Heat-Induced Redistribution of Disulfide Bonds in Milk Proteins. 2. Disulfide Bonding Patterns Between Bovine β-Lactoglobulin and κ-Casein
Published in Journal of Agricultural and Food Chemistry, Volume 52, Issue 25, December 15, 2004, pages 7669-7680.
Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with κ-casein (κ-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys121 of β-lactoglobulin (β-LG) has been implicated. The reaction at 60°C between β-LG A and an activated κ-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [β-LG(102-124)] and κ-CN Cys88 and others between κ-CN Cys88 or κ-CN Cys11 and β-LG Cys160. Only the latter two novel disulfide bonds were identified in heated (90°C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to β-LG(102-135): κ-CN(11-13) and β-LG A(61- 69): κ-CN(87s97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, β-LG(1-40) and β-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.