Fractionation of β-Lactoglobulin Tryptic Peptides by Ampholyte-Free Isoelectric Focusing

 Université Laval.

 California Polytechnic Institute.

 Author to whom correspondence should be addressed [telephone (418) 656-5988; fax (418) 656-3353; e-mail Yves.Pouliot@aln.ulaval.ca].

Solutions of tryptic hydrolysate of bovine β-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 (β-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 β-LG. The separation of acidic (β-LG 84−91) and basic (β-LG 76−82) peptides was achieved by IEF, whereas uncharged peptides (β-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; β-lactoglobulin; tryptic hydrolysate; peptides; peptide−peptide interactions; isoelectric focusing; nanofiltration