Changes in the energy distribution in mutant thylakoid membranes of pea with modified pigment content. II. Changes due to magnesium ions concentration

Low-temperature (77K) steady-state chlorophyll fluorescence emission spectra, room temperature fluorescence and light scattering of thylakoid membranes isolated from pea mutants were studied as a function of Mg2+ concentration. The mutants have modified pigment content and altered structural organization of the pigment-protein complexes, distinct surface electric properties and functions. The analysis of the 77K emission spectra revealed that Mg2+-depletion of the medium caused not only an increased energy flow toward photosystem I in all investigated membranes but also changes in the quenching of the fluorescence, most probably by internal conversion. The results indicated that the macroorganization of the photosynthetic apparatus of mutants at supramolecular level (distribution and segregation of two photosystems in thylakoid membranes) and at supermolecular level (stacking of photosystem II supercomplexes) required different Mg ion concentrations. The data confirmed that the segregation of photosystems and the stacking of thylakoid membranes are two distinct phenomena and elucidated some features of their mechanisms. The segregation is initiated by changes in the lateral microorganization of light harvesting complexes II, their migration (repulsion from photosystem I) and subsequent separation of the two photosystems. Most likely 3D aggregation and formation of macrodomains, containing only photosystem II antenna complexes, play a certain precursory role for the increasing degree of the membrane stacking and the energy coupling between the light harvesting complexes II and the core complexes of photosystem II in the frame of photosystem II supercomplexes.     

ORGANIZATION OF PIGMENT-PROTEIN COMPLEXES INTO MACRODOMAINS IN THE THYLAKOID MEMBRANES OF WILD-TYPE and CHLOROPHYLL fo-LESS MUTANT OF BARLEY AS REVEALED BY CIRCULAR DICHROISM

The organization of pigment-protein complexes into large chiral macrodomains was investigated in wild-type and chlorophyll b-less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular-dependence of circular intensity differential scattering showed that in wild-type chloroplasts, the formation of macrodomains was governed by interactions of the light-harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing-density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f-2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild-type thylakoid membranes. Circular dichroism is a valuable probe for examining the long-range interactions between pigment-protein complexes which participate in the formation and stabilization of membrane ultrastruc-ture. A functional role of macrodomains in long-range energy migration processes is proposed.     

Light-harvesting and structural organization of Photosystem II: from individual complexes to thylakoid membrane

Photosystem II (PSII) is responsible for the water oxidation in photosynthesis and it consists of many proteins and pigment-protein complexes in a variable composition, depending on environmental conditions. Sunlight-induced charge separation lies at the basis of the photochemical reactions and it occurs in the reaction center (RC). The RC is located in the PSII core which also contains light-harvesting complexes CP43 and CP47. The PSII core of plants is surrounded by external light-harvesting complexes (lhcs) forming supercomplexes, which together with additional external lhcs, are located in the thylakoid membrane where they perform their functions. In this paper we provide an overview of the available information on the structure and organization of pigment-protein complexes in PSII and relate this to experimental and theoretical results on excitation energy transfer (EET) and charge separation (CS). This is done for different subcomplexes, supercomplexes, PSII membranes and thylakoid membranes. Differences in experimental and theoretical results are discussed and the question is addressed how results and models for individual complexes relate to the results on larger systems. It is shown that it is still very difficult to combine all available results into one comprehensive picture.     

THE 75°C THERMOLUMINESCENCE BAND OF GREEN TISSUES: CHEMILUMINESCENCE FROM MEMBRANE-CHLOROPHYLL INTERACTION

Abstract— Exposure of thylakoid membranes of green plants to high temperature promotes the appearance of free radicals resulting in a thermoluminesccnce (TL) band peaking around 75°C. The occurrence of this band with the same intensity in prcilluminated and in dark-adapted samples demonstrates that, contrary to several other TL bands, it is not a result of charge recombination. The high temperature TL band is oxygen dependent. Parallel to TL emission singlet oxygen is formed, as demonstrated by spin trapping EPR measurements and by the decrease of TL intensity in the presencc of sodium-azide, a singlet oxygen scavenger.
We suggest that the 75°C TL band is a result of a temperature-enhanced interaction between molecular oxygen and the photosynthetic membrane, possibly involving lipid peroxidation. The spectral maximum of the emission (around 720 nm) implics that light emission occurs upon energy transfer from an excited product to chlorophyll molecules destablized from pigment-protein complexes.     

Effect of ultraviolet-B radiation on intact cells of the cyanobacterium Spirulina platensis: characterization of the alterations in the thylakoid membranes

Intact trichomes of Spirulina platensis are exposed to ultraviolet- B (UV-B) radiation (270-320 nm; 1.9 mW m(-2)) for 9 h. This UV-B exposure results in alterations in the pigment-protein complexes and in the fluorescence emission profile of the chlorophyll-protein complexes of the thylakoids as compared with thylakoids isolated from control dark-adapted Spirulina cells. The UV-B exposure causes a significant decrease in photosystem II activity, but no loss in photosystem I activity. Although there is no change in the photosystem I activity in thylakoids from UV-B-exposed cells, the chlorophyll a emission at room temperature and at 77 K indicates alterations associated with photosystem I. Additionally, the results clearly demonstrate that the photosystem II core antennae of chlorophyll proteins CP47 and CP43 are affected by UV-B exposure, as revealed by Western blot analysis. Furthermore, a prominent 94 kDa protein band appears in the sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of UV-B-exposed cell thylakoids, which is absent from the control thylakoids. This 94 kDa protein appears not to be newly induced by UV-B exposure, but could possibly have originated from the UV-B-induced cross-linking of the thylakoid proteins. The exposure of isolated Spirulina thylakoids to the same intensity of UV-B radiation for 1-3 h induces losses in the CP47 and CP43 levels, but does not induce the appearance of the 94 kDa protein band in SDS-PAGE. These results clearly demonstrate that prolonged exposure of Spirulina cells to moderate levels of UV-B affects the chlorophyll a-protein complexes and alters the fluorescence emission spectral profile of the pigment-protein complexes of the thylakoid membranes. Thus, it is clear that chlorophyll a antennae of Spirulina platensis are significantly altered by UV-B radiation.     

Separation of Rubisco in Extracts of Plant Leaves by Capillary Electrophoresis with Sieving Polymers

ABSTRACT

Rubisco (Ribulose-1, 5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) was separated in extracts of plant leaves by capillary electrophoresis with replaceable polymers. Rubisco extracted from plant leaves was fully denatured into small (15.8 kDa) and large (52.9 kDa) subunits in the presence of excess sodium dodecylsulfate (SDS). SDS-Rubisco complexes were separated by using an uncoateda fused silica capillary filled with a replaceable polymer solution, with detection at 220 nm. A calibration plot of Rubisco concentration in plant extract versus peak area was linear over the concentration range of 0.09-1.2 mg mL^?1 with a correlation coefficient (r²) of 0.995 and a detection limit of 0.05 mg mL^?1. The reproducibility of migration times was less than 0.27% RSD (n=5) and reproducibility of peak area was better than 6.9% The recovery of Rubisco in leaf tissues through the extraction and analysis procedure was 78.2% (±4.8) and through the analysis procedure alone was 96.9% (±5.1). The proposed method was used for the quantification of Rubisco in protein extracts from leaves of a variety of plant species.     

Factors controlling the mobility of photosynthetic proteins

Protein diffusion in and around the photosynthetic membrane must play a crucial role in photosynthetic functions including electron transport, regulation of light-harvesting, and biogenesis, turnover and repair of membrane components. Protein mobility is controlled by a complex web of specific interactions, plus the viscosity of the environment and the extent of macromolecular crowding. I discuss the techniques that can be used to measure protein mobility in photosynthetic membranes. I then summarize what we know about the constraints on protein mobility imposed by macromolecular aggregation and crowding in and around the thylakoid membranes of green plants and cyanobacteria, with particular reference to the fluidity of the thylakoid membrane and the aqueous phases on either side of the membrane (the stroma/cytoplasm and the thylakoid lumen). Current indications are that the stroma/cytoplasm is a relatively fluid environment, whereas protein mobility in the lumen may be extremely restricted. The thylakoid membrane itself has an intermediate fluidity: some protein complexes are virtually immobile, probably due to their incorporation into large, stable macromolecular aggregates. However, there is sufficient free space to allow the long-range diffusion of some complexes. Finally, I discuss some future directions for research in this area.     

Surfactant Effects on the Permeability of Photosynthetic Membrane from Rhodobacter sphaeroides 2.4.1 Probed by Electrochromic Shift of Endogenous Carotenoids

Four surfactants, sodium cholate(SC), n-dodecyl-β-D-maltopyranoside(DDM), lauryldimethylamine oxide(LDAO) and Triton X-100(TX), which are generally used in photosynthetic pigment-protein complexes preparation, were studied on their interaction with photosynthetic membrane from Rhodobacter sphaeroides 2.4.1 by electrochromic absorption band-shift of endogenous carotenoids and by vesicle size measurements as well. The surfactant critical micelle concentration(cmc) was found to be negatively correlated with the capability of enhancing the permeability of photosynthetic membranes to proton, and more elaborated model of surfactants interacting with membranes was obtained. The electrochromic absorption band-shift measurement might develop into a useful tool to evaluate the effects of surfactants on various membranes.     

Influence of Interaction Between Heavy Oil Components and Petroleum Sulfonate on the Oil–Water Interfacial Tension

The purpose of this study is to obtain the interaction between heavy oil components and petroleum sulfonate (NPS). In this article, the effects of pH, NaCl concentration, and NPS on the oil–water interfacial tension (IFT) of Gudao crude oil and its polar components were investigated. The results show that the NPS concentration corresponding to turning point of IFT is 0.001 g·mL^?1. This is lower than the CMC of NPS (0.0015 g·mL^?1) as there is a positive synergetic effect between NPS and the active substances of crude and its components, and the strength of their interaction depends on the interfacial activity of crude components. In simulated system of crude and polar components with 0.1 wt% NPS, at basic condition, the acidic substances in the polar components create naphthenates (the component whose acid number is higher creates more naphthenates), leading to lower IFT, so the interaction between heavy oil components and NPS is stronger in the basic condition. Proper concentration of NaCl in the stimulated systems improved the hydrophile-lipophile balance of emulsifier (NPS), accelerated the well-regulated adsorption of NPS in oil–water interface, and increased the interfacial activity of NPS, the interaction between heavy oil components and NPS was also enhanced.     

Potentiometric Anion Selectivity and Analytical Applications of Polymer Membrane Electrodes Based on Novel Mn(III)‐ and Mn(IV)‐Salophen Complexes

New Mn(III)‐L and Mn(IV)‐L complexes were prepared from the highly lipophilic salophen ligand (L): phenol 2,2′‐[(4,5‐dimethyl‐1,2‐phenylene)bis[(E)‐nitrilomethylidyne]]bis[4,6‐bis(1,1‐dimethylethyl). The prepared complexes were fully characterized and used for the construction of thiocyanate membrane electrodes. Optimized membrane electrodes contained 33.0 mg PVC, 66.0 mg o‐nitrophenyloctylether, 50 or 5 (mole %) tetrakis(trifluoromethyl)phenyl borate and 1 mg Mn(III)‐L (sensor 2) or Mn‐(IV)‐L (sensor 12), respectively. Such electrodes exhibited linear responses toward thiocynate in a concentration range of 10^?1–10^?5 M and detection limits of 8.3×10^?6, 8.9×10^?6 M for sensor 2 and 12, respectively. Optimized membrane electrodes exhbited high selectivty toward thiocayante compared to more lipophilic anions. The observed thiocyanate selectivity of the optimized membranes was confirmed by formation constant calculations for Mn(III)‐L and Mn(IV)‐L with SCN^?, β=10^14.1 and 10^12.5, which was measured potentiometrically using the sandwich membrane method. Furthermore, computational study using DFT calculations was performed to at DFT/B3LYP level of theory to confirm the observed selectivity data. The response times were 3 and 0.5 min for low and high concentrations. The lifetimes of the optimized electrodes were ～4–6 weeks. The analytical utility of the optimized membrane electrodes was demonstrated by the analysis of thiocyanate level in different saliva samples.     

ENERGY DISSIPATION AND PHOTOPROTECTION MECHANISMS DURING CHLOROPHYLL PHOTOBLEACHING IN THYLAKOID MEMBRANES

The kinetics of chlorophyll photobleaching were followed in whole thylakoid membranes as well as in photosystem I and photosystem II submembrane fractions. The onset of photobleaching was characterized by a slow rate which indicated the presence of energy traps implicated in the photoprotection of the bulk pigments. The pigments in photosystem I submembrane fractions bleached at a faster rate than those in photosystem II counterparts, the latter being more sensitive towards photoinhibition. An analysis of the pigment-protein complexes isolated from whole thylakoid membranes during the course of a photobleaching experiment has shown that the core-antenna complexes, including CP29, are more sensitive to illumination than the peripheral complexes. The absorption spectra of the CPI and CP29 complexes presented a blue shift of the red absorption maximum after partial photobleaching, indicative of a non-homogeneous bleaching of the holochromes in these complexes. An analysis of these data points towards the involvement of CP29 in a photoprotection mechanism at the level of photosystem II. The weaker resistance of photosystem I to photobleaching relative to photosystem II and its stronger resistance to photoinhibition is discussed in terms of an energy dissipation pathway in thylakoid membranes.     

Selective and Efficient Removal of Fluoride from Water: In Situ Engineered Amyloid Fibril/ZrO2 Hybrid Membranes

We report a new strategy for efficient removal of F^? from contaminated water streams, and it relies on carbon hybrid membranes made of amyloid fibril/ZrO₂ nanoparticles (<10 nm). These membranes exhibit superior selectivity for F^? against various competitive ions, with a distribution coefficient (K_d) as high as 6820 mL g^?1, exceeding commercial ion‐exchange resins (IRA‐900) by 180 times and outdoing the performance of most commercial carbon‐activated aluminum membranes. At both low and high (ca. 200 mg L^?1) F^? concentrations, the membrane efficiency exceeds 99.5 % removal. For real untreated municipal tap water (ca. 2.8 mg L^?1) under continuous operating mode, data indicates that about 1750 kg water m^?2 membrane can be treated while maintaining drinking water quality, and the saturated membranes can be regenerated and reused several times without decrease in performance. This technology is promising for mitigating the problem of fluoride water contamination worldwide.     

Speciation and analysis of corrosion products in the primary coolant of pressurized water reactors

The procedure involves separate sampling and determination of the insoluble, cationic and anionic species of corrosion products (Fe, Ni, Cr, Mn, Co, Zn, Cu) in the primary coolant of pressurized water reactors (PWRs) with concentrations in the range 0–2000 mg l^?1 boron and 0–5 mg l^?1 lithium. Samples of coolant (0.2–1 l) are passed through packs consisting of one 0.45-μm filter paper, one cation-exchange membrane (Whatman SA-2) and one anion-exchange membrane (Whatman SB-2). The membranes are examined by wavelength-dispersive x-ray spectrometry. Selection of the ion-exchange membranes and the influence of the boron and lithium concentrations (and pH) on retention of soluble species are discussed. With sample volumes of 0.5 l, the detection limits are between 0.05 and 0.3 μg l^?1 for undissolved species and from 0.03 to 0.14 μg l^?1 for ions. Data collected during a PWR shutdown procedure are summarized.     

Instability of thin liquid films between membranes

The conditions for instability of the thin liquid film between two plane-parallel membranes were derived taking into account the influence of the membrane tension, the membrane bending elasticity, the film viscosity and the disjoining pressure. It was shown that the liquid film could be unstable if the negative (attractive) disjoining pressure is predominant. The characteristic timeτ _m of growth of perturbation due to thermal or other fluctuations of the membrane shape increases with increasing the film viscosity, the membrane tension and the membrane bending elasticity, and decreasing the film thickness and the negative disjoining pressure. It is of the order of 10^?2÷10³ sec. When the membranes approach each other at certain value of the average film thicknessh _cr called critical, the fastest growing perturbations lead to formation of a liquid film with smaller (or zero) thickness. It was found that the critical thickness increases with increasing the negative disjoining pressure and the membrane area and decreasing the membrane tension and the bending elasticity having typical values of the order of 10^?6÷10^?5 cm. The case of a membrane approaching a solid plane was also considered. Excluding the small differences in numerical coefficients the results are similar to the case of two identical membranes.     

Novel Biomedical Sensors for Flow Injection Potentiometric Determination of Creatinine in Human Serum

Coated‐wire (CW) and tubular (Tu) type membrane sensors for creatinine are developed. These consist of creatinine tungstophosphate(CTP), creatinine molybdophosphate (CMP) and creatinine picrolonate (CPC) ion‐pair complexes as electroactive materials dispersed in plasticized poly(vinyl chloride) matrix membranes. Electrochemical evaluation of these sensors under static (batch) mode of operation reveals near‐Nernstian response with slopes of 62.9, 58.1, and 55.2 mV decade^?1 over the concentration range 1×10^?2–5.0×10^?6, 1×10^?2–7.5×10^?5, and 1×10^?2?3.1×10^?5 mol L^?1. The lower detection limits are 0.39, 3.49, and 2.20 μg mL^?1 creatinine with CTP, CMP and CPC membrane based sensors plasticized with o‐NPOE, respectively. Tubular and coated wire CTP membrane sensors are incorporated in flow‐through cells and used as detectors for flow injection analysis (FIA) of creatinine. The intrinsic characteristics of the detectors under hydrodynamic mode of operation in a low dispersion manifold are determined and compared with data obtained under static mode of operation. With 10^?2 mol L^?1 phosphate buffer of pH 4.5 as a carrier solution, the tubular and coated wire CTP detectors exhibit rapid response of 58.9 and 50.7 mV decade^?1 over the concentration range 1×10^?2–1×10^?5 mol L^?1 and detection limits of 0.39 μg mL^?1 and 0.85 μg mL^?1, respectively. Validation of the assay methods with the proposed sensors by measuring the lower detection limit, range, accuracy, precision, repeatability and between‐day‐variability reveals good performance characteristics confirming applicability for continuous determination of creatinine. The sensors are used for determining creatinine in human blood serum at an input rate of 40 samples per hour. No interferences are caused by creatine, most common anions, cations and organic species normally present in biological fluids. The results favorably compare with data obtained using the standard spectrophotometric method.     

Influence of variation in eumelanin content on absorbance spectra of liquid skin-like phantoms

The attenuation behavior of two different types of skin‐like phantoms representing the range of Fitzpatrick skin Types I–VI was investigated and compared with real human skin. Intralipid (IL) and Pheroid^? artificial lipid membrane vesicles, respectively, were added to synthetic eumelanin concentrations ranging from 0.0044 to 0.13 mg mL^?1 to produce skin‐like phantoms. Spectrophotometric absorbance and transmittance measurements were performed. Results indicated some of the nonmonotonic trends observed in real human skin, albeit shifted more toward the visible wavelength range. There exists, however, an underlying difference in interaction between the melanin and the Pheroid^? and IL skin‐like phantoms.     

Sub‐10 nm Polyamide Nanofiltration Membrane for Molecular Separation

To separate small molecules from the solvent with high permeability and selectivity, the membrane process is thought to be highly effective with much lower energy consumption when compared to the traditional thermal‐based separation process. To achieve high solvent permeance, a sub‐10 nm thick polyamide nanofiltration membrane was synthesized through interfacial polymerization of ethidium bromide (EtBr) and trimesoyl chloride (TMC). Thanks to the extremely low solubility of the EtBr monomer in the organic phase, the polymerization process was strictly limited at the interface of the water and hexane, leading to an ultrathin polyamide membrane with a thickness down to sub‐10 nm. When used in nanofiltration, these ultrathin membranes display ultrafast water permeation of 40 liter per square meter per hour per bar (L m^?2 h^?1 bar^?1), and a high Congo red rejection rate of 93 %. This work demonstrates a new route to synthesize ultrathin polyamide membranes by the traditional interfacial polymerization.     

Single robust RP-HPLC analytical method for quantification of curcuminoids in commercial turmeric products,Ayurvedic medicines,and nanovesicular systems

A single robust reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated as per International Conference on Harmonization guidelines for the accurate quantification of curcuminoids in commercial turmeric products, Ayurvedic medicines, and nanovesicular systems. The proposed chromatographic method was found to be specific, linear (r²?≥?0.999), precise at intra- and inter-day levels (percentage relative standard deviation <2.0%), accurate (percentage recovery 99.14–102.29%), and robust. The limits of detection and quantification were found to be 7.40 and 24.70?ng?mL^?1 for curcumin, 9.24 and 30.80?ng?mL^?1 for demethoxycurcumin, and 6.48 and 21.61?ng?mL^?1 for bisdemethoxycurcumin, respectively. Among different commercial turmeric products and Ayurvedic medicines tested, the contents of curcumin (3.54?±?0.06–25.8?±?0.08?mg?g^?1), demethoxycurcumin (1.28?±?0.02–9.97?±?0.03?mg?g^?1), and bisdemethoxycurcumin (0.50?±?0.01–5.97?±?0.01?mg?g^?1) varied significantly. The developed method was effectively applied to the determination of encapsulation efficiency of curcuminoids (ranged between 84.33?±?3.50 and 96.59?±?2.53%) in the nanovesicular systems. In conclusion, the reported method is suitable for the analysis of curcuminoids in a wide variety of turmeric products and used for the quality control of products that contain curcuminoids.     

Sensitive Detection of Capsaicin by Adsorptive Stripping Voltammetry at a Boron‐Doped Diamond Electrode in the Presence of Sodium Dodecylsulfate

In the present paper, a sensitive electroanalytical methodology for the determination of capsaicin using adsorptive stripping voltammetry (AdSV) at a boron‐doped diamond (BDD) electrode is presented. The voltammetric results indicate that in the presence of sodium dodecylsulfate (SDS) the BDD electrode remarkably enhances the oxidation of capsaicin which leads to an improvement of the peak current with a shift of the peak potential to less negative values. A linear working range of 0.05 to 6.0 µg mL^?1 (0.16–20 µM) with a detection limit of 0.012 µg mL^?1 (0.034 µM) has been obtained using BDD electrode by AdSV.     

SURFACE-ENHANCED RESONANCE RAMAN SCATTERING SPECTROSCOPY AS A SURFACE TOPOGRAPHY PROBE IN PLANT PHOTOSYNTHETIC MEMBRANES

Strong resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) signals from carotenoids were detected from thylakoid (stromal-side out) vesicles and inside-out (lumenal-side out) vesicles isolated from spinach chloroplasts. The intensity of the signals from both types of membranes was comparable, indicating that plant carotenoids are exposed on or close to both surfaces or sides of the thylakoid membrane. This is in contrast to previous studies with bacterial photosynthetic membranes (Picorel et al., 1988, J. Biol. Chem. 263 , 4374–4380; and 1990, Biochemistry 29 , 707–712) that show carotenoids selectively located on the cytoplasmic side. In addition_; strong RR and SERRS signals were detected from stacked and unstacked photosystem-II-enriched membrane fragments, demonstrating that carotenoids are also exposed on both surfaces of the appressed region of the thylakoid membrane. Antibodies against the photosystem (PS) II extrinsic proteins blocked SERRS signals from stacked PS II membrane fragments, but only partially affected the SERRS signals from unstacked membranes. The results indicate that these antibodies, which preferentially cover the surface of the original lumenalside of the appressed region, act as spacers between the membrane and SERRS electrode surfaces. The original stromal-side of the appressed region is unaffected. These findings verify the distance sensitivity of the SERRS technique and underscore the above conclusion about the location of carotenoids in the appressed regions. Finally, SERRS signals are sensitive to membrane aging and storage temperature; caution is suggested to those applying SERRS spectroscopy to intact membrane systems.