Fractionation of β-Lactoglobulin Tryptic Peptides by Ampholyte-Free Isoelectric Focusing
Published in Journal of Agricultural and Food Chemistry, Volume 50, Issue 3, January 30, 2002, pages 578-583.
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 (p/ 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.