Characterization of protein-bound metal species by bioanalytical and neutron activation techniques

Several bioanalytical techniques have been employed in conjunction with instrumental neutron activation analysis (INAA) to study metalloproteins and protein-bound trace elements in bovine kidneys. The accuracy of the INAA method developed has been evaluated by analyzing certified reference materials. Up to 29 elements can be reliably determined in biological tissue samples by INAA. Dialysis of the bovine kidney homogenate shows that more than 90% of Ca, Cd, Cu, Fe, Mg, Mn, Mo, Se, V and Zn, and about 20% of Br are bound to macromolecules, mainly proteins. A combination of gel filtration and ion exchange chromatography, chromatofocusing, electrofocusing, ammonium sulphate precipitation and INAA of the bovine kidney microsomecytosol subcellular fraction further indicates that much of the copper is associated with a single protein of an isoelectric point around 5 and a molecular weight of about 30 000 daltons. Some results on manganese proteins are also reported.