Determination of molecular weight of a purified fraction of colloidal calcium phosphate derived from the casein micelles of bovine milk

Colloidal calcium phosphate (CCP) plays a key role in the formation and integrity of casein (CN) micelles. However, limited information is available on the molecular weight (Mw) of CCP. Recently, we theoretically derived the Mw of CCP and the objectives of this study were to experimentally determine the Mw of CCP. We used 2 methods to prepare CCP fractions: skim milk was enzymatically digested with either trypsin or a combination of papain and proteinase enzymes to remove most CN. The CN phosphopeptides are resistant to trypsin hydrolysis. Digestion was carried out in a membrane tube that was dialyzed against the same bulk milk used in sample preparation to remove small peptides and to minimize perturbation of CCP. After digestion, the protein contents of the enzyme-treated milks were 0.92 and 0.36% for the trypsin and papain-proteinase treatments, respectively. Size-exclusion chromatography, coupled with multi-angle laser light scattering, was used to separate the CCP-phosphopeptide fraction from the digested mixture. Simulated milk ultrafiltrate was used as a mobile phase during size-exclusion chromatography separation to try to preserve the integrity of CCP. Size-exclusion chromatography peaks, which had higher Ca and P contents than the baseline, were identified as the likely fractions containing the phosphopeptide-stabilized CCP; this peak eluted with retention times of 100 to approximately 110 min for trypsinated samples. The papain-proteinase treatment caused excessive loss of CN that were needed to stabilize CCP, which resulted in no obvious peak that had elevated Ca and P contents. Debye plots at these retention times indicated that the weight-average Mw for the fraction prepared by trypsin was 17,450 g/mol. Attempts to estimate the Mw of the phosphopeptides associated with CCP using sodium dodecyl sulfate-PAGE were not successful, as we did not observe any peptide bands in these gels, presumably because of their low concentration in the isolated, unconcentrated fraction. Assuming that 4 CN phosphopeptides stabilized each CCP and if the Mw of each of these phosphopeptides was about 2,500 g/mol, then the Mw of CCP would be around 7,450 g/mol. This experimental value was close to the theoretically-derived Mw of 4,897 and 9,757 g/mol for tetrahedron and bi-pyramid shaped objects, respectively, when using the brushite form of calcium phosphate.