SummaryAbout 90% of the Zn in bovine skim milk was sedimented by ultracentrifugation at 100000 g for 1 h. About half of the non-sedimentable Zn was non-dialysable, indicating that it was associated with protein, probably non-sedimented casein micelles. Casein micelles incorporated considerable amounts of Zn added to skim milk as ZnCl2, and at Zn concentrations ≥ 16 mM coagulation of casein micelles occurred. Ca was displaced from casein micelles by increasing ZnCl2 concentration and ˜ 40% of micellar Ca was displaced by 16 mM-ZnCl2. Micellar Zn, Ca and P1 were gradually rendered soluble as the pH of milk was lowered and at pH 4·6 > 95% of the Zn, Ca and P1 were non-sedimentable. These changes were largely reversible by readjustment of the pH to 6·7. About 40% of the total Zn in skim milk was non-sedimentable at 0·2 mM-EDTA and most of the remainder was gradually rendered soluble by EDTA over the concentration range 1–50 mM. This indicates that there are two distinct micellar Zn fractions. No micellar Ca or P1 was solubilized at EDTA concentrations up to 1·0 mM, indicating that both colloidal calcium phosphate (CCP) and casein micelles remained intact under conditions where the more loosely bound micellar Zn fraction dissolved. Depletion of casein micelles of colloidal Ca and P1 by acidification and equilibrium dialysis resulted in removal of Zn, and in colloidal Pi-free milk non-dialysable Zn was reduced to ·-2 mg/1 (˜ 32% of the original Zn). Thus, ˜ 32% of the Zn in skim milk is directly bound to caseins, while ˜ 63% is associated with CCP. Over 80% of the Zn in colloidal Pi-free milk was rendered soluble by 0·2 mM-EDTA, indicating that the casein-bound Zn is the loosely bound Zn fraction in casein micelles. A considerable fraction of the Zn in acid whey (pH 4·6) co-precipitated with Ca and Pi on raising the pH to 6·7 and heating for 2 h at 40 °C, indicating that insoluble Zn phosphate complexes form readily under these conditions. Studies on dialysis of milk against water, or dilution of milk or casein micelles with water, showed that CCP and its associated Zn is very stable and dissolves only very slowly at pH 6·6. The nature of Zn binding in casein micelles may help to explain the lower nutritional bioavailability of Zn in bovine milk and infant formulae compared with human milk.
The histidine-rich loop in the extracellular domain of ZIP4 binds zinc and plays a role in zinc transport
