Fluorescence changes in isolated broken chloroplasts and the involvement of the electrical double layer

We studied the effects of a variety of cations on chlorophyll fluorescence yield of broken chloroplasts prepared under carefully controlled ionic conditions. In the absence of light-induced electron transport and associated proton pumping, two types of cation-induced chlorophyll fluorescence changes could be distinguished in broken chloroplasts. These are termed "reversible" and "irreversible" fluorescence yield changes. Reversible fluorescence yield changes are characterized by antagonistic effects of monovalent and divalent cations and are prevented by the presence of 5 mM Mg2+ in the suspending media. Reversible-type fluorescence yield changes show little or no dependence on the structure, lipid solubility, or coordination number of the cation, but depend strictly on the net positive charge carried by the ion. It is proposed that these fluorescence changes are brought about through the interaction of monovalent or divalent cations with an electrical double layer at the interface of the outer surface of the thylakoid membrane and the surrounding aqueous solution. The results are interpreted in terms of the Gouy-Chapman theory of the diffuse double layer, indicating that the thylakoid outer surface bears an excess fixed negative charge density of about 2.5 muC/cm2, or approximately 1 negative charge per 640 A2 of membrane surface. Chlorophyll fluorescence quenching in isolated broken chloroplasts suspended in media containing 5 mM MgCl2 is also observed on addition of certain polyvalent cations to the medium. This type of cation-induced fluorescence change appears to be largely irreversible and may occur through specific binding of the cation to the thylakoid as a result of the high electrostatic attraction exerted by the negatively charged membrane surface.