Thermodynamic study of the dimerization of 8-anilino-1-naphthalenesulfonic acid by isothermal titration calorimetry Montserrat Andujar-Sanchez, Ana Cámara-Artigas, Vicente Jara-Perez * Dpto. Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, La Cañada de San Urbano, 04120 Almería, Spain article info Article history: Received 8 May 2009 Received in revised form 20 July 2009 Accepted 2 September 2009 Available online 7 September 2009 Keywords: Dimerization ANS Isothermal titration calorimetry abstract Dimerization of the fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS) in aqueous media have been studied by isothermal titration calorimetry (ITC). ITC experiments carried out at different pHs show that dimerization constants are highly pH dependent, decreasing their values with increasing pH. No dimerization is detected over pH 7. Analyzing the dependence of K dim on pH, using a model that only considers dimers between zwitterionic molecules of ANS, a value of 5.6 for the pK of anilinium moi- ety is obtained. It is in agreement with the pK determined spectrophotometrically. The dimerization pro- cess is enthalpically disfavored and entropically driven at all pH and temperatures studied, indicating that hydrophobic effect has an important role on the formation of dimers. Although dimerization con- stants are low, dimerization equilibria must be taken into account when the energetics of the interaction of ANS to a protein is studied at pH below 7. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Molecules with fluorescence spectra, quantum yields, and life- times that are sensitive to their environment have been used as probes for structure studies of biological macromolecules for many years. 8-anilino-1-naphthalenesulfonic acid (ANS) is an amphi- pathic dye, with hydrophobic naphthalene and phenyl groups, and a charged sulfonate group. This compound is one of the most frequently used fluorescence probes for the investigations of struc- tural properties of protein molecules. Stryer [1] showed that the fluorescence of ANS, bound to the known non-polar sites of apo- myoglobin and apohemoglobin, was equal to the fluorescence of an equivalent amount of ANS in a number of hydrophobic organic solvents. The extensive use of this fluorophore is due to the dra- matic enhancement of its fluorescence [2] and the shift of the emission maximum to shorter wavelengths when surrounded by non-polar aminoacid residues of proteins. ANS has been used as a back titration fluorescence indicator for lipid molecule displace- ment from lipid carrier proteins [3], for detection of ‘‘molten glob- ules” [4–6], for investigations of non-polar surface patches of proteins [7], and many more. ANS has also been used to investigate the structural and physicochemical characteristics of the aggre- gates of transthyretin to provide insights into the mechanism of protein misfolding and misassembly [8]. Currently, ANS is being used as a standard probe for investigating the population of com- pact partially folded intermediate states of proteins [9–16]. Although early works considered hydrophobicity the only determinant of the fluorescence enhancement and the driving force of ANS binding to proteins, the importance of electrostatic interactions between the sulfonate group of ANS and positive charges on the polypeptide has been clearly demonstrated [17]. To study the nature of the forces responsible for the binding of ANS, the energetics of the interaction ANS–protein has been stud- ied for different proteins by isothermal titration calorimetry [16,18–23]. In all these works, the thermograms are corrected with the dilution heat of ANS obtained in blank experiments injecting the dye into buffer. Uversky et al. [24] performed investigations of the effect of the ANS concentration and solvent composition on spectral properties of this dye and they concluded that aggrega- tion of ANS molecules takes place in aqueous media. This process must be taken into account to interpret ITC experiments between ANS and proteins properly. In this work we have studied dimeriza- tion of ANS at different pHs, and temperatures by isothermal titra- tion calorimetry, obtaining thermodynamic data of this process and information about the forces involved in the interaction of ANS molecules. 2. Experimental 2.1. Spectrophotometrical pK determination Absorption spectra at 298.13 K and different pH were recorded in a Beckman DU 7400 spectrophotometer. Molar extinction coef- ficients of the acidic and basic forms of ANS were determined at 350 nm using solutions of ANS in HCl 0.01 M and NaOH 0.01 M, 0021-9614/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jct.2009.09.002 * Corresponding author. Tel.: +34 50 015316; fax: +34 50 015008. E-mail address: vjara@ual.es (V. Jara-Perez). J. Chem. Thermodynamics 42 (2010) 337–341 Contents lists available at ScienceDirect J. Chem. Thermodynamics journal homepage: www.elsevier.com/locate/jct