Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra were recorded for spinach thylakoids and for isolated, aggregated chlorophyll a / b light-harvesting pigment-protein complex, in random and magnetically aligned states of orientation at room and low temperatures. The shape and magnitude of the C D signal of most bands strongly depended on the orientation of the thylakoid membranes or the aggregated pigment-protein complex. In both thylakoids and aggregated light-harvesting complexes, however, the MCD spectra of the two different orientations were almost identical. Random and magnetically aligned samples exhibited anomalous, large C D signals outside the bands of pigment absorbance. Lack of similarity between the corresponding M C D and C D spectra showed that the large C D signals are not produced as a distortion of CD of absorbance by light scattering. Instead, these anomalous spectral features are believed to originate in differential selective scattering of circularly polarized light. Our results lead to the conclusion that the light-harvesting pigment-protein complex in thylakoid grana forms a helical macroarrav with dimensions commensurate with the wavelengths of the anomalous circular dichroism signals. A hypothesis is put forward suggesting a role for C i r c u l a r dichroism (CD)' is a powerful technique, yielding structural information on biological systems. CD of absorbance (CDA), an intrinsic property of chiral molecules, may also arise from short-or long-range coupling between chromophores of

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Selective scattering spectra of granal and agranal chloroplasts were measured in the red spectral region and compared with calculations based on the Mie theory. The spectra were influenced considerably by the intactness and ultrastructural pattern of the chloroplasts. It was demonstrated that the spectra consist of two components: one attributable to grana and the other, to single lamellae. The dependence of the selective scattering spectra on the ultrastructural characteristics offers a convenient method for monitoring the quality of chloroplast preparations by a procedure much faster than electron microscopy. Selective scattering spectra of algal cells such as Chlorella and Anacystis, as well as of spinach chloroplasts and fragments, have been reported in the literature (1)(2)(3)(4)(5)(6)(7)(8). In these studies the photosynthetic lamellar system was considered as a whole with no regard to the ultrastructural pattern characteristic of the chloroplasts. As shown under the electron microscope, chloroplasts consist of two types of lamellae: single lamellae that appear never to fuse with each other, and stacked lamellae that build up the highly ordered piles called "grana." The individual contributions of single and stacked lamellae in the selective scattering spectra have not been distinguished in previous reports.The aim of the present work was to study the selective scattering spectra of chloroplasts containing various amounts of grana and to compare intact class A and class B chloroplasts (9) in order to obtain information on the relationship between ultrastructure and selective scattering. Selective spectra of chloroplasts were shown to be dependent on their intactness and ultrastructure. This phenomenon offers a convenient method for checking the quality of chloroplast preparations. MATERIALS AND METHODSChloroplast preparations with high and low granum contents were obtained from maize leaves (Zea mays L. var. MV SC 660). The plants were grown in a greenhouse for 9-l1 days.Class A chloroplasts were isolated from a suspension of mesophyll protoplasts (10) or bundle sheath cells. Mesophyll protoplasts were prepared in a digestion medium containing 0.6 M d-sorbitol, 0.1 M potassium phosphate, 0.5% (wt/vol) Macerozyme R-10, 2.0% (wt/vol) cellulase Onozuka R-10, and 0.5% (wt/vol) potassium dextran sulfate, pH 5.8. Bundle sheath cells were separated in a maceration medium containing 0.4 M d-sorbitol, 0.1 M potassium phosphate, 0.5% Macerozyme R-10, 1.0% cellulase Onozuka R-10, and 1.0% Helicase, pH 5.8. Macerozyme and cellulase preparations were purchased from Kinki Yakult MFG Co. Ltd., Nishinomiya, Japan; the Helicase was obtained from Industrie Biologique Frangaise, Gennevilliers, France; and potassium dextran sulfate was from Meito Sangyo Co. Ltd., Nagoya, Japan. The incubation was for 90 min at 37°. Protoplasts and cells were washed three times in 0.6 M d-sorbitol and transferred to the medium of Anderson and Boardman (11) containing 0.3 M sucrose, 0.05 M potassium phosphate, and 0.01 M potassium chlor...