low temperature”, and protocol B, “all flashes at low temperature”. Comparison of the temperature-dependence curves obtained by these two protocols revealed a marked difference particular for the three-flash experiments. The difference was attributed to the formation of a low-temperature sensitive precursor state between S₂ and S₃. The state is formed by two flash illumination given at −5 to −50°C, spontaneously transforms to normal S₃ on dark warming, and is not converted to S₀ by the 3rd flash. The precursor state was tentatively assigned to an S₃ in which H⁺ release is not completed.     

Control of excitation transfer in photosynthesis. IV. Kinetics of chlorophyll a fluorescence in Porphyra yezoensis

The kinetics of chlorophyll a fluorescence were measured at 685 nm in intact cells of Porphyra yezoensis during alternate illumination of the organism with two colors of light, one absorbed by phycoerythrin and the other by chlorophyll a. Two components of fluorescence change overlapping each other in time were separated; the fast component may be controlled by the rate of Photoreaction II which competes with the fluorescence emission process, and the slow component by the light-induced change in excitation transfer between two pigment systems as suggested in our previous study⁶. The kinetics of the slow change in fluorescence yield were extensively investigated.

Terms, “State I” and “State II” are used to describe the state of excitation transfer. In the State I a lesser amount of excitation energy is delivered in Pigment System I and greater to Pigment System II than in the State II. The conversion of the states is achieved by the selective illumination of pigment systems.

The conversion from the State I toward the State II occurred under Light II (light absorbed by Pigment System II) with a half time of about 10 sec, and it saturated at a light intensity of less than 1000 ergs×cm⁻²×sec⁻¹. The reverse conversion occurred under Light I (light absorbed by Pigment System I) with a half time of about 5 sec, and it saturated at about 10 000 ergs×cm⁻²×sec⁻¹.

Light I and Light II competed with each other in the interconversion of the states.     

Reserpine as an uncoupling agent

1. Reserpine, like the uncoupling agent, 2,4-dinitrophenol prevents oxidative phosphorylation but stimulates the rate at which oxygen is reduced.

2. Both reserpine and 2,4-dinitrophenol fail to stimulate oxygen uptake by isolated mitochondria in the presence of arginine.

3. Both 2,4-dinitrophenol and reserpine induce proton permeability in the mitochondrial membrane so that H⁺ is absorbed from the suspending medium.

4. When the reaction system contains reserpine, accumulation of Ca²⁺ by mitochondria is inhibited.

5. Reserpine decreases both ADP:O and P:O ratios which strongly suggest that reserpine is an uncoupling agent.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Light requirements for proton movement by isolated chloroplasts as measured by the bromocresol purple indicator

ATP formation by isolated chloroplasts is due to the proton gradient phenomena, according to Mitchell. The number of protons moved per electron pair transported and per photon absorbed is related to the number of protons required to produce each ATP. Thus, a critical test of the Mitchell hypothesis is the quantum yield of H⁺ transport. Bromocresol purple, a pH indicator, can be used to measure the pH external to isolated chloroplasts accurately and rapidly. The action spectrum (with pycocyanine as the electron acceptor) appears to be that of a System I-linked reaction (high above 700 nm). The quantum yield has been calculated to be 3.5 ± 0.1 H⁺/hv from 640 nm to 690 nm and 6.7 ± 0.4 H⁺/hv above 700 nm. The action spectrum of the efflux of H⁺ occurring in the dark, which is usually identified as being equivalent to the steady-state influx, has the same shape as that of the influx. The quantum yield, however, is reduced by 0.5. Therefore, Photosystem II seems to affect both the initial influx and dark efflux. The H⁺/photon and H⁺/e₂ for the initial influx are too high for the Mitchell hypothesis. Only the H⁺ efflux in the dark from 640–690 nm has an H⁺/hv of 1.6 which agrees with the theory of Mitchell.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown “petite-negative” yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40°C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles.

2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerolgrown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae.

3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the “petite-positive” yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126.

4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 μM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 μM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor.

5. We conclude that “petite-positive” and “petite-negative” yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Réoxydation du quencher de fluorescence “Q” en présence de 3-(3,4-dichlorophényl)-1,1-diméthylurée

Reoxidation of the fluorescence quencher “Q” in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Reoxidation of the fluorescence quencher Q in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea shows the following properties:

It is sensitive to very low concentrations of hydroxylamine (a few μM).

It corresponds to a back reaction between Q⁻ and the primary oxidant Z⁺ formed in the light. A part of this back reaction gives rise to luminescence emission.

Within the range we studied the kinetic of reoxidation is second order with regards to Q⁻.     

Localization of photophosphorylation and proton transport activities in various regions of the chloroplast lamellae

Membrane fragments released by French pressure cell treatment of whole chloroplasts and isolated by differential centrifugation have been characterized structurally and with respect to phosophorylating and proton transport activities. In agreement with results of other workers, the heavy fraction released by pressure treatment was found by electron microscopy studies to be made up of mostly intact grana stacks while the light fraction was comprised of vesicles derived from the stromal lamellae. Both fractions were found to carry out rapid rates of cyclic photophosphorylation catalyzed by phenazine methosulfate (PMS). However, only the grana membranes demonstrated active proton accumulation in the presence of PMS. No light induced H⁺ uptake could be detected in the stromal lamellae fraction; and as expected, proton gradient dissipating agents such as NH₄Cl, nigericin in the presence of K⁺, and gramicidin were only slightly inhibitory to phosphorylation at concentrations which were very inhibitory in the grana membrane fraction.

Further evidence that stromal lamellae do not have active proton transport in the intact chloroplast was obtained by comparing various chloroplasts having different amounts of stromal and grana membranes. Comparative studies on young and old chloroplasts from lettuce, mesophyll and bundle sheath cell plastids from sorghum, and greening plastids from etiolated corn seedlings revealed a direct correlation between the extent of grana formation and the amount of proton transport activity. Samples which had larger amounts of stromal lamellae had high rates of ATP formation but a reduced capacity for H⁺ accumulation.     

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg²⁺ dependent; Co²⁺ and Mn²⁺ but not Ca²⁺ could replace Mg²⁺ to some extent; the activation by Mg²⁺ was slightly antagonized by Ca²⁺. Even in the presence of Mg²⁺, Na⁺ or K⁺ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]_{0.5 V} = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg²⁺. Solubilization, however, led to instability of the enzyme.

The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10^-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN₃ had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.

Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H⁺ translocation. A H⁺-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H⁺-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

A versatile colony assay based on NADH fluorescence

Direct visualization of the activity of enzymes expressed by bacterial colonies attached to a solid support, often referred to as “filter assay”, is a powerful strategy for the identification of new or improved biocatalysts. In this work we demonstrate the usefulness of NAD⁺/NADH coupled enzymatic reactions as visualization tool in such experimental setups. Dehydrogenases, capable of oxidizing or reducing the reaction product released from the bacterial colony were supplemented to the screening solution, together with the screening substrate and a sufficient amount of NAD⁺ or NADH, respectively. We also examined the screening of directly NAD⁺/NADH coupled reactions. The release or consumption of NADH in the area of colonies was monitored on behalf of its fluorescence at 450 nm. Excitation was achieved by standard “black-light” UV tubes (340–360 nm). The visible fluorescence signal was recorded using a CCD-camera. We got excellent results for the screening of threonine aldolases and esterases and were able to show the principle utility for amidase, nitrilase, nitrile hydratase, hydroxynitrile lyase and benzaldehyde dehydrogenase active colonies.     

Evidence for chemiosmotic coupling of electron transport to ATP synthesis in spinach chloroplasts

In spinach chloroplasts it has been shown that (1) the size of the proton gradient under phosphorylating conditions is smaller than under non-phosphorylating conditions; (2) ADP, ATP or Dio-9, added under non-phosphorylating conditions, decrease the rate of electron transport but increase the size of the proton gradient; (3) ADP, ATP or Dio-9 inhibit not only electron transport but also the rate of decay of the proton gradient; (4) the H⁺/e⁻ ratio under non-phosphorylating conditions is 1.0. It is not affected by ADP, ATP or Dio-9.

These results show that protons pass out of the thylakoids at the site of ATP synthesis and that this leakage is inhibited by ADP, ATP or Dio-9, compounds that interact with the site of ATP synthesis. As these compounds do not alter the H⁺/e⁻ ratio the formation of the proton gradient must be an intermediate between electron transport and ATP synthesis. These data are in support of the chemiosmotic theory of coupling of electron transport to ATP synthesis.     

Evidence for the identity of P430 of Photosystem I and chloroplast-bound iron-sulfur protein

The EPR spectrum of Photosystem-I subchloroplast particles, which had been pre-illuminated in the presence of methyl viologen, showed a large P700⁺ signal whereas bound iron-sulfur proteins were not detected. This observation is consistent with a “one-way” electron discharge by the primary electron acceptor, P430⁻, subsequent to the primary photochemical charge separation, and an accumulation of photooxidized P700⁺. Subsequent illumination of the same sample at 77 °K did not change the EPR spectrum. However, if the pre-illuminated subchloroplast particles were allowed to recover at room temperature by standing in the dark for 10 min or by addition of a chemical reductant, subsequent illumination of the sample at 77 °K yielded an EPR spectrum consisting of signals due to both P700⁺ and reduced iron-sulfur protein.     

The effect of infra-red radiation on rectal temperature and respiration rate of unacclimated female new zealand white rabbits

1. 1.|An experiment was carried out to examine the effects of various levels of infra-red (i.r.) radiation on rectal temperature (RT) and respiration rate (RR) in New Zealand While rabbits.

2. 2.|A 4 × 3 × 6 factorial design was employed in which the factors were: four intensities of i.r. radiant heating of 0.0, 1.9, 2.1 and 2.4 MJ/m²/h, three replicates and six rabbits.

3. 3.|rectal temperature differed (P < 0.05) between treatments and were highest at the “high” level of i.r. radiation (1°C higher than for controls). At the “medium” and “low” levels of i.r. heating RTs were respectively 0.3 and 0.2°C higher than in controls.

4. 4.|At different levels of radiation RR were different (P < 0.05), with the highest (422.7 ± 218.1 breaths/min) at 2.4 MJ/m²/h i.r. radiant heating. This RR was almost 2.5 times that in controls, while at the “low” and “medium” i.r. levels RR values were respectively 1.5 and 2 times those of controls.

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

DNA interaction with biologically active divalent metal ions: binding constants calculation

In our previous work we have shown that under the action of Cu²⁺, Mn²⁺ and Ca²⁺ ions DNA is able to transit into a compact state in aqueous solution. In the present work we carried out calculations of binding constants for divalent metal ions interacting with DNA in terms of the macromolecule statistical sum. The formula for calculation of the binding constants and cooperativity parameters was proposed. It was shown that on the “coil state”–“compact (globule) state” transition a single DNA molecule may undergo the first-order phase transition while the transition of the assembly of average DNA chains is of sigmoidal character typical of the cooperative and continuous transition.