Chlorophyll, isolation from spinach

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... 

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. ... 

Table I. Long-term light acclimation in the antenna of PS II. Thyla-koids were isolated from spinach grown at different light intensities and subfractionated to yield stroma lamellae vesicles. Chlorophyll proteins were resolved by mild SDS-PAGE.

Thylakoids and chlorophyll-protein complexes were isolated from spinach Spinacia oleracea). PSI and PSII were as isolated by Satoh (1982), LHCII and LHC736 were as isolated by Fuad et al.(1983), aged LHCII was obtained by leaving LHCII exposed to air and light for approximately 48h at 22°C. Thylakoids were isolated in the presence of 10 mM MgCl2. 

A new chlorophyll, designated chlorophyll RC I (Chi RC I), with absorption and fluorescence characteristics different fron all other known chlorophylls has been isolated from spinach chloroplasts and Scenedesmus cells (Domemann and Senger 1981b/1982). Its enrichment in PS I-particles and the identity of its molar ratio with that of P-700 (Dbmemann and Senger 1981a) suggests its close correlation with the reaction center of PS I, if not its identity with the chrcmophore of P-700 itself. 

Anderson JM and Broadman NK (1966) Fractionation of the photochemical systems of photosynthesis.I.Chlorophyll contents and photochemical activities of particles isolated from spinach chloroplasts, Biochim. Biophys, Acta 112, 403-421. 

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... 

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... 

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... 

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).

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...

Isolation of inside-out thylakoid vesicles (B3) from spinach and their subfractionation by sonication and phase partition was done essentially as previously described (2,3). The fractions named 180s, 540s and BS according to the nomenclature described in (2,3) were obtained. All these fractions derive from the B3 fraction, and hence from the grana partitions, and have a high PSII activity (2,3). Their chlorophyll a/b ratios are given in Table 1. 

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. 

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