Problems associated with the use of exclusion-diffusion chromatography for identification of zinc ligands in human...

The effect of operating conditions used in gel filtration, i.e. the type of gel and elution buffer, on the qualitative and quantitative distribution of zinc in human milk was studied. The gels used were Sephadex G10 and G15, Biogel P2 and Trisacryl GF05. The three elution buffers used were 0.05 M Tris-HCl, pH = 7.4, 0.05 M Tris-acetate, pH = 7.4 and 0.1 M ammonium acetate, pH = 6.5. Ultrafiltration was used as an additional technique for the verification of a number of the results obtained. Zinc ligands in human milk are, possibly, serum albumin and citric acid. The results showed a gel effect, which was greater on the identification and quantitation of the low molecular-weight ligand (the percentage of zinc associated with citric acid was lower with Biogel P2). The elution buffer had a considerable effect, particularly when quantifying the protein-bound fraction. The percentage of zinc coeluted with proteins decreased when the elution buffer contained acetate ions. The combination of Sephadex G15 and 0.05 M Tris-HCl, pH = 7.4 was found to be the best compromise. Zinc distribution with this system was 35-44% of zinc coeluted with proteins and 18-22% with citric acid.