Proteomic quantification of disulfide-linked polymers in raw and heated bovine milk.
Disulfide bond formation between milk protein molecules was quantified in raw and heated bovine milk using reducing and nonreducing two-dimensional electrophoresis. Analysis of protein profiles in raw milk indicated that 18% of alpha(S2)-casein, 25% of beta-lactoglobulin, and 46% of kappa-casein molecules were involved in disulfide-linked complexes (calculated through differences in spot volumes on two-dimensional electrophoretograms under reducing and nonreducing conditions), whereas levels of alpha(S1)- and beta-caseins were similar under both conditions. Following heat treatment at 90 degrees C for 30 min, spot volumes of serum albumin, beta-lactoglobulin, and kappa-casein decreased by 85%, 75%, and 75%, respectively, with the formation of several spots on nonreducing gels corresponding to polymers. Homopolymers and heteropolymers of kappa-casein and alpha(S2)-casein were identified by mass spectrometry in raw milk samples; polymers involving only alpha(S2)-casein or only kappa-casein accounted for 43% and 12% of the total polymers present, respectively. In addition, 45% of polymers in raw milk involved alpha(S2)-casein in association with other proteins as heteropolymers, indicating the key role of this protein in intermolecular disulfide bridging between proteins in raw milk. The intensity of monomeric kappa-casein spots decreased progressively with heating time at 90 degrees C, with greatest changes in spots with acidic isoelectric points. Interactions and association of milk proteins via disulfide bridges are discussed in relation to the proteins involved and their potential protective function against formation of fibril aggregates.
Chevalier
François
F
School of Food and Nutritional Sciences, University College Cork, Cork, Ireland. francois.chevalier@cea.fr
Kelly
Alan L
AL
D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006358:Hot Temperature; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D040901:Proteomics
D004220:Disulfides; D008894:Milk Proteins; D011108:Polymers