Free zinc concentration in bovine milk measured by analytical affinity chromatography with immobilized metallothionein

A new analytical affinity chromatography method was developed for measuring the free Zn²⁺] concentration in bovine milk. The column was generated by immobilizing avidin and attaching biotinylated metallothionein (MT) on controlled-pore glass beads. Zinc bound to the MT column at physiological free Zn²⁺] concentration and was dissociated again in an elution buffer of pH 2. The distributions of extrinsically added⁶⁵Zn and native zinc in different fractions of milk were virtually identical, validating the use of extrinsic labeling in studies of the free Zn²⁺] concentration in milk. Extrinsically labeled whey fractions were mixed with standard solutions whose free Zn²⁺] concentrations were calculated by computer model.⁶⁵Zn retained by the column provided an indication of free Zn²⁺] concentration in the mixture, and by interpolation, in the original milk. The free Zn²⁺] concentration measured by the affinity chromatography method in the milk of a group of six cows was 90.4±29.7 pM. This value is similar to estimates of free Zn²⁺] concentrations in other biological fluids by entirely different methods. Measurement of free Zn²⁺] may be helpful in understanding the physiology and biochemistry of zinc metabolism.