273 Bioelectrochemistry and Bioenergetics, 16 (1986) 273-285 A section of J. Electroanal. Chem., and constituting Vol. 212 (1986) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands 849 - THE SIGNIFICANCE OF INTERFACIAL CHARGE AND PROTON DISPLACEMENTS FOR THE MECHANISM OF ENERGY TRANSDUCTION IN BIOMEMBRANES * R. KRAAYENHOF, F.A. DE WOLF, H.S. VAN WALRAVEN and K. KRAB Biological Laboratory, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam (The Netherlands) (Revised manuscript received January 20th 1986) SUMMARY Energization of chloroplast membranes induces a significant increase of negative surface charge density, as appears from measurements of the electrokinetic potential and the adsorption of cationic probes. ‘Ibis phenomenon is correlated with the displacement of protons from the external interfacial layer. The estimations of the interfacial potential were compared to the actual surface potential of liposomes with known surface charge density, and large discrepancies were apparent. Transient interfa- cial pH changes were observed in subchloroplast vesicles in response to single-turnover energixation by short light flashes. Reconstituted AT&se proteoliposomes with internally trapped and externally added pH indicators were used for kinetic analysis of the sequential steps of ATP-driven proton influx. A clear discrimination of external and internal, as well as interfacial and bulk proton displacements, was possible. These findings are accommodated in a simple model for energy-linked proton displacements and changes of surface charge, which takes into account the significance of both localized and delocalized charge reorientations and proton gradients. ABBREVIATIONS ** ACMA, 9-amino-6-chloro-2-methoxyacridine; CEPA, 9-chloroethylaminopropy- lamino-6-chloro-2-methoxyacridine; DHP, dihexadecyl phosphate (dicethyl phos- phate): N&MA, 9-azido-6-chloro-2-methoxyacridine; oxonol VI, bis(3-propyl-5- oxoisoxazol-4-yl)pentamethine-oxonol; PEA, 9-propylaminoethylaminomino-6-chloro-2- methoxyacridine; PC, phosphatidylcholine; S-13, 5-chloro-3-t-butyl-2’-chloro-4’- nitrosalicylanilide; MES, morpholinoethane sulphonic acid; TES, N-tris(hydroxy- methyl)methyl-2-aminoethane sulphonic acid; HEPES, N-2-hydroxyethylpipera- zine-N’-Zethanesulphonic acid; NR+, neutral red; CR-, cresol red; Val, valinomy- tin. * Contribution presented at the VIIIth International Symposium on Bioelectrochemistry and Bioen- ergetics, Bologna, June 24th-29th 1985. ** The symbol + is used here for electric potentials rather than the more usual +, due to conventions of this journal. 0302-4598/86/$03.50 8 1986 Elsevier Sequoia S.A.