Abstract
Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.
Highlights
Many techniques have been used to investigate the ultrastructure of the casein micelles
We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses
The ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides
Summary
Preparation of Casein Phosphopeptides—The casein phosphopeptides -CN[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25] and ␣S1-CN(59 –79) were selectively precipitated from a tryptic digest of casein using calcium chloride and ethanol and further purified by anion exchange fast protein liquid chromatography and reversed phase HPLC [13]. The calcium concentrations of the original reaction mixture after centrifugation to demonstrate no precipitation and of the ultrafiltrate were determined, and the peptide-bound calcium was calculated as the difference between these values. To confirm that no precipitation had occurred during incubation, the samples were centrifuged at 17,000 ϫ g for 5 min, and the calcium and phosphate concentrations were determined prior to ultrafiltration. The ion activity products for various phases of calcium phosphate were determined from the free calcium and phosphate concentrations and pH using an iterative computational procedure that calculates the ion activity coefficients using the expanded DebyeHuckel equation This procedure takes into account ion pairs CaHPO40, CaH2PO4ϩ, and CaPO4Ϫ; the dissociation of H3PO4 and H2O; and the ionic strength [32, 34]. Observations were made with a field emission SEM instrument (Philips XL 30 FEG, Eindhoven, The Netherlands) operating at 20 kV using the secondary electron mode
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
