THE EFFECT OF TREATMENT OF CHLOROPLAST MEMBRANES WITH GUANIDINE HC1 AND AQUEOUS ACETONE ON THE FLUORESCENCE OF BOUND ANS AND CHLOROPHYLL-a†

Abstract The fluorescence intensity of the extrinsic chromophore 1-anilino-naphthalene-8-sulfonate (ANS) bound to pea chloroplast fragments shows a sigmoidal rise as the pH of the suspending medium is decreased by the addition of HC1. The abrupt increase occurs at pH – 4.5. A 70% decrease in the maximal fluorescence intensity (pH range 3.5-4.5) of bound ANS was observed when soluble chloroplast proteins were removed by washing with water. Extraction of chloroplast membranes with 6 M guanidine-HC1 abolishes the acid–induced enhancement of ANS fluorescence. However, the subsequent removal of lipids (by 80% acetone extraction) from the guanidine-HC1-extracted naked membranes restores the acid-induced fluorescence increase. These results suggest that ANS binds mainly to the surface of the chloroplast membrane and the fluorescence changes of ANS by acidification mainly reflect the changes in the associated proteins. The lack of enhancement of the fluorescence of ANS by acidification of the guanidine-HCl treated membranes and the recovery of the acid-induced fluorescence rise after extraction of the lipids from the guanidine-HCl treated membranes suggest that the boundary lipids somehow prevent the entry of the ANS molecules into the hydrophobic interior of the naked membrane. The lipid-depleted, guanidine-HCl extracted naked membrane fragments do not show any shift in the position of the peak of emission of ANS (λ= 470 nm) upon acidification as the lipid-depleted preparations without guanidine-HCl treatment do (shift from 460 to 470 nm). Divalent cations (Mn²⁺, Ca²⁺, Mg²⁺) also increased ANS fluorescence intensities when added to both types of lipid-depleted chloroplast preparations. A comparative analysis of ANS fluorescence bound to the lipid-depleted and guanidine-HCl treated chloroplast fragments with that of just lipid-depleted fragments shows that the acidification of the latter brings about a greater change in the value of the relative binding sites (n) and the dissociation constant k_d of ANS than the protonation of the former. The role of chloroplast protein and lipid components in the structural changes of the thylakoid membrane imposed by external perturbations is discussed.