Spinach chlorophyll isolation

Diets high in red meat and low in green vegetables have been associated with increased colon cancer risk and the opposite has been postulated for diets rich in green vegetables. A plausible explanation for an increased colon cancer risk is that dietary haem is metabolized in the gut to a factor that increases colonic cytotoxicity and hyperproliferation, which are considered important risk factors in the development of cancer. In this sense, it has been shown that spinach and isolated natural chlorophyll, but not sodium-copper chlorophyUin, prevented the proliferation of colonic cells and may therefore reduce colon cancer risk. It has been speculated that haem and chlorophylls, due to their hydrophobicity, form a complex, thus preventing the metabolism of haem. ... 

Figure 5. Photograph of a Li-dodecylsulfate polyacrylamide electrophoresis gel (10-15%) and radioactivity distribution herein of spinach thylakoids isolated by 20 nmol/mg chlorophyll [ cj azido-triazinone.

Chloroplasts. Intact chloroplasts were isolated from freshly harvested growth chamber-grown spinach (Splnacla oleracea L.) as described by Lilley and Walker (10). Thylakoids were prepared by the method of Armond t al. (11). Chlorophyll concentrations were determined by the method of MacKinney (12). Photochemical reactions yere conducted at 25°C with a photon fluence rate of 750... 

Chloroplasts will be isolated by careful extraction of spinach leaves, using tricine buffer containing sucrose. The crude extract contains both whole and fragmented chloroplasts, but both contain all the necessary photosyn-thetic components and are capable of photophosphorylation. The preparation described in this experiment retains almost all of the chlorophyll in the chloroplasts. The total chlorophyll content of the chloroplasts will be determined by extracting the pigment with aqueous acetone and measuring the absorption at A. = 652 nm. The chlorophyll concentration is calculated according to Equation E9.3 (Arnon, 1949),... 

Dunahay, T. and L A. Staehelin. 1986. Isolation and characterization of a new minor chlorophyll a/b-protein complex (CP24) from spinach. Plant Physiol. 80,429 134. 

Isolation of Chlorophyll and Carotenoid Pigments from Spinach... 

To examine the effects of chemicals on thylakoid membranes, chloroplasts were prepared from commercially purchased spinach leaves as described by Shigematsu et al (1989 ). Concentrations of chlorophyll were calculated from the equations of Mackinney (1941). Isolated thylakoid membranes were stored at -80 °C until use. 

B Andersson and JM Anderson (1980) Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593 427-440 P-" Albertsson (1985) Partition of Cell Particles and Macromolecules (3rd edition) John Wiley H-E "kerlund, B Andersson and P-" Albertsson (1976) Isolation of photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition. Biochim Biophys Acta 449 525-535 P Grber, A Zickler and H-E "kerlund (1978) Electric evidence for the isolation of inside-out vesicles from spinach chloroplasts. FEES Lett 96 233-237... 

JP Thornber, CA Smith and JL Bailey (1966) Partial characterization of two chlorophyll-protein complexes isolated from spinach-beet chloroplasts. Biochem J 100 14-15... 

NK Boardman and JM Anderson (1964) Isolation from spinach chloroplasts of particles containing different proportions of chlorophyll a and chlorophyll b and their possible role In the light reactions of photosynthesis. Nature 203 166-167... 

Ruban AV and Horton P(1995) An investigation ofthe sustained component of nonphotochemical quenching of chlorophyll fluorescence in isolated chloroplasts and leaves of spinach. Plant Physiol 108 721-726... 

Fig. 3. Kinetics of chlorophyll fluorescence quenching in leaves, thylakoids and LHClIb from spinach. Leaf samples were dark adapted (viol) or pre-ilUiminated (zea) as described by Noctor et al. (1991), and the thylakoids and LHClIb isolated from the leaves as described by Rtiban et al. (1994b). For leaves and thylakoids, quenching was initiated by illumination, for LHClIb by dilution to low pH (Ruban et al., 1998a,b). Solid lines are a second order rate equation. Data are redrawn from Ruban and Horton (1998).

Markers to follow membrane degradation are chlorophyll and carotenoid content of the culture (Vl ) or volatile short-chain hydrocarbon formation during peroxidation of fatty acids (1 2,37). The kinetics of all three parameters in oxyfluorfen-treated algal cells is demonstrated in Figure 3. Ethane formation was also observed with isolated spinach chloroplasts (27). As was further demonstrated Figure 3), photosynthetic... 

Figure 5. Addition of nitrofen to isolated spinach chloroplasts in the light and in the dark. Thylakoids equivalent to 100 fig of chlorophyll were incubated at pH 7.4 in the presence of 50 /nM C-labeled nitrofen for 15 min. Aliquots of this preloaded material were then treated with increasing concentrations of cold nitrofen as indicated (37).

Stock solutions (20 mol m ) of substituted 9,10-anthraquinones (Aldrich Chemical Company) were prepared in dimethyl sulfoxide or 1,2-dimethoxyethane. Previous studies [7-10] determined that these solvents induced no quenching of chlorophyll fluorescence at the concentrations employed in diluted chloroplast samples. Spinach chloroplasts were isolated as in previous studies [7-10] and suspended in a buffer of 10 mol m sucrose/50 mol m KEPES-NaOH (pH 1.5)/5 mol m NaCl. Fluorescence samples contained [chlorophyll] = 0.010 kg m"" and [quinone] = 0 to 0.200 mol m . Quinones were incubated in chloroplast solutions for 5 minutes at 5 C in the dark to permit diffusion to the site of action [7-10]. 

Fig. 2 shows the fluorograms of various labeling experiments with isolated spinach thylakoids (5 nmol inhibitor/mg chlorophyll). [ H]2-Acetoxymethyl-l,4-naphtho-quinone binds covalently to nucleophilic groups of its receptor protein without prior activation (5). The azido-derivatives of inhibitors have to be illuminated in order to generate the nitrene which is responsible for covalent binding. The labeled proteins were separated by 10-15 % PAGE. The gels were incubated with Amplify... 

Photosynthetic Electron Transport. Thylakoids were isolated from spinach leaves by the method of Whitmarsh (13). Chlorophyll content was measured according to Arnon (14). The reaction medium contained 0.1 M sorbitol, 10 mM MgCl2, 10 mM NaCl, 10 mM Tricine-NaOH (pH 7.8) 0.05 mM Dichlorophenol-indophenol (DCPIP), 1 mM NH4C1, 1 pM Gramicidin D and thylakoid membranes equivalent to 5 pg chlorophyll/ml. Triplicate readings were taken 0, 15 and 30 s after illumination at 580 nM and initial rates were calculated over a range of inhibitor concentrations. ... 

Incubation of leaf discs in LY181977 causes an increase in chlorophyll fluorescence similar to that caused by atrazine and diuron (Figure 2). Similarly the IC50 for DCPIP reduction by isolated spinach thylakoids is similar for LY181977 (0.73 pM) and atrazine (0.36 pM). This indicates that LY181977 inhibits photosynthetic electron transport through photosystem II with efficacy similar to that of atrazine. 

Chloroplasts were isolated mechanically from p>eas and spinach. Pea chloroplasts were used intact (75-80%), while spinach chloroplasts were osmotically shocked before assay in a medium containing 25 mM MgCl2. Chlorophyll fluorescence quenching coefficients were calculated as in (2). The transthylakoid ApH and internal proton concentration were... 

Fig. 3 Relationship between qE and pH for chloroplasts isolated from dark-adapted spinach leaves wi th negligible levels of zeaxanthin (open circles) and from pre-illuminated leaves (30 mins at 1000 Umol.mT, s ) with high zeaxanthin levels (closed circles). Chlorophyll...

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