Compared to nondenaturing gel elec-trophoresis, ultracentrifugation through sucrose density gradients is a more gentle method for isolating labile LHC. Moreover, this method allows the isolation of sufficient material for further analyses and has the advantage that the green band collected from the centrifuge tube can be used immediately without the need to extract it from a gel.
Sucrose gradients can be formed either by means of a gradient mixer in combination with a peristaltic pump or, more conveniently, by the freeze-thaw method described by Bassi and Simpson (6). For the latter method, a solution with 0.5 M sucrose, 5 mM Tricine-NaOH (pH 7.8), and 0.1% (wt/vol) LM is filled in the centrifuge tubes. The tubes are placed in a -20°C freezer. Three hours before sample application, the tubes are transferred to a refrigerator and kept there until completely thawed. Subsequently, the upper tenth of the gradient solution is carefully removed, which results in gradients with a sucrose concentration of about 0.1 to 1 M sucrose. Depending on the rotor used, centrifuga-tion at 4°C is performed at 450 000x g for 16 hours (SW60, Beckman Coulter) or 260 000x g for 23 hours (SW40 or 41, Beckman Coulter). Subjecting a reconstitution mixture containing the 2 apopro-teins of LHCI-730 to density-gradient ultracentrifugation results in a separation as is shown in Figure 2. The resolution of zones with free pigments, monomeric complexes, and the heterodimeric LHCI-730 is clearly visible. The bands of interest are collected with a flat-tipped syringe needle from the top.
As a consequence of the low isoelectric points of the higher plant pigment proteins, ranging from 4 to 5 (21), anion
Figure 2. Sucrose gradient fractionation of reconstitution mixtures containing the two apoproteins of LHCI-730. FP, free pigments; m-LHCI, monomeric LHCI; d-LHCI, dimer-ic LHCI (LHCI-730).
exchange chromatography is suitable for the isolation of these proteins. This method is a good choice if one intends large-scale isolation. Furthermore, the advantages described for sucrose density gradients also apply to this method.
Diethylaminoethyl (DEAE)-cellulose is mostly used as stationary phase for column development by gravity, fast flow Q-Sepharose® or Poros Q (both from Amer-sham Pharmacia Biotech, Piscataway, NJ, USA) for pump-mediated column processing (41,91,97). The mobile phase is usually composed of a slightly alkaline buffer (e.g., phosphate buffer, Tris), and a detergent such as LM, both in low concentrations [10 mM and 0.05% (wt/vol), respectively]. Prior to sample application, the column is washed first with 4 column volumes of, e.g., 10 mM sodium phosphate buffer (pH 7.4) and then with 2 volumes of the buffer supplemented with the detergent, which is used for the solubilization of the pigment—protein complexes, e.g. 0.05% (wt/vol) LM. Then the sample, adjusted to the same phosphate buffer (pH 7.4) and detergent concentration, is applied. After the sample has completely entered the column bed, the column is washed with 2 column volumes of buffer including detergent. Elution is achieved by the buffer plus detergent supplemented with NaCl. Mostly, a gradient of 0 to 400 mM NaCl works well. The steepness of the NaCl gradient has to be determined individually. Eluted bands can be characterized with regard to apoprotein composition (section 3.4.1), pigment composition (section 2.2.1), and spectral properties (section 2.2.2).
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