Different Thermal Unfolding Pathways of Catalase in the Presence of Cationic Surfactants Elena Blanco, Juan M. Ruso,* Gerardo Prieto, and Fe ´ lix Sarmiento Group of Biophysics and Interfaces, Department of Applied Physics, Faculty of Physics, UniVersity of Santiago de Compostela, E-15782 Santiago de Compostela, Spain ReceiVed: September 27, 2006; In Final Form: December 15, 2006 In this paper we have corroborated the usefulness of spectroscopic techniques, such as UV-visible, in the study and thermodynamic characterization of the thermal unfolding of catalase as a function of the concentration and alkyl chain length of n-alkyltrimethylammonium bromides (C n TAB, n ) 8, 10, and 12). For this reason, a thermodynamic model was used which included experimental data corresponding to the pre- and posttransition into the observable transition. It has been found that n-alkyltrimethylammonium bromides play two opposite roles in the folding and stability of catalase. They act as a structure stabilizer at a low molar concentration and as a destabilizer at a higher concentration. The maximum of the unfolding temperature has been found to decrease with the alkyl chain. The reason for this difference has been suggested to be the side chains involved. In the presence of C8TAB and C10TAB, Gibbs energies of unfolding (ΔG(T)) decrease with concentration, whereas for C12TAB an increase has been observed. These findings can be explained by the fact that when differences in the hydrophobic nature of the surfactants exist, different pathways of unfolding may occur. Also, the presence of surfactants has been observed to affect the cold denaturation of catalase. Thermodynamic results suggest that the thermal denaturation of catalase in the presence of n-alkyltrimethyl- ammonium bromides is a perfect transition between two states. Introduction The stability of proteins is the result of residue-residue and residue-solvent interactions. The fact is that all interactions are essentially electrostatic in origin, and traditionally they have been classified as van der Waals, electrostatic, hydrogen bonds, hydrophobic, and disulfide bridges. 1 Water-soluble proteins, which are composed of 25-30% hydrophobic amino acids, can self-assemble to form a defined structure. In general, hydro- phobic amino acids are located in the interior region of the protein to avoid exposure to water. 2 Due to their amphiphilic character, the interaction between a surfactant and a protein results in changes in the natural state of the protein. The recent huge advances in biomacromolecular assembly and its applica- tions have improved our understanding of the physical processes of these complex systems. 3 In previous papers, we have studied the interactions between different proteins and amphiphilic ligands (lysozyme-n-alkyl- trimethylammonium bromides, 4 lysozyme-n-alkyl sulfates, 5 catalase-nafcillin, 6 ovalbumin-SDS, 7 zein-SDS 8 ) using a variety of physical methods including equilibrium dialysis, microcalorimetry, light scattering, and electrophoretic mobilities. The results have permitted us to obtain a thermodynamic picture of the nature of the protein-amphiphilic interaction (the binding interaction is exothermic and dominated by large increases in entropy). Recently, we have reported an investigation of the interaction of hydrogenated and fluorinated surfactants with human serum albumin (HSA). 9 Sodium octanoate, sodium perfluorooctanoate, and sodium dodecanoate were found to bind extensively to HSA in an aqueous solution. The changes in the slope of Gibbs energies were identified with the saturation of the first binding set. The surfactants were found to have different favorite adsorption sites along the protein, and the adsorption processes of perfluorooctanoate and dodecanoate were similar. In the present study we have examined the nature of the interaction of selected surfactants with a globular protein. This was done with several purposes in mind. First, we wanted to study the surfactant-protein interactions by optical methods which are less invasive. Second, there is a necessity for new models to quantify the thermodynamic parameters of these processes. A third consideration was to obtain information about the effect of the surfactants on the thermal stability of the proteins. The compounds used as ligands included three cationic surfactants, C 8 TAB, C 10 TAB, and C 12 TAB n-alkyltrimethylam- monium bromides, which allowed us to observe the effect of the alkyl chain length. The globular protein chosen was catalase. Catalase (hydrogen peroxide:hydrogen peroxide-oxidoreductase, EC 1.11:1.6), present in the peroxisomes of nearly all aerobic cells, serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. The protein exists as a dumbbell-shaped tetramer of four identical subunits; each subunit is formed by a single polypeptide chain with hemin as a prosthetic group. Catalase was one of the first enzymes to be purified to homogeneity and has been the subject of intense study by several physical methods. Experimental Section Materials. Crystalline bovine liver catalase (Product No. C-9322) was obtained from Sigma. The surfactants octyltri- methylammonium bromide (C8TAB), decyltrimethylammonium bromide (C10TAB), and dodecyltrimethylammonium bromide (C12TAB) were obtained from Lancaster MTM Research Chemicals Ltd. All materials were of analytical grade, and solutions were made using doubly distilled and degassed water. Difference Spectroscopy. Difference spectra were measured using a Beckman spectrophotometer (Model DU 640), with six microcuvettes, which operates in the UV-visible region of the electromagnetic spectrum wavelength. All measurements were * Corresponding author. Telephone: +34 981 563 100. Fax: +34 981 520 676. E-mail: faruso@usc.es. 2113 J. Phys. Chem. B 2007, 111, 2113-2118 10.1021/jp066343m CCC: $37.00 © 2007 American Chemical Society Published on Web 02/07/2007