Effect of NO Å on the adhesion–spreading of DMPC and DOPC liposomes on electrodes, and the partition of NO Å between an aqueous phase and DMPC liposomes Michael Hermes a , Clemens Czesnick a , Stefanie Stremlau b , Christine Stöhr b , Fritz Scholz a,⇑ a Institut für Biochemie, Universität Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany b Institut für Botanik und Landschaftsökologie, Universität Greifswald, Grimmer Str. 88, 17487 Greifswald, Germany article info Article history: Received 12 January 2012 Received in revised form 25 January 2012 Accepted 1 February 2012 Available online 3 March 2012 Keywords: NO Å radical Liposome Chronoamperometry Mercury electrode Membrane Fluidity abstract The effect of NO Å radicals on the adhesion and spreading of unilamellar DMPC and DOPC liposomes on mercury electrodes were studied by chronoamperometry. NO Å was either generated in situ from NOC- 18 or supplied as an admixture of nitrogen gas. These measurements reveal details of the kinetics of the adhesion and spreading process and provide access to information on the membrane fluidity of lipid bilayer membranes. The results indicate that NO Å stabilizes the membranes of DMPC liposomes. The effect of NO Å radicals is most probably caused by their physical association with the polar head groups of DMPC, as indicated by 31-P NMR spectroscopy. This interaction also explains the partition coefficient K NO DMPCjH 2 O of NO Å between water and DMPC liposomes of 10.0 ± 2.1. Ó 2012 Elsevier B.V. All rights reserved. 1. Introduction Membrane fluidity (MF) (reciprocal value of the microviscosi- ty) is essential for the functions of cellular membranes. Thus, the MF of red blood cells has been found to be related to cardio- vascular and metabolic disorders, as hypertension, diabetes melli- tus, and others [1–3]. Diffusion measurements in cell membranes support the idea that the MF is linked to membrane compartmen- talisation [4]. The MF is mainly regulated by the phospholipid and cholesterol content of the cellular membrane, and it is essentially modulated by temperature changes. Lipid rafts inside the plasma membrane are causally linked with MF [5–8]. Microdomain dynamics and MF can be elegantly studied by ESR [9] and fluores- cence correlation spectroscopy [10], fluorescence recovery after photobleaching [11], single particle tracking [11], NMR [9], and dual colour FRAP [11]. Electrochemistry complements the spec- troscopic techniques [12]. Recently a new electrochemical ap- proach has been introduced based on measuring and analyzing the adhesion–spreading events of liposomes on a mercury elec- trode [13–18]. Such measurements reveal the detailed kinetics of this complex vesicle rupture process [19,20]. This approach has been also used to study thrombocyte vesicles [21] and intact mitochondria [22] and for studies of the effects of foreign mole- cules on the MF of liposomes. Here we report a study of the ef- fects of NO Å radicals on DMPC and DOPC liposomes. The study is part of a larger project focussed on the interaction of free rad- icals with lipid mono- and bilayers [23]. 2. Experimental DMPC and DOPC were from Lipoid GmbH, Ludwigshafen, Germany and KCl (Suprapur™ grade) from Merck, Darmstadt, Germany. Giant unilamellar vesicles (GUVs) were prepared with the mod- ified rapid evaporation method suggested by Moscho et al. [24]: 3 mg of DMPC or DOPC were dissolved in a round bottom flask con- taining 2.2 ml of chloroform (>99.8%) and 220 ll of pure methanol. 30 ml of 0.1 M KCl solution were added along the flask wall. The or- ganic solvent was removed (rotary evaporator) under reduced pressure at 40 °C for DMPC and at 45 °C for DOPC, with 30 rpm (final pressure: 40 mbar). After evaporation for 3 min, the pump was stopped and the flask continued to rotate for another 5 min. The resulting aqueous solution contained GUVs at a concentration of 0.1 g l 1 . Each measurement series was started from the vesicle preparation temperature of 40 °C, and measured downwards in 3 K steps. Upon each crossing of the phase transition temperature (of 23.6 °C for DMPC) the vesicles are stressed; low-temperature range can thus better be measured when the suspension after prepara- tion is cooled down to 2 °C, and from there heated up to the PTT. Before each series, the suspensions were deaerated for 20 min with N 2 . 1572-6657/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2012.02.001 ⇑ Corresponding author. Tel.: +49 (0)3834 864450; fax: +49 (0)3834 864451. E-mail address: fscholz@uni-greifswald.de (F. Scholz). Journal of Electroanalytical Chemistry 671 (2012) 33–37 Contents lists available at SciVerse ScienceDirect Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem