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Immobilized Thylakoids in a Cross-Linked Albumin Matrix

Effects of Cations Studied by Electron Microscopy, Fluorescence Emission, Photoacoustic Spectroscopy, and Kinetic Measurements

Laboratoire de Technologie Enzymatique (ERA No. 338 CNRS), Université de Technologie de Compiègne, B.P. 233—60206 Compiègne, France, Service d'Analyse Physico-Chimique, Université de Technologie de Compiègne, B.P. 233—60206 Compiègne, France, Laboratoire de Photobiologie, Université de Liège, Sart Tilman (B 22) B 4000 Liège, Belgium

Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O₂ evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O₂ production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mM Na⁺ (or K⁺), 1 mM Mg²⁺, and 0.1 mM Mn²⁺ are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F₇₃₅ to F₆₉₅ ratio increases and the PA₆₇₆ to PA₄₄₀ ratio decreases.