Thermodynamic Parameters of -Lactoglobulin-Pectin Complexes Assessed by Isothermal Titration Calorimetry MAUDE GIRARD,SYLVIE L. TURGEON,* AND SYLVIE F. GAUTHIER STELA Dairy Research Centre, Faculte ´ des sciences de l’agriculture et de l’alimentation, Pavillon Paul-Comtois, Universite ´ Laval, Que ´bec, Canada, G1K 7P4 Isothermal titration calorimetry (ITC) was used to determine the binding constant, stoichiometry, enthalpy, and entropy of -lactoglobulin/low- and high-methoxyl pectin (-lg-LM- and HM-pectin) complexes at 22 °C and at pH 4. The binding isotherms revealed the formation of soluble intrapolymer complexes (C1) further followed by their aggregation in interpolymer complexes (C2). The interaction between -lg and LM- or HM-pectin in C1 and C2 occurred spontaneously with a Gibbs free energy around -10 kcal/mol. The C1 were enthalpically driven, whereas enthalpic and entropic factors were involved in the C2 formation. Because ITC did not allow the dissociation of different enthalpic contributions, the values measured as pectin and -lg interacted could partially be attributed to conformational changes. The C1 had a binding stoichiometry of 8.3 and 6.1 -lg molecules complexed per LM- or HM-pectin molecule, respectively. The C2 had about 16.5 and 15.1 -lg molecules complexed per LM- and HM-pectin, respectively. KEYWORDS: -Lactoglobulin; pectin; interactions; isothermal titration calorimetry INTRODUCTION The -lactoglobulin (-lg) is a small globular protein of 18 350 Da. The -lg has a well-known structure containing mainly -sheets, some -turns, and one R-helix (1). Its quaternary structure is influenced by pH, mineral content, and temperature. This protein is monomeric at pH below 3 or above 8, dimeric at neutral pH, and found in both states between pH 4 and 6 (2). The -lg may form a few octamers around pH 4.5 (3), but some authors are doubtful about the existence of such an assembly (2, 4). Pectin is a cell-wall polysaccharide, generally extracted from apple pomace or citrus fruits. This anionic polysaccharide is essentially made up of D-galacturonic residues (D-galA). High-methoxyl (HM) pectin occurs when more than 50% of its carboxylic groups are esterified, whereas low- methoxyl (LM) pectin is found when less than 50% of these groups are esterified (5). The interactions between proteins and polysaccharides can lead to the formation of complexes. These complexes, or coacervates, have many applications, including fat substitution (6), protein separation (7), microencapsulation (8), and enzyme immobilization (9). The nature of protein/polysaccharide com- plexes is influenced by entropic factors, such as the structure, and the molecular weight of biopolymers. The complexes are also influenced by enthalpic forces, which are regulated by the protein/polysaccharide ratio and the nature and density of charges on the biopolymers. Many studies on protein/polysac- charide interactions have been carried out (10-13). Ultrafil- tration used with destabilizing agents showed that interactions between -lg and pectin occurred through ionic and hydrogen bonds, whereas the hydrophobic interactions were negligible (14). The measurement of thermodynamic parameters, such as binding constant, enthalpy, entropy, and binding stoichiometry of interactions, is essential to accurately evaluate binding conditions. Binding parameters from biopolymer systems are sometimes hard to quantify because of the heterogeneity of the natural material. Consequently, few quantitative studies have been carried out on the interactions between -lactoglobulin (- lg) and polysaccharides (15). Thermodynamic quantities in- volved in biopolymer interactions are often indirectly determined through noncalorimetric experimental methods (16). These methods involve the calculation of thermodynamic parameters from theoretical relationships. Enthalpy variations (i.e., gain and loss) are the easiest thermodynamic parameters to measure during complexation. Isothermal titration calorimetry (ITC) is currently the only technique used to directly measure the enthalpy for many ternary mixtures (16). ITC was used to study the interaction between -lg and LM- or HM-pectin at pH 4 in 5 mM of sodium phosphate buffer. To our knowledge, this technique has not yet been used to carry out a binding study involving two biopoly- mers having heterogeneous molecular weights. MATERIALS AND METHODS Materials. Bovine -lg A and B (3X crystallized) was purchased from Sigma-Aldrich Canada Ltd (Oakville, On, Canada) as 95% pure protein. Low-methoxyl pectin (DE 28.3%, apparent molecular weight 94.3 kDa) and high-methoxyl pectin (DE 73.4%, apparent molecular weight 118 kDa) were donated by Hercules Copenhagen A/S (Copen- hagen, Denmark). All reagents were of analytical grade and used as received. * To whom correspondence should be addressed. Tel.: (418) 656-2131 ext. 4970. Fax: (418) 656-3353. E-mail: Sylvie.Turgeon@aln.ulaval.ca. 4450 J. Agric. Food Chem. 2003, 51, 4450-4455 10.1021/jf0259359 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/18/2003