Fractionation of -Lactoglobulin Tryptic Peptides by Ampholyte-Free Isoelectric Focusing PAULE EMILIE GROLEAU, ² RAFAEL JIMENEZ-FLORES, ‡ SYLVIE F. GAUTHIER, ² AND YVES POULIOT* ,² Centre de Recherche STELA, Pavillon Paul-Comtois, Universite ´ Laval, Que ´bec, Canada G1K 7P4, and Dairy Products and Technology Centre, California Polytechnic State University, San Luis Obispo, California 93407 Solutions of tryptic hydrolysate of bovine -lactoglobulin were fractionated by liquid-phase IEF in a preparative Rotofor cell at constant power for 2 h without ampholytes in order to identify interactions between peptides. The 20 peptide fractions collected were analyzed by capillary electrophoresis and SDS-PAGE under native, denaturing, and reducing conditions. The hydrolysate was shown to be composed mainly of acidic peptides (pI 2-5, 62%) of molecular mass below 6 kDa, and numerous disulfide bonds were detected. Purified peptides (-LG 15-20, 71-75, 76-82, and 84-91) were also focused individually and in mixtures and matched to components of the IEF fractions obtained from the tryptic hydrolysate of -LG. The separation of acidic (-LG 84-91) and basic (-LG 76-82) peptides was achieved by IEF, whereas uncharged peptides (-LG 15-20 and 71-75) were poorly separated due to their low electrophoretic mobility. Because no peptide-peptide interaction could be identified by IEF fractionation, it is suggested that electrical fields may decrease electrostatic interactions between charged peptides. KEYWORDS: Whey proteins; -lactoglobulin; tryptic hydrolysate; peptides; peptide-peptide interactions; isoelectric focusing; nanofiltration INTRODUCTION The emerging market for nutraceuticals and functional foods has stimulated the production of enzymatic hydrolysates from whey proteins (1) with improved functional properties and biologically active peptides (2, 3). The levels of bioactive peptides in such hydrolysates are low, however, creating a demand for techniques capable of providing rapid and efficient isolation of these molecules. Nanofiltration (NF) membranes may be used to separate peptides according to mass and charge and have been used to separate amino acids in model systems (4, 5), peptides (6), and enzymatic hydrolysates from whey proteins (7). The mechanism underlying NF separation is a molecular sieve effect or a charge effect or both, depending on membrane characteristics (8). Although model solutions have helped to characterize selectivity, it remains difficult to predict the permeation of peptides from a complex mixture such as a hydrolysate. The selectivity of membrane separation techniques such as nanofiltration in the fractionation of enzymatic hydrolysates of proteins is believed to be impaired by peptide-peptide interactions. Pouliot et al. (7) have reported that the same peptide was transferred differently depending on whether it was produced by tryptic or chymotryptic hydrolysis. Similar differences have been observed for the transfer of specific peptides when the ionic strength of the solution was increased before filtration (9). Selectivity thus appears to be dependent on peptide properties, on surrounding peptides, and on processing conditions. Competition between peptides at pores and peptide-peptide interactions are both believed to modify individual peptide permeation through membranes. The hydrolysis of proteins by enzymes produces substances of lower molecular mass with increased numbers of ionizable groups and increased exposure of hydrophobic groups (10, 11), creating reactive peptides that are more likely to interact. Cassaens et al. (12) demonstrated that peptides obtained from -lactoglobulin (-LG) hydrolysis with trypsin and Staphylo- coccus aureus V8 protease may associate via hydrophobic interactions and disulfide bonds but that these associations are especially prevalent among plasmin-derived peptides. Chen et al. (13) and Otte et al. (14) have also reported that peptides derived from partially hydrolyzed -LG interact via non-covalent bonds, mainly by electrostatic and hydrophobic interactions, and form stronger gels than intact -LG. According to Otte et al. (15), peptides obtained from the N-terminal region of -LG have a greater tendency to aggregate due to the clustering of hydrophobic and hydrophilic groups. The nature of the peptides and the physicochemical characteristics of their surroundings thus both influence peptide behavior in mixtures such as protein * Author to whom correspondence should be addressed [telephone (418) 656-5988; fax (418) 656-3353; e-mail Yves.Pouliot@aln.ulaval.ca]. ² Universite ´ Laval. ‡ California Polytechnic Institute. 578 J. Agric. Food Chem. 2002, 50, 578-583 10.1021/jf010772u CCC: $22.00 © 2002 American Chemical Society Published on Web 12/22/2001