Short communication Interaction of merocyanine 540 with charged membranes Anton Mateas ˇik a , Libus ˇa S ˇ ikurova ´ b, * , Dus ˇan Chorva ´t Jr. a a International Laser Center, Ilkovicova 3, Bratislava 812 19, Slovak Republic b Department of Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska ´ dolina, 842 48 Bratislava, Slovak Republic Received 1 June 2001; received in revised form 9 October 2001; accepted 12 October 2001 Abstract In this work, phospholipid liposomes were used to investigate the influence of lipid negative charge on the interaction of merocyanine 540 (MC540) with model membranes. Liposomes were prepared from a mixture of neutral dimyristoyl lecithin (DMPC) and negatively charged dimyristoyl phosphatidic acid (DMPA). A strong dependence between the presence of charges on the membrane and dye association was found. The affinity of the dye to liposomes was decreased with an increasing content of DMPA in liposomes. Changes in absorption spectra of MC540 suggest that the decrease in affinity of MC540 to charged membranes is accompanied by a hypsochromic solvatochromic shift and changes in monomer/dimer equilibrium of MC540 incorporated in the membrane. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Merocyanine 540; Charged liposomes; Absorption spectroscopy 1. Introduction The fluorescent dye merocyanine 540 (MC540) prefer- entially photosensitizes leukemia cells, lymphoma cells [1] and enveloped pathological viruses [2]. It has been shown that the highly susceptible cells to MC540-sensitized photo- inactivation tend to bind more dye and to saturate their dye- binding sites more rapidly than MC540-resistant cells. Therefore, the preferential binding of a sensitizer to the malignant species is suggested to be a key factor in the efficiency of MC540-sensitized photodynamic impact [3]. MC540 incorporates to the outer leaflet of the phospho- lipid bilayer of a cellular membrane [4]. Considering this, the difference between high and low affinity binding should reflect differences in composition and/or structural organ- ization of the membrane. In model experiments with lip- osomes, it has been proposed that MC540 resides in membrane slightly above domain of the glycerol backbone of phospholipids and it has been shown that MC540 binding is very sensitive to lipid packing of phospholipid bilayer [5]. Due to the presence of a negative charge on the MC540 molecule, localized charges on the surface of membrane may play an important role in the process of incorporation of MC540 into the membrane. Up to now, investigations have shown that there is some correlation between binding of MC540 and the presence of the charged structure on the membrane [6,7]. The aim of our work was to determine the influence of the lipid negative charge on the interaction of MC540 with membranes. 2. Experimental MC540 was purchased from Molecular Probes (the Netherlands) and was used without further purification. The dye stock solution of MC540 of 10 À 3 M was prepared in bidistilled water and kept in the dark until use. A mixture of two synthetic phospholipids, 1,2-dimyristoyl-sn-glycero- 3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero- 3-phosphatidic acid (DMPA) from Fluka (Germany), was used in the preparation of small unilamellar liposomes by sonicating their water dispersion (14 Â 10 À 6 mol of DMPA –DMPC mixture in 1 ml) in a temperature-controlled sonicating bath (T = 25 °C) for 1 h. The molar ratio of both lipids DMPA/DMPC in the suspension was 0:100, 10:90 and 20:80, respectively. The liposome suspension of molar concentration of 14 Â 10 À 3 M was used as stock solution. The bidistilled water, pH 6.2 F 0.1, was used in all sample preparations. 1567-5394/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S1567-5394(01)00140-2 * Corresponding author. Tel.: +421-2-6029-5124; fax: +421-2-6541- 2305. E-mail address: sikurova@fmph.uniba.sk (L. S ˇ ikurova ´). www.elsevier.com/locate/bioelechem Bioelectrochemistry 55 (2002) 173 – 175