Partition of amphiphilic molecules to lipid bilayers by isothermal titration calorimetry Maria João Moreno a, * , Margarida Bastos b , Adrian Velazquez-Campoy c,d a Biological Chemistry Group, Department of Chemistry—FCTUC, University of Coimbra, 3004-535 Coimbra, Portugal b Department of Chemistry, Faculty of Sciences, University of Porto, P-4169-007 Porto, Portugal c Institute of Biocomputation and Physics of Complex Systems, University of Zaragoza, 50009 Zaragoza, Spain d Fundación ARAID, Diputación General de Aragón, 50004 Zaragoza, Spain a r t i c l e i n f o Article history: Received 9 September 2009 Received in revised form 10 November 2009 Accepted 11 November 2009 Available online 16 November 2009 Keywords: Isothermal titration calorimetry Biomembranes Lipid bilayers Amphiphiles Partition coefficient Nonideal behavior a b s t r a c t The partition of the amphiphile sodium dodecyl sulfate (SDS) between an aqueous solution and a 1-pal- mitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was followed by isothermal titration calo- rimetry (ITC) as a function of the total concentration of SDS. It was found that the obtained partition coefficient is strongly affected by the ligand concentration, even after correction for the charge imposed in the bilayer by the bound SDS. The partition coefficient decreased as the total concentration of SDS increased,with this effect being significant for local concentrations of SDS in the lipid bilayer above 5 molar%. At those high local concentrations, the properties of the lipid bilayer are strongly affected, lead- ing to nonideal behavior and concentration-dependent apparent partition coefficients. It is shown that with the modern ITC instruments available, the concentrations of SDS can be drastically reduced while maintaining a good signal-to-noise ratio. The intrinsic parameters of the interaction with unperturbed membranes can be obtained from the asymptotic behavior of the apparent parameters as a function of the ligand concentration for both nonionic and ionic solutes. A detailed analysis is performed, and a spreadsheetis provided to obtain the interaction parameters with and without correction for electrostatics. Ó 2009 Elsevier Inc. All rights reserved. During recent decades,the scientific research in biological chemistry has been focused mainly on the study of genes and pro- teins. Currently, the biological membranes of cells, and in particu- lar their lipid bilayers, is receiving considerable attention,and lipidomics is becoming a hot subject [1]. The current shift in the attention toward the biological membranes is justified by the important role that this class of supramolecular structures plays in the control of cell structure and function working as a semiper- meable and dynamic boundary of cells and cell organelles. In par- ticular in the case of organs delimited by tight endothelia (e.g., the blood–brain barrier),the permeation through the membranes of the surrounding cellmonolayer is a process that determines the availability of small molecules in the tissue and is a major bottle- neck in the development of new drugs. Therefore,the knowledge of the partition of small molecules between aqueous media and the lipid bilayer is of extreme importance. Calorimetry is a well- established technique in ligand-binding studies between small molecules (ligands) and proteins [2] but has been less often used in the characterization of the interaction with lipid bilayers. This is due both to the small heats expected for an interaction that in many cases is driven mostly by the hydrophobic effect and to the fact that the data analysis procedures are less wellestablished and not commercially available. Recent developments in isother- mal titration calorimetry (ITC) 1 have resulted in instruments with very high sensitivity, allowing the use of this technique in the char- acterization of the interaction of some nonpolar ligands with lipid bilayers [3]. It has also been found that the interaction of a large vari- ety of ligands with lipid bilayers in the liquid-disordered state is accompanied by relatively large and negative molar enthalpies [4– 10] . Membranes are binding agents with a high plasticity, and with- out well-defined binding sites,that change their structure signifi- cantly (and in some cases irreversibly) due to the presence of high concentrations of ligands [11–15]. Therefore, the usual protocols fol- lowed for the characterization of binding to proteins, both in exper- imental design and in data analysis, are not adequate. In the absence of specific interactions between the ligand and the lipid assemblies, the local concentration of ligand in biological systems is usually small and the relevant parameter is the intrinsic partition coefficient between the aqueous phase and unperturbed lipid bilayers.This 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.11.015 * Corresponding author.Fax: +351 239 827703. E-mail address: mmoreno@ci.uc.pt (M.J. Moreno). 1 Abbreviations used: ITC, isothermal titration calorimetry;SDS,sodium dodecyl sulfate; POPC,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; EDTA, ethylenedi- aminetetraacetic acid. Analytical Biochemistry 399 (2010) 44–47 Contents lists available at ScienceDirect Analytical Biochemistry j o u r n a l homepage: w w w . e l s e v i e r . c o m / l o c a t e / y a b i o