RESUMEN
The structure of the xanthophyll pigments lutein and zeaxanthin differs in the position of one double bond and refers to one of the ionon rings. Specific antibodies to zeaxanthin were used to analyse the localisation and orientation of these two xanthophyll pigments in lipid membranes formed with egg yolk lecithin. Bimolecular and monomolecular layers were used. Antibody-antigen interaction was demonstrated and analysed by the bathochromic shift of the absorption spectra of both pigments and by the increase of light-scattering of the pigmented liposome suspension. It appeared that the extent of the spectral effects accompanying the interaction of the antiserum to zeaxanthin, injected to the liposome suspension which was pigmented with either zeaxanthin or lutein, was different in spite of their similar molecular structures. The results are interpreted in terms of a localisation and distribution of lutein, in the hydrophobic phase of liposomes within two essentially different pigment pools, one oriented horizontally and the other vertically with respect to the membrane plane. This interpretation is supported by the analysis of isotherms of the compression of monomolecular layers of lutein and zeaxanthin formed at the air-water interface and of mixed xanthophyll-lipid monolayers as well as by analysis of the penetration of antibody proteins dissolved in the subphase into the mixed xanthophyll-lipid films.
RESUMEN
Monomolecular layers of the largest light-harvesting pigment-protein complex of Photosystem II (LHCII) were formed at the argon-water interface. The molecular area of the LHCII monomer in monomolecular layers determined from the isotherms of compression is found to be close to 14 nm2, which corresponds well to the molecular dimensions of the protein evaluated on the basis of crystallographic studies. Monolayers of LHCII were deposited on a glass support by means of the Langmuir-Blodgett technique and subjected to spectroscopic studies: electronic absorption spectrophotometry and spectrofluorometry. The fluorescence excitation spectra of chlorophyll a in monolayers of LHCII were analysed using gaussian deconvolution. Comparison of the absorption and fluorescence excitation spectra enabled calculation of the rate of excitation energy transfer in the system. Excitation energy was found to be transferred to chlorophyll a from chlorophyll b with 97% efficiency, from neoxanthin with 85%, from lutein with 62% and from violaxanthin with at least 54% efficiency. The analysis of the position of the 0-0 absorption band of the xanthophylls revealed that neoxanthin is located in the same protein environment as lutein but in a different environment than violaxanthin. The analysis of fluorescence excitation spectra of chlorophyll a in LHCII, recorded with the excitation light beam polarised in two orthogonal directions, enabled the determination of the mean orientation angle of the accessory xanthophyll pigments with respect to the plane of the sample. The mean orientation of lutein found in this study (approx. 51 degrees ) corresponds well to the crystallographic data. Neoxanthin was found to adopt a similar orientation to lutein. The transition dipole moment of violaxanthin was found to form a mean angle of 71 degrees with the axis spanning two polar regions of the protein, perpendicular to the plane of the monolayer, suggesting planar orientation of this pigment with respect to the plane of the thylakoid membrane. These experimentally determined xanthophyll orientations are discussed in terms of importance of peripheral xanthophyll pigments in supramolecular organisation of LHCII and the operation of the xanthophyll cycle within the thylakoid membrane.