Identification of peptides released from casein micelles by limited trypsinolysis

Bovine casein micelles were treated with low concentrations of trypsin before and after heating. Peptides present in the supernatant solutions of time course samples after high-speed centrifugation were separated by reversed-phase HPLC and identified principally by mass spectrometry. beta-Casein was hydrolyzed rapidly, the initial cleavages occurring in the region of the plasmin-sensitive bonds. alpha(s1)-Casein was hydrolyzed more slowly, the initial cleavage points being in the N-terminal region, but some trypsin-sensitive bonds in this part of the molecule appeared to be shielded. alpha(s2)-Casein was hydrolyzed slowly from the C-terminal. Hydrolysis of kappa-casein was insignificant. No peptides containing phosphoserine clusters were released. Although heating increased the rate of hydrolysis of all of the caseins, the HPLC profiles were generally similar to those obtained with unheated micelles. The results are discussed in relation to the possible structure of the surface of the casein micelle.