6 The Open Macromolecules Journal, 2008, 2, 6-18 1874-3439/08 2008 Bentham Open Open Access Interaction Between n-octyl--D-thioglucopyranoside and Bovine Serum Albumin C. Carnero Ruiz*, J. M. Hierrezuelo, J. M. Peula-García and J. Aguiar Grupo de Fluidos Estructurados y Sistemas Anfifílicos, Departamento de Física Aplicada II, Escuela Universitaria Poli- técnica, Universidad de Málaga, Campus de El Ejido, 29071 - Málaga, Spain Abstract: The binding of the nonionic surfactant n-octyl--D-thioglucopyranoside (OTG) to the globular protein bovine serum albumin (BSA) has been investigated by using experimental techniques such as surface tension, steady-state fluo- rescence and dynamic light scattering. It was observed that the surfactant micellization is delayed in the presence of pro- tein; this was interpreted as a consequence of the fact that part of the surfactant is not available for the formation of mi- celles, because it is partitioned into the protein hydrophobic sites. This was taken as an evidence of the interaction be- tween surfactant and protein. The fluorescence emission spectra of intrinsic tryptophans revealed that the protein is par- tially denatured in the presence of high surfactant concentrations. The analysis of the binding features as obtained by two different methods, (i) one based on surface tension measurements, and (ii) another based on the behaviour of the intrinsic BSA fluorescence, indicated that the binding process is non-cooperative at low surfactant concentration, but becomes co- operative when it is high enough. The reduction of the average aggregation number in the presence of protein was inter- preted as a sign of the formation of clusters of surfactant adsorbed on the protein surface. A slight conformational change in the protein structure at low surfactant concentration was revealed by resonance energy transfer from tryptophan resi- dues to 8-anilinonaphthalene-1-sulfonate. A treatment of the autocorrelation functions as obtained by dynamic light scat- tering measurements, based on the application of appropriate fitting techniques, allowed for the discrimination between two kinds of structures in the OTG/BSA system: surfactant-protein complexes, with a “pearl necklace” structure, in equi- librium with the free micelles of OTG. Keywords: Protein-surfactant interactions, binding studies, BSA, OTG. INTRODUCTION The occurrence of interactions between proteins and low molecular weight surfactants has relevance in a number of biotechnological disciplines. The role of these interactions in applications such as drug delivery, cosmetics, or solubilizing processes used for the extraction, isolation and purification of biological macromolecules is decisive. In addition, since the general principles underlying the formation of protein- micellar assembly are common to others like reverse mi- celles, bilayers, liposomes, and biological membranes [1, 2], a proper understanding on the surfactant-protein interactions is very important because it can be used to our advantage in the choice of the surfactant [3, 4]. Therefore, the characteri- zation of the interactions responsible for the binding of sur- factants to proteins has been a subject of extensive study for many years by using a wide range of physical methods [4-6]. Serum albumins are the most abundant proteins in blood plasma and have the function of incorporating and transport- ing lipids such as fats, cholesterol and derivatives, into the lymph or bloodstream. Bovine serum albumin (BSA) is a protein that is frequently used for research purposes and as a reference in clinical analyses and biochemistry research due *Address correspondence to this author at the Grupo de Fluidos Estruc- turados y Sistemas Anfifílicos, Departamento de Física Aplicada II, Escuela Universitaria Politécnica, Universidad de Málaga, Campus de El Ejido, 29071 - Málaga, Spain; E-mail: ccarnero@uma.es to its stability, water solubility, and versatile binding capac- ity [7, 8]. The binding of surfactants to BSA depends on the surfactant nature. For example, it is well-known that anionic surfactants interact strongly with BSA and other proteins causing their denaturation. This effect has been attributed to the ability of these surfactants to associate micelle-like ag- gregates on the surface of the protein promoting unfolding and, consequently, protein structure alterations [9]. For this reason, many studies have focused on the binding of sodium dodecyl sulfate (SDS) and other anionic surfactants, the studies involving cationic and nonionic surfactants being relatively fewer [8-18]. Because of their favourable physicochemical properties, nonionic surfactants are extensively used in many fields of technology and research [19]. From the point of view of the protein-surfactant interaction the nonionic surfactants are generally less effective than ionic ones, however, there are applications which require to preserve the protein functional- ity and, hence, the use of nonionic surfactants is preferred. The nature of the interactions responsible for the binding of ethoxylated nonionic surfactants to proteins is being charac- terized. These investigations indicate that the hydrophobic moiety of surfactants can bind to the non-polar amino acids, whereas the hydrophilic ethyleneoxide chain can interact with the peptide bond and with one or more polar amino acids residues, probably by electrostatic interactions and hydrogen bonding [19, 20]. In many applications in the biomembrane field, which imply solubilization and purifica-