Effect of FCCP on the interaction of ANS with DOPC liposomes.

CUTRO ANDREA; ROVERI OSCAR A.

Los Cocos. Córdoba

Congreso; • XXXVIII Reunión Anual de la Sociedad Argentina de Biofísica.; 2009

sociedad argentina de biofísica

•Effect of FCCP on the interaction of ANS with DOPC liposomes. Cutró, A.C., Roveri, O.A. Departamento de Quimica Biológica, Área Biofisica, Universidad Nacional de Rosario, Suipacha 531,S2002LRK Rosario, Argentina. e-mail: acutro@fbioyf.unr.edu.ar 1-anilino-8-naphthalene sulfonate (ANS) is a hydrophobic anion whose interaction with a phospholipid membrane is accompanied by an increase in its fluorescence. The time course of such an increase can be resolved in at least two kinetic phases (Haines, D.H., Simkovitz, P. (1977) J. Membrane Biol. 33, 63 – 108). When studying the effect of several compounds on the binding of ANS to bilayers, it has been observed an unexpected effect of the protonophore uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). Hence we decided to thoroughly study the effect of FCCP on the ANS fluorescence enhancement upon binding to 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) liposomes. FCCP inhibited the fluorescence enhancement observed when ANS is added to a suspension of DOPC liposomes. The effect on the amplitude of the fast phase was complete (IC50 = 12,0 ± 0,8 µM FCCP). This first kinetic phase mainly reports the binding of ANS to the bilayer. FCCP decreases the apparent affinity of the bilayer for ANS (Kd(app) = 15,8 ± 1,6 and 25,5 ± 1,1 µM, in the absence and in the presence of 5 µM FCCP). Also, the same [FCCP] Fluorescence decreases 14% ΔFmax (the value of fluorescence enhancement extrapolated to infinite [ANS]). These results suggest that FCCP distorts the phospholipid lattice in the bilayer, in such a way that decreases the number of ANS binding sites and/or the quantum yield of the bound ANS. Also the second, slow kinetic phase, is affected by FCCP. Despite we have not been yet able to clearly characterize such slow phase, our experiments show that FCCP decreases its initial velocity, effect that can be explained either by a decrease of the amplitude of the slow phase or the kinetic constant for the ANS transport through the bilayer. ACKNOWLEDGEMENTS: This work has been carried out with grants from ANPCyT, CONICET and support from UNR. ACC has a postgraduate Fellowship from ANPCyT and OAR is Member of the Carrera del Investigador (CONICET), Argentina.