184 J. Agric. Food Chem. lQQ2, 40, 184-190 Acylation and Alkylation of Bovine @-Lactoglobulin in Organic Solvents Carole Creuzenet, A. Touati, E. Dufour, Y. Choiset, J. M. Chobert, and T. Haertl6' LEIMA, Institut National de la Recherche Agronomique, BP 527, 44026 Nantes Cedex 03, France Bovine @-lactoglobulin (BLG)was solubilized in solvent systems of varying polarity and hydrophobicity. An amount of 2 mg/mL initially added could be solubilized up to 70% (1.4 mg/mL) in CHC13/CH30H (7/3, v/v) acidified with HC1 and up to 40% (0.8 mg/mL) in CHC13/CH30H (3/7, v/v) in the presence of triethylamine. After solubilization in organic solvents, the yield of reductive alkylation is much higher (98%) than that in aqueous conditions (75%). Acylation of BLG with stearic acid anhydride was also performed in binary mixtures of methanol and chloroform. The efficiency of substitutions of c-amino groups in studied organic systems amounted to 80% compared to none in water. Besides, the emulsifying activity of 40% stearylated BLG was 3-6-fold superior (depending on the pH) than that of unmodified BLG, even after a 30-min heating at 80 "C. INTRODUCTION &Lactoglobulin (BLG) is a compact globular protein found in the milk of several mammal species. It represents as much as 50 % of the total whey proteins in bovine milk. Depending on conditions, BLG (162 amino acids) exists in an oligomeric form or as a monomer (MW 18 200). Two orthogonal @-sheets, one of which is flanked by an a-helix, shape a hydrophobic pocket (Papiz et al., 1986; Monaco et al., 1987) which is probably involved in the binding of various ligands (Futterman and Heller, 1972;Brown, 1984; O'Neill and Kinsella, 1987; Dufour and Haertl6, 1990a; Dufour et al., 1990). A significant amount of already accumulated structural information and the availability of BLG make it a model of choice to study chemical modifications of globular proteins and their influence on the protein functional properties. Since the pioneering works of Fraenkel-Con- rat and Olcott (1945) and Fraenkel-Conrat and Feeney (1950),many studies have been done in this field, namely lipophilization of glycinin (Haque et al., 1982)or of BLG (Akita and Nakai, 1990a,b), amidation and esterification of BLG (Mattarella and Richardson, 1983; Mattarella et al., 19831,alkylation of @-casein and &casein tryptic pep- tides (Chobert et al., 1990; Touati et al., 1990), and gly- cosylation of BLG (Bertrand-Harb et al., 1990; Waniska and Kinsella, 1988). Most of these protein modifications were performed to improve their foaming and/or emul- sifyingproperties. The outcome of these studies supports the idea that the hydrophilic/lipophilic balance and hydrophobic/hydrophilic interactions are the most im- portant factors of the surface activity of proteins (Akita and Nakai, 1990a;Kinsella, 1979;Kat0 et al., 1981;Chobert et al., 1987). Numerous studies on the conformation of proteins sol- ubilized in hydroorganic media have demonstrated that proteins undergo changes in their secondary structure, the extent of which depends on the solvent system used. Initially, the structural changes of BLG under the influence of weakly protic solvents in acid pHs were studied by Tan- ford et al. (1960) and Townend et al. (1967). Then, Inoue and Timasheff (1967) demonstrated that BLG acquired a highly a-helical conformation in 2-~hloroethanol/water (114 v/v) or in methanol/water (2/3 v/v). More recently, Dufour and Haertl6 (1990b) showed that in hydroalco- holic mixtures BLG undergoes a reversible structural * To whom correspondence should be addressed. transition depending on the polarity and the dielectric constant (t) of the solvent. In aqueous media, native BLG contains 52% @-sheets and 8% a-helices. It refolds in a hydroalcoholic mixture with t = 50, reaching 50 % a-helices. According to Nozaki and Tanford (19711,the hydrophilic- ity of the peptide backbone is one of the factors that may explain why proteins acquire a highly a-helical structure when dissolved in organic solvents. It has been demonstrated that alkylation could increase the strength of interactions between BLG and some of its ligands (Dufour and Haertl6, 1990a,b) but that it was incomplete (75 % ) when performed in aqueous solutions, unless a large excess of reagents is applied during a long reaction time. Consequently, it was tempting to submit the globulin structure to strong solvent perturbations to disorganize its folding, with the hope that randomized side chains were going to be less protected against full and fast substitution. The present work establishes conditions of reasonable solubility of BLG in binary organic mixtures (methanol and chloroform) of varying hydrophobicity and polarity. Then it describes alkylation and acylation yields obtained in such solvent systems, as well as interface properties of some BLG derivatives. MATERIALS AND METHODS Materials. Bovine 8-lactoglobulin was a kind gift of Dr. J. Faucant and J. C. Maubois, INRA (Rennes,France). It has been isolated from lactoserum by ultrafiltration, as a mixture of two variants: A (38.5%) and B (35.7%). It also contained a-lactai- bumin (2.5 %) as the main protein contaminant, as judged from reversed-phaseHPLC (RP-HPLC) chromatograms (Figure 2A) and electrophoresis data (Figure 3). Except for the a-lactalbu- min band, the electrophorogram (Figure 3) only shows a faint band (MW = 37 OOO), which is probably a BLG dimer. Hence, the purity of the used BLG preparation appears, on the gels, to exceed 75% calculated on the basis of HPLC data. Stearic anhydride was from Fluka (Mulhouse,France). Suc- cinic and glutaric anhydrides as well as acetaldehydeand amines were from Aldrich (Strasbourg, France). The solvents,methanol and chloroform, were from Carlo Erba (Milan, Italy). Solubility Assays. Solubility studies were performed in binary mixtures of chloroform and methanol containing 0,10, ..., 100% (v/v)methanol, with the addition of an acid (5 N HCl, 10 wL/mL of solvent) or a tertiary amine (triethyl-, tripropyl-, or tributylamine (TEA, TPA, TBA), 25 pmol/mg of BLG). During the solubility tests, 10 mg of BLG (mi = 10 mg) was dissolved in 5 mL of solvent mixtures (Vi = 5 mL). After 4 h of stirring at room temperature, a known volume (Vf) of solution was filtered 0 1992 American Chemical Society