Isolation and lipid composition of spinach chloroplast envelope membranes

The quenching of the Chl a² fluorescence from spinach chloroplasts and chloroplast fragments by nitroaromatic compounds and the effect of added metal cations on the quenching rate is investigated. The extent of the quenching with nitrobenzene and 1,3-dinitrobenzene was found to be independent of whether Chl a is excited directly, or through Chl b by means of electronic energy transfer. On the basis of this, the contribution from a purely static mechanism is considered as unlikely.Nitroaromatics substituted with ionizable groups are almost equally effective quenchers for the fluorescence of Chl ain vivo and in methanol. On the other hand, nitroaromatics which are slightly soluble, or nearly insoluble, in water quench more strongly the fluorescence of Chl ain vivo. The overriding factor that determines the relation between the apparent and the true quenching constant appears to be the partition of the quencher in the lipid and the aqueous phases of the membrane suspension.Divalent metal cations enhance the quenching by nitrobenzene dramatically, most likely by increasing the hydrophobic character of the chloroplast membranes. This enhancement occurs at cation concentrations higher than those corresponding to the maximal turbidity increase of the membrane suspension; hence, it is attributed to ultrastructural changes of the membrane rather than to volume changes of the thylakoid. These changes may affect the extent of the quenching both by an increase in the local concentration of the nitroaromatic, and by an enhanced rate of excitation exchange among the chlorophylls.