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PubMed2006

Isolation of chlorophylls a and b from spinach by counter-current chromatography.

A method for the isolation of chlorophylls from spinach by counter-current chromatography was developed. An initial extraction protocol was devised to avoid the notorious sensitivity of chlorophylls to degradation by light, heat, oxygen, acids and bases. Further purification and separation of chlorophylls a and b were achieved using counter-current chromatography. Chlorophyll structures and purities were established by HPLC, fast atom bombardment mass spectrometry and nuclear magnetic resonance. Purity was estimated to be >95% (100% by HPLC). Typical yields from 30g of freeze-dried spinach were 300mg of chlorophyll a and 100mg of chlorophyll b.

Chlorophyll↗
PubMed2006

Chlorophyll-a in the rivers of eastern England.

Chlorophyll-a concentration variations are described for two major river basins in England, the Humber and the Thames and related to catchment characteristics and nutrient concentrations across a range of rural, agricultural and urban/industrial settings. For all the rivers there are strong seasonal variations, with concentrations peaking in the spring and summer time when biological activity is at its highest. However, there are large variations in the magnitude of the seasonal effects across the rivers. For the spring-summer low-flow periods, average concentrations of chlorophyll-a correlate with soluble reactive phosphorus (SRP). Chlorophyll-a is also correlated with particulate nitrogen (PN), organic carbon (POC) and suspended sediments. However, the strongest relationships are with catchment area and flow, where two straight line relationships are observed. The results indicate the importance of residence times for determining planktonic growth within the rivers. This is also indicated by the lack of chlorophyll-a response to lowering of SRP concentrations in several of the rivers in the area due to phosphorus stripping of effluents at major sewage treatment works. A key control on chlorophyll-a concentration may be the input of canal and reservoir waters during the growing period: this too relates to issues of residence times. However, there may well be a complex series of factors influencing residence time across the catchments due to features such as inhomogeneous flow within the catchments, a fractal distribution of stream channels that leads to a distribution of residence times and differences in planktonic inoculation sources. Industrial pollution on the Aire and Calder seems to have affected the relationship of chlorophyll-a with PN and POC. The results are discussed in relation to the Water Framework Directive.

Agriculture↗
PubMed2002

A structural investigation of the central chlorophyll a binding sites in the minor photosystem II antenna protein, Lhcb4.

Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressing modified apoproteins in Escherichia coli, and reconstituting them in the presence of chlorophyll and carotenoid cofactors. This method has allowed, in particular, the engineering of mutant LHCs in which each of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinating amino acids [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10056-10061]. The availability of these mutants is of particular importance for determining the precise position of absorption bands for the different chlorophyll molecules, as well as the sequence of energy transfer events that occur within LHC complexes, provided that the structural impact of each mutation is precisely evaluated. Using resonance Raman spectroscopy, we have characterized the pigment-protein interactions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of the four central chlorophyll a molecules has been removed by such mutations. By comparing the spectra of these mutants with those of the wild-type protein, the state of interaction of the carbonyl group, the coordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediate environment in the binding pocket of the chlorophyll a molecule were defined for each cofactor binding site. In addition, the structural impact of the absence of one chlorophyll a molecule and the quality of protein folding were evaluated for each of these mutated polypeptides.

Binding Sites↗
PubMed1999

Exchange of pigment-binding amino acids in light-harvesting chlorophyll a/b protein.

Four amino acids in the major light-harvesting chlorophyll (Chl) a/b complex (LHCII) that are thought to coordinate Chl molecules have been exchanged with amino acids that presumably cannot bind Chl. Amino acids H68, Q131, Q197, and H212 are positioned in helixes B, C, A, and D, respectively, and, according to the LHCII crystal structure [Kühlbrandt, W., et al. (1994) Nature 367, 614-621], coordinate the Chl molecules named a(5), b(6), a(3), and b(3). Moreover, a double mutant was analyzed carrying exchanges at positions E65 and H68, presumably affecting Chls a(4) and a(5). All mutant proteins could be reconstituted in vitro with pigments, although the thermal stability of the resulting mutant versions of recombinant LHCII varied significantly. All complexes reconstituted with the mutant proteins contained fewer chlorophyll molecules per two lutein molecules than complexes reconstituted with the wild-type protein. However, the chlorophyll-binding amino acids could not be unambiguously assigned to binding either chlorophyll a or b, as in most cases more than one chlorophyll molecule was lost due to the mutation. The changes in Chl stoichiometries suggest that in LHCII some chlorophyll positions can be filled with either Chl a or b. Only some of the point mutations in LHCII affected the ability of the apoprotein to assemble into trimeric LHCII upon insertion into isolated thylakoid membranes. Among these were exchanges of H68 with either F or L, suggesting that the stability of the LHCII trimer significantly depends on this amino acid or the Chl molecule named a(5) that is attached to it and is located close to the center of the trimeric complex. The ion pair bridge between E65 and R185 in LHCII does not appear to be essential for the proper folding of the protein.

Amino Acid Sequence↗
PubMed1999

Kinetics of chlorophyll degradation and color loss in heated broccoli juice.

Degradation of chlorophyll in broccoli juice occurred at temperatures exceeding 60 degrees C. Chemical analysis revealed that degradation of chlorophyll a and b to pheophytin a and b, respectively, followed first-order kinetics and that chlorophyll a was more heat sensitive than chlorophyll b. Temperature dependencies of chlorophyll a and b degradation rate constants could be described by Arrhenius equations with activation energies (E(a)) of 71.04 +/- 4.89 and 67.11 +/- 6.82 kJ/mol, respectively. Objective greenness measurements, using the -a value as the physical property, together with a fractional conversion kinetic analysis, indicated that green color degradation followed a two-step process. Kinetic parameters for the first degradation step were in accordance with the kinetic parameters for pheophytinization of the total chlorophyll content, as determined by chemical analysis (E(a) approximately 69 kJ/mol). The second degradation step, that is, the subsequent decomposition of pheophytins, was characterized by an activation energy of 105.49 +/- 4.74 kJ/mol.

Beverages↗
PubMed2005

Phytoplankton community and chlorophyll a as trophic state indices of Lake Skadar (Montenegro, Balkan).

BACKGROUND, AIMS AND SCOPE: Phytoplankton, as a first step in trophic cascades of lakes, can be a good indicator of trophic states, considering that every environmental change affects this community and many species of this community are sensitive to changes, and that they response very quickly. In this study, we tried to assess and predict the trophic state of Lake Skadar according to phytoplankton data. METHODS: Water samples were collected using Ruttner sampling bottle. Temperature, dissolved oxygen, ph, conductivity and transparence were measured in situ using portable equipment. Nutrients and chlorophyll a were measured using standard spectrophotometric methods. A determination of phytoplankton species was performed using relevant keys and the counting of cells was performed using sedimentation methods. RESULTS AND DISCUSSION: The species composition of Lake Skadar revealed 95 taxa, with Chlorophyceae and Bacillariophyceae being represented best. According to an average chlorophyll a concentration of 5.9 pg/l, Lake Skadar belongs to the mesotrophic level of the trophic scale. Developed prediction equation for chlorophyll a revealed a good prediction (R2 = 0.71) and the parameter Secchi depth was primarily correlated with chlorophyll a concentration. Trophic state indices derived from chlorophyll a and transparency, were close together, but both were below the phosphorous index. Values of trophic state indices rank the Lake Skadar as being mesotrophic. This study also showed that indices of diversity based on phytoplankton are weak indicators of trophic status and that they can well characterize only differences between assemblages and associations. According to calculated saprobic indices (ranging from 1.5 to 2.15), Lake Skadar is on betamesosaprobic level of saprobity, which means that it is moderately polluted with organic compounds. CONCLUSIONS: Total phosphorus is not the main limiting factor for the phytoplankton community in Lake Skadar. Disagreements between chlorophyll and the transparency index, on the one hand, and the total phosphorus index, on the other, suggest that the phytoplankton in Lake Skadar is probably limited by other factors than phosphorus, such as nitrogen, toxic substances or intense zooplankton grazing. According to the majority of investigated parameters and indices derived from phytoplankton data, Lake Skadar is mesotrophic, with tendencies toward eutrophic levels during the summer period. RECOMMENDATIONS AND OUTLOOK: Long-term monitoring is required for a better estimation of state and the conditions of Lake Skadar. Further studies on factors influencing the phytoplankton community, especially zooplankton grazing and toxic substances, which were not included in this study, should be continued in the future to improve the efficiency of phytoplankton usage in estimating the ecological and trophic conditions of Lake Skadar.

Albania↗
PubMed1997

On the presence and role of a molecule of chlorophyll a in the cytochrome b6 f complex.

Highly purified preparations of cytochrome b6 f complex from the unicellar freshwater alga Chlamydomonas reinhardtii contain about 1 molecule of chlorophyll a/cytochrome f. Several lines of evidence indicate that the chlorophyll is an authentic component of the complex rather than a contaminant. In particular, (i) the stoichiometry is constant; (ii) the chlorophyll is associated with the complex at a specific binding site, as evidenced by resonance Raman spectroscopy; (iii) it does not originate from free chlorophyll released from thylakoid membranes upon solubilization; and (iv) its rate of exchange with free, radioactive chlorophyll a is extremely slow (weeks). Some of the putative functional roles for a chlorophyll in the b6f complex are experimentally ruled out, and its possible evolutionary origin is briefly discussed.

Animals↗
PubMed1953

Chlorophyll monolayers in chloroplasts.

1. The data on the fine structure of the chloroplasts and the chlorophyll analyses, in two algal flagellates, Euglena gracilis and Poteriochromonas stipitata, are consistent with the assumption that the chlorophyll molecules are arranged in monomolecular layers at the interfaces between the lipid (dense) and the aqueous protein complex layers. 2. The cross-sectional area available to each chlorophyll molecule in the chloroplasts of Euglena and Poteriochromonas was found to be 222 x 10(-16) cm(2). and 246 x 10(-16) cm(2). By comparison, the cross-sectional area of the hydrophilic head of the chlorophyll molecule is approximately 240 x 10(-16) cm(2). 3. The model of the molecular network depicted in Text-fig. 1 is purely speculative. It predicts a lower limit for the number ratio of chlorophyll to other pigment molecules of about 1:1 and a weight ratio of 2:1. A loose packed structure such as that shown in Text-fig. 1 predicts a weight ratio of 4:1 to 6:1. These values bracket the large majority of experimental values found for this ratio. The relative constancy of the number of chlorophyll molecules per chloroplast and the volume of the chloroplast indicated by the data in Table III suggest that chloroplasts probably possess a similar structural arrangement in a variety of photosynthetic microorganisms and plants.

Chlorophyll↗
PubMed1996

Substrate specificity of chlorophyll(ide) b reductase in etioplasts of barley (Hordeum vulgare L.).

Enzyme activity of chlorophyll(ide) b reductase is present in etioplasts. Recently the conversion of chlorophyllide b to chlorophyll a via 7(1)-hydroxychlorophyll a was demonstrated in barley etioplasts. We used zinc pheophorbide b for a detailed investigation of the reduction of the 7-formyl group to the 7(1)-hydroxy compound in intact barley etioplasts. The reaction proceeded likewise before esterification and after esterification with phytyl diphosphate. The metal-free pheophorbide b, that is not accepted by chlorophyll synthase for esterification, is reduced to 7(1)-hydroxypheophorbide a to a small extent. The zinc (13(2)S)-pheophorbide b is at least equally well accepted for reduction as the epimer with the 13(2)R configuration of natural chlorophyll b. The reaction requires NADPH or NADH, although the latter is less effective. ATP is not required for the first step to the 7(1)-hydroxy compound. The significance of chlorophyll b reduction for acclimation from shade to sun leaves and for chlorophyll degradation is discussed.

Adenosine Triphosphate↗
PubMed1999

Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley etioplasts.

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.

Chlorophyll↗
PubMed2003

[Distribution features of chlorophyll a and primary productivity in high frequency area of red tide in East China Sea during spring].

The distributions of chlorophyll a and primary productivity were determined during April to May 2002 in the East China Sea. The results showed that the average concentration of chlorophyll a was 1.086 mg.m-3 at surface layer, and that nano- and pico-phytoplankton (< 20 microns) dominated the phytoplankton biomass in this sea region during Spring (up to 64% of total chlorophyll a content). Ultra-phytoplankton (< 5 microns) consisted 27% of total phytoplankton biomass. Nutrients and feeding pressure of zooplankton affected the distribution of chlorophyll a and its size-fractionation. The average primary productivity was 10.091 mg.m-3.h-1, while that of red tide tracking stations R-03, RL-01 and RG-01 was 399.984 mg.m-3.h-1. Light and nutrients were the main factors affecting the distributions of chlorophyll a and primary productivity. The station DC-11 had a high concentration of phytoplankton biomass. The surface layer concentration of chlorophyll a and primary productivity were up to 9,082 mg.m-3 and 128,79 mg.m-3.h-1, respectively, but the color of the seawater was normal.

Animals↗
PubMed2003

[Multianalysis between chlorophyll-a and environmental factors in Qiandao Lake water].

In this paper, the regression equation between chlorophyll-a (Chla) and some environmental factors was calculate by multiple stepwise regression, based on the monthly monitoring data in the Qiandao Lake from Jan. 1999 to Dec. 2000, and modified Carlson trophic state index for all the sampling sites was also analyzed. The results indicated that the spatial and temporal distribution of chlorophyll a in the sampling sites were varied strongly, the concentration of chlorophyll a in the Xin'an river changed from 50 mg.m-3 to 30 mg.m-3, but that in the lake was under 5 mg.m-3. The modified Carlson trophic state index (TSIM) for the sampling sites in southeast of the lake was under 37 (belonging to olligotrophic), and the others were under 53 (belonging to mesotrophic). In different sampling sites, the significant environmental affecting factors for chlorophyll a were different. However, the relationships between chlorophyll a and water temperature and total phosphorus were positive, and that between chlorophyll a and diaphaneity was negative.

Chlorophyll↗
PubMed1996

Clastogenic activity of pure chlorophyll and anticlastogenic effects of equivalent amounts of crude extract of Indian spinach leaf and chlorophyllin following dietary supplementation to mice.

Dietary consumption of green vegetables has been associated with protection against mutagenic and clastogenic activity of genotoxicants. Chlorophyll, being present in all green plants, had earlier been suggested to be the principal factor involved. Mice were administered (i) crude aqueous extract of leaf of Indian spinach, Beta vulgaris L. var.benghalensis Hort., and equivalent amounts of (ii) chlorophyll extracted from the leaf; (iii) purified chlorophyll, (iv) chlorophyllin, a sodium-copper derivative of chlorophyll; daily for 7 days. On day 7, one set of mice from each treatment was administered potassium dichromate-a known metallic clastogen. The mice were sacrificed after 24 hours. Chromosome preparations were made from bone marrow following the usual colchicine-air dry-Giemsa schedule. The cytogenetic endpoints scored were chromosomal aberrations and damaged cells. Crude leaf extract and chlorophyllin were nonclastogenic and reduced the clastogenic effects of potassium dichromate to the control distilled water level. Chlorophyll alone, whether extracted from the leaf or obtained in commercially purified form, was clastogenic and could reduce the effects of the chromium salt only to its own level. The protective action of the crude leaf extract may be attributed to the total effect of the interaction between the different components within the leaf extract, in which the clastogenicity of chlorophyll had been neutralized.

Animals↗
PubMed1994

Chlorophyll a biosynthetic heterogeneity.

Chlorophyll a biosynthesis is presently interpreted in terms of two different biochemical pathways. According to one pathway, chlorophyll a is made via a single linear chain of reactions starting with divinylprotoporphyrin IX and ending with monovinylchlorophyll a. The experimental evidence for this pathway is marred by incompletely characterized intermediates that were detected in Chlorella mutants. The second pathway considers chlorophyll a to be made via multiple and parallel biosynthetic routes that result in the formation and accumulation of monovinyl- and divinylchlorophyll a chemical species. Two of these routes, namely the di/monocarboxylic monovinyl and divinyl routes, are responsible for the biosynthesis of most of the chlorophyll a in green plants. The experimental evidence for these two routes consists of: (a) the detection and spectroscopic characterization of intermediates and end products; (b) the demonstration of precursor-product relationships between various intermediates in vivo and in vitro; and (c) the detection of 4-vinylreductases that appear to be mainly responsible for the observed biosynthetic heterogeneity. The biological significance of chlorophyll a biosynthetic heterogeneity is becoming better understood. On the basis of the prevalence of the di/monocarboxylic monovinyl-and divinylchlorophyll a biosynthetic routes, green plants have been classified into three different greening groups. It now appears that the major chlorophylls in the euphotic zone of tropical waters are divinylchlorophyll a and b. It also appears that the di/monocarboxylic monovinyl and divinyl biosynthetic routes lead to the formation of different pigment proteins in different greening groups of plants, and that the more highly evolved monovinylchlorophyll a biosynthetic route is associated with higher field productivity in wheat.

Chlorella↗
PubMedDate not supplied

Properties of chlorophyll on plasticized polyethylene particles.

There are several reasons for suspecting that there is a specific interaction between chlorophyll and galactolipids in the chloroplast. The model system described is intended to detect association of chlorophyll with polar lipids and other surfactants at a hydrocarbon-water interface. It consists of chlorophyll and other lipids or surfactants absorbed to the surface of polyethylene particles, which have been swelled with undecane to allow the lipophilic parts of these molecules to be anchored firmly in the hydrocarbon substrate. The absorption spectrum of adsorbed chlorophyll is usually modified by the presence of surfactant, and usually in the direction of decreased order of aggregation. Spectra in the presence of glycolipids in particular seem peculiar to the surfactant. The particles are strongly fluorescent, at room temperature as well as at 77K, and emission bands from aggregated chlorophyll species are observed along with fluorescence of monomeric chlorophyll.

Chlorophyll↗
PubMed1983

Isolation of PS II reaction centre and its relationship to the minor chlorophyll-protein complexes.

Evidence is presented for the identification of the chlorophyll- protein complex CPa-1 (CP 47) as the reaction centre of photosystem II (PS II). We have developed a simple, rapid method using octyl glucoside solubilization to obtain preparations from spinach and barley that are highly enriched in PS II reaction centre activity (measured as the light-driven reduction of diphenylcarbazide by 2,6-dichlorophenolindophenol). These preparations contain only the two minor chlorophyll-protein complexes CPa-1 and CPa-2. During centrifugation on a sucrose density gradient, there is a partial separation of the two CPa complexes from each other, and a complete separation from other chlorophyll-protein complexes. The PS II activity comigrates with CPa-1 but not CPa-2, strongly suggesting that the former is the reaction centre complex of PS II. Reaction centre preparations are sensitive to the herbicide 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but only at much higher concentrations than those required to inhibit intact thylakoid membranes. A model of PS II incorporating our current knowledge of the chlorophyll-protein complexes is presented. It is proposed that CPa-2 and the chlorophyll a + b complex CP 29 may function as internal antenna complexes surrounding the reaction centre, with the addition of variable amounts of the major chlorophyll a + b light-harvesting complex.

Chlorophyll↗
PubMed1986

Immunological characterization of photosystem II chlorophyll-binding proteins from the cyanobacterium, Aphanocapsa 6714.

Chlorophyll-binding proteins from the cyanobacterium Aphanocapsa 6714 were identified by immunoblotting procedures. Three chlorophyll-binding complexes, CPIII', CPIIIa, and CPIIIb, were associated with PSII. CPIII' likely serves as an' antenna to PSII in Aphanocapsa since it could be removed from active PSII core preparations without loss of activity. The CPIII' proteins cross-reacted to antibodies prepared against the maize PSII light-harvesting complex, LHC-II. The CPIIIa polypeptides cross-reacted to antibodies raised against the Chlamydomonas PSII chlorophyll-proteins 5 and 6, indicating that this complex contains the major chlorophyll-binding species of the cyanobacterial PSII core. Lastly, an antibody prepared against the canobacterial 36-kDa chlorophyll-binding protein [Pakrasi, H., Riethman, H., and Sherman, L. (1985). Proc. Natl. Acad. Sci. USA 82, 6903-6907] recognized only the 36-kDa IIIb apoprotein, indicating that CPIIIb represents a distinct chlorophyll-protein complex.

Chlorophyll↗
PubMed1990

Decrease of the chlorophyll fluorescence ratio F690/F730 during greening and development of leaves.

Using the two-wavelength chlorophyll fluorometer the fluorescence induction kinetics (Kautsky effect) were measured simultaneously in the 690 nm and 730 nm region for ten common tree species during the greening period (April to July). The chlorophyll-fluorescence ratio F690/F730 (i.e. ratio of fluorescence intensity at the two maxima near 690 and 730 nm) was calculated from the laser-induced induction kinetics (He/Ne-laser 632.8 nm) at the fluorescence maximum and the steady state. The ratio F690/F730 decreases with increasing chlorophyll content of developing leaves. Its dependence on the chlorophyll content can be fairly well expressed by a power function which has a general validity for leaves, pigment extracts and chloroplast suspensions. The ratio F690/F730 is somewhat higher at maximum (fm) than at steady-state fluorescence (fs), but there is a very good correlation between both values. The ratio F690/F730 is a good indicator of the chlorophyll content and can be used as a non-destructive measure of the chlorophyll content of leaves. It also appears to be a suitable fluorescence parameter in the future remote sensing of the physiological state of the vegetation by laser-equipped airborne systems.

Chlorophyll↗