chlorophyll-b and pheophytin-a

In view of the folklore use of green leaves to treat inflammation, the anti-inflammatory property of chlorophylls and their degradation products were studied. Chlorophyll a and pheophytin a (magnesium-free chlorophyll a) from fresh leaves showed potent anti-inflammatory activity against carrageenan-induced paw edema in mice and formalin-induced paw edema in rats. Chlorophyll a inhibited bacterial lipopolysaccharide-induced TNF-α (a pro-inflammatory cytokine) gene expression in HEK293 cells, but it did not influence the expression of inducible nitric acid synthase and cyclooxygenase-2 genes. Chlorophyll b only marginally inhibited both inflammation and TNF-α gene expression. But both chlorophyll a and chlorophyll b showed the same level of marginal inhibition on 12-O-tetradecanoyl-phorbol-13-acetate-induced NF-κB activation. Chlorophylls and pheophytins showed in vitro anti-oxidant activity. The study shows that chlorophyll a and its degradation products are valuable and abundantly available anti-inflammatory agents and promising for the development of phytomedicine or conventional medicine to treat inflammation and related diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Chlorophyll; Chlorophyll A; Chromolaena; Cyclooxygenase 2; Edema; Eupatorium; Formaldehyde; HEK293 Cells; Humans; Inflammation; Lipopolysaccharides; Mice; Moraceae; NF-kappa B; Nitric Oxide Synthase Type II; Pheophytins; Plant Extracts; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

We report a new device for the estimation of the content of chlorophyll a pigment in water samples as an indicator of water quality. The extraction of the pigment from water was also optimized. 10 mL of water was filtered through a nylon filter (45 microm pore size and 13 mm of diameter), after the chlorophylls were dissolved by immersing the filter in 1 mL of a low non-hazardous solvent as ethanol. An in-valve in-tube SPME device coupled to capillary liquid chromatography with diode array detection was designed. A capillary column of 70 cm in length (0.32 mm i.d. coated with 5% diphenyl-95% polydimethylsiloxane, 3 microm coating thickness) was used as the loop of the injection valve for preconcentration and a Zorbax SB C(18) (SiO(2)-based) 150 mm x 0.5 mm i.d., 5 microm column (Agilent) was used as analytical column. The achieved detection limit was 0.05 microg L(-1) and the working range of concentrations was 0.1-1 microg L(-1). % RSD values between 2 and 11 were obtained. Chlorophyll a in several water matrices was determined with good results in presence of other pigments such as chlorophyll b, pheophytin a and pheophytin b.

Topics: Chlorophyll; Filtration; Limit of Detection; Pheophytins; Quality Control; Solid Phase Extraction; Solvents; Water Pollutants, Chemical

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.

Topics: Chlorophyll; Cyanobacteria; Electron Transport; Energy Metabolism; Kinetics; Light-Harvesting Protein Complexes; Luminescent Measurements; Oxidation-Reduction; Oxygen; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Proteins; Spectrometry, Fluorescence; Temperature

The resolution of quaternary mixtures of chlorophylls a and b and pheophytins a and b has been accomplished by partial least-squares (PLS) multivariate calibration, applied to the fluorescence signals of these pigments. The total luminescence information of the compounds has been used to optimize the spectral data set to perform the calibration. After preliminary studies, a method is described in acetone media, to avoid emulsions with the olive oil samples. Different scanning paths have been selected for each method. For the simultaneous determination of the pigments in olive oil samples, a comparative study of the results found by using excitation, emission, and synchronous spectral data, as analytical signal, was performed. The excitation spectra were selected as the better analytical signals for the determination of the pigments in olive oil samples. The optimum wavelength range to record the excitation spectra (lambda(em) = 662 nm) was selected to minimize the contribution of pheophytin a and to maximize the contribution of the other pigments, which are the minor constituents in olive oil. Determination of these pigments in olive oil samples was effected from the excitation spectra of dissolutions o suitable aliquots in acetone. Recovery values from olive oil, spiked with chlorophylls a and b and pheophytins a and b, were in the ranges of 70-112, 71-111, 76-105, and 82-109%, respectively.

Topics: Chlorophyll; Chlorophyll A; Olive Oil; Pheophytins; Plant Oils; Spectrometry, Fluorescence

The reduction of chlorophyllide b and its analogue zinc pheophorbide b in etioplasts of barley (Hordeum vulgare L.) was investigated in detail. In intact etioplasts, the reduction proceeds to chlorophyllide a and zinc pheophorbide a or, if incubated together with phytyldiphosphate, to chlorophyll a and zinc pheophytin a, respectively. In lysed etioplasts supplied with NADPH, the reduction stops at the intermediate step of 7(1)-OH-chlorophyll(ide) and Zn-7(1)-OH-pheophorbide or Zn-7(1)-OH-pheophytin. However, the final reduction is achieved when reduced ferredoxin is added to the lysed etioplasts, suggesting that ferredoxin is the natural cofactor for reduction of chlorophyll b to chlorophyll a. The reduction to chlorophyll a requires ATP in intact etioplasts but not in lysed etioplasts when reduced ferredoxin is supplied. The role of ATP and the significance of two cofactors for the two steps of reduction are discussed.

Topics: Adenosine Triphosphate; Alcohol Oxidoreductases; Chlorophyll; Chlorophyll A; Ferredoxin-NADP Reductase; Ferredoxins; Hordeum; Intracellular Membranes; Oxidation-Reduction; Pheophytins; Plastids; Subcellular Fractions; Zinc

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

Topics: Chlorophyll; Chlorophyll A; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plants; Zinc

Photosystem II, the photosynthetic water-oxidizing complex, can be isolated from both plants and cyanobacteria. A variety of methods have been developed for purification of this enzyme, which can be isolated in several functional and structural forms. Knowledge of the pigment content of photosystem II preparations is important for precise spectroscopic, biochemical, and functional analysis. We have determined pigment stoichiometries in oxygen-evolving photosystem II preparations from plants and cyanobacteria. We have employed a solvent system for the isocratic elution of a reverse phase HPLC column in which we have determined the extinction coefficients of the relevant pigments. Pigments were extracted from four photosystem II preparations. These preparations included spinach photosystem II membranes [Berthold, D. A., Babcock, G. T., & Yocum, C. F. (1981) FEBS Lett. 134, 231-234], spinach photosystem II reaction center complexes [Ghanotakis, D. F., & Yocum, C. F. (1986) FEBS Lett. 197, 244-248], spinach photosystem II complexes [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856], and photosystem II particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803 [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Pigment stoichiometries were determined using two different methods of data analysis and were based on the assumption that there are two pheophytin a molecules per photosystem II reaction center. The pigment stoichiometries obtained were comparable for the two methods of data analysis and agreed with previous biophysical and biochemical characterizations of the preparations. The average pigment stoichiometries (chlorophyll:plastoquinone-9 per 2 pheophytin a) determined using the two data analysis methods were as follows: photosystem II membranes, 274:3.2; photosystem II reaction center complexes, 78:2.5; Synechocystis PS II particles, 55:2.4; photosystem II complexes, 121:2.0.

Topics: Chlorophyll; Chlorophyll A; Chromatography, High Pressure Liquid; Cyanobacteria; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plastoquinone; Spectrophotometry; Spinacia oleracea

Topics: Chemistry Techniques, Analytical; Chlorophyll; Chlorophyll A; Pheophytins; Research; Water